Treating Cancer Through Metastasis Neutralization and Possible Activation

While cancer in any form is potentially dangerous to a patent, it is widely acknowledged that only a small percentage of primary tumors are threatening to the life of a patient in the interim. A vast majority of cancer patient deaths occur due to cancer metastasis. Metastasis is a complex pathway of molecular interactions that produce an end result that involves the departure of a group of tumor cells from the primary tumor into the bloodstream and eventual invasion into other tissues resulting in the formation of additional tumors. Not surprisingly the process is governed by a number of complex pathways that are not fully understood. Despite this lack of knowledge regarding metastasis, is it clear that one of the best strategies for addressing cancer would be to create a therapeutic regimen that would prevent metastasis from occurring in the first place allowing physicians ample time to eradicate the primary tumor with no legitimate threat of reoccurrence.

Of the agents thought to be involved in cancer metastasis chemokine receptor CXCR4 is a promising agent of study and potential therapeutic target. Chemokines are a group of low molecular weight cytokines that induce chemotaxis, most of the time as chemoattractants, largely in leukocytes, endothelial and epithelial cells.1 Chemokines are commonly classified into CC, XC, CXC or CX3C designations based on the positioning of their respective conserved cysteine residues.1 One of the key normal functions of chemotaxis is to facilitate the movement of pro-inflammatory cells to the site of inflammation, including immune cells, normally after some form of injury. CXCR4 is an attractive target because of both strong anecdotal and experimental evidence regarding its overall expression and role in tumor malignancy and metastasis.1-5

CXCR4 functions as a G protein-coupled receptor (GPCR) that principally binds stromal cell-derived factor 1 (SDF-1), which is also known as CXCL12. With regards to cancer, CXCR4 plays a significant role in directional migration though activation of actin polymerization6,7 as well as invasion and adhesion, which influence the overall level of aggression for a tumor. There is also some evidence suggesting that CXCR4 plays a role in angiogenesis as well.4,5

CXCR4 can undergo four major changes to influence its functionality: homo or heterodimerization (with CCR2, CCR5, CXCR7 or CD4), phosphorylation, glycosylation, or sulfation.8-14 Unfortunately limited information is known about functional changes associated with dimerization in cancer for almost all studies involving CXCR4 dimerization relate to HIV, but it is thought that such changes enhance CXCL12 binding. It is also believed that dimerization typically occurs internally before CXCR4 is expressed on the cell surface, typically as oligomers. This oligomeric structure persists in the plasma membrane.14

Phosphorylation occurs principally at serine residue number 339 (Ser339) after exposure to either CXCL12, epidermal growth factor (EGF), or phorbol ester and it is believed that phosphorylation may also occur to a much smaller extent at Ser324, Ser325, and Ser330.12 Phosphorylation is important for increasing the probability of receptor internalization and secondary messenger activation. On a side note mono-ubiquitination occurs at Lys327, Lys331 or Lys333.13 Glycosylation of human born CXCR4 only appears to occur at Asn11 and seems to serve no unique function other than stabilizing CXCL12 binding (lack of glycosylation reduces binding efficiency).1

Sulfation, which takes place primarily at Tyr21 and does not appear to occur on two other potential sites (Tyr7 and Tyr12),15 may the most interesting modification regarding CXCL12 binding probability and functionality.16 When CXCL12 binds to CXCR4 there is a specific site interaction between sulfated Tyr21 on CXCR4 and Arg47 on CXCL12.15 One of the reasons that sulfation appears important is that one study demonstrated that while highly metastatic NPC cells and non-metastatic NPC cells expressed similar levels of CXCR4, both via mRNA and protein, only high levels of sulfinated CXCR4 resulted in high metastatic potential.17

While there are a number of tyrosine sulfation pathways, with regards to CXCR4 one of the more prominent interactions involves the action of the latent membrane protein 1 (LMP1). While LMP1 concentration changes are not universal to CXCR4 activity increases, LMP1 interacts with EGF receptors, which is thought to be one of the early steps in inducing Tyrosylprotein sulfotransferase-1 (TPST-1) dependent tyrosine sulfation of CXCR4.15 However, there is the lingering question of whether sulfation is the chicken or the egg, is it a chief agent in how CXCR4 activation influences the potency of cancer or is it a result of that activation?

CXCR4 activation also significantly increases the overall expression of matrix metalloproteinases (MMP), especially MMP-2, MMP-3, MMP-7 and MMP-9.18,19 One of the major triggers for this action could involve the activated CXCR4 guiding Bone marrow-derived cells (BMDCs) to their pre-metastatic niche, which then triggers BMDCs to initiate the release various metastatic positive elements including various MMPs most notably MMP-7 and MMP-9.20,21 CXCR4 is also involved in the homing of cells into the endosteal HSC,22 which facilitates the expression of SUMO-specific protease 1 that regulates MMP-9 as well.23

As stated above one of the key steps in metastasis is the proteolytic degradation of the extracellular matrix (ECM) in which various MMPs are critical agents. Initially MMPs were thought to be degenerative proteases that were limited to cleaving matrix components, but that role has expanded to include the release of growth factors and other bioactive peptides localized at cleaved extracellular matrices.24-26 While there are up to 26 known MMPs (MMP-1, MMP-2, MMP-3, …) only a few have demonstrated significant roles in both cancer growth and cancer metastasis. Of these select few MMPs that play a prominent role in cancer, MMP-3 and MMP-7 appear to be the most important.

MMP-7, (a.k.a. matrilysin) is the smallest MMP and is commonly expressed in epithelial tumor cells instead of interstitial cells27 and has numerous substrates in the ECM namely collagen fibers, laminin, gelatin, proteoglycan and elastin, etc.28,29 MMP-7 is commonly over-expressed in various types of cancer including, but not limited to non-small cell lung, pancreatic, oral squamous cell carcinoma, colorectal, prostate, stomach and papillary thyroid carcinoma.28,30-34 In addition to its ECM degradation role, MMP-7 can also breakdown cell surface proteins, which aids cancer cell proliferation through the regulation of apoptosis and angiogenesis as well as help evade immune system detection.34-36

Furthermore, as mentioned above, MMP-7 is thought to increase expression of MMP-2 and MMP-9 to aid in ECM degradation and other pro-cancer actions.37,38 Finally not surprisingly MMP-7 levels increase in response to decreased blood glucose levels for this increase is tied to a low quality or deteriorating principal environment for the tumor, which should trigger elements responsible for assisting metastasis like MMP-7. However, while MMP-7 appears to be the most active MMP in most cancers, its activation may only be a downstream event caused by MMP-3.

An early action taken by a member of the MMP family typically involves MMP-3 cleaving decorin, releasing growth factor-beta and cleaving transforming growth-factor-alpha (TGF-a), which activates the MAP-kinase pathway.39 This activation can later activate MMP-7, which as previously mentioned leads to the activation of MMP-2 and MMP-9.40 In addition to activating other MMPs like -2, -7 and -9, MMP-3 can also promote genomic instability and epithelial–mesenchymal transition (EMT) through the activation of Rac1b, which stimulates both the production and release of intercellular mitochondrial superoxide.41,42 MMP-3 appears to be the dominant expression route for Rac1b.41

Due to the important role MMPs play in inducing both metastasis and possible anti-apoptosis protection, a number of researchers have thought of MMP inhibition as a promising treatment option. However, clinical trials investigating the viability of MMP inhibitors, commonly known as tissue inhibitors of metalloproteinases or TIMPs, have not proven successful.43 One of the major theories behind this failure is that during tumor development MMPs have different roles depending on tumor progression and the other molecules present in the tumor microenvironment. Some information has demonstrated anti-tumor effects for certain MMPs, most notably MMP-3, MMP-8, MMP-9, MMP-12, and a newer MMP, MMP-26, which may be a natural protectorate MMP.43-47

Clearly this dual behavior makes targeting MMPs directly difficult for therapeutic reasons, as demonstrated clinically, thus it could be more important to focus on important MMP triggers like CXCR4, which appear to activate MMPs during a time when their interaction with the tumor microenvironment will produce a net negative for the patient.

CXCR4 also can activate the P110-beta isoform of PI3K resulting in the eventual synthesis of phosphatidylinositol (3,4,5)-triphosphate, which leads to the phosphorylation of protein kinase B/Akt and mTOR pathways most notably activation of p70S6K and 4E binding protein 1.7,48,49 Not surprisingly mTOR inhibitor, rapamycin, reduces the extent of p70S6K and 4E binding protein 1 activation in a CXCL12/CXCR4 environment.7,19,50 Furthermore CXCR4 also activates elements in the Src family of protein tyrosine kinases, which aid the activation of focal adhesion elements like Crk, paxillin, and tyrosine kinase/Pyk2.51

For a long time CXCR4 was thought to be a unique target for CXCL12 until CXCR7 was identified, potentially complicating the role of CXCR4. Similar to CXCR4, CXCR7 is expressed at a much higher rate in malignant cancer cells versus normal cells and binds CXCL12 with high affinity.10,52 However, despite the significant similarities between CXCR4 and CXCR7, CXCR7 does not appear to play a meaningful role in cancer development or metastasis.53,54 The role of CXCR7 appears to involve the migration of primordial germ cells or interneurons.55 Its increased expression simply may be the result of dramatically increased levels of CXCL12 in the localized environment. Interestingly enough CXCR7 may prove to be a possible therapeutic element as an indirect natural CXCL12 competitive inhibitor of sorts for every CXCL12 molecule that binds to CXCR7 is no longer available to bind to CXCR4.

Earlier it was mentioned that CXCR4 might have a relationship in tumor growth and/or angiogenesis. If true, then this result is complicated because cancer growth at established tumor sites is more rapid in the absence of CXCR4 rather than its presence.53 Therefore, it may be that while CXCR4 assists growth immediately after invasion, its continued presence becomes detrimental for the tumor because it helps induce metastasis, thereby diverting resources like recruited and differentiated endothelial cells or progenitors from the original tumor towards elements that will be involved in the metastasis or even the attraction of metastatic elements already in the bloodstream.

CXCR4 also appears to interact with another potential important factor in cancer metastasis, macrophage migration inhibitory factor (MIF). MIF is a pro-inflammatory cytokine that plays an important role in inflammation and immune response and is expressed at a higher than normal rate during numerous cancer stages like cell proliferation, angiogenesis and anti-tumor immune interaction.56-58 High MIF concentrations have also been associated with poor outcomes in lymphoma, melanoma and colon cancer.59,60 CXCR4 interacts with MIF through the formation of a MIF receptor complex with CD74, which further enhances MIF-stimulated AKT activation.61

There is some thought that when MIF lacks its traditional activation pathway it requires caspase-1 activity for proper secretion.62 Also Golgi-associated protein p115 may be essential for the transport of MIF from the perinuclear ring to the plasma membrane and then out of the cell.63 In addition to aiding metastasis, MIF is also thought to apply some level of apoptosis resistance to cancer cells, favoring those with androgen-dependency over those with androgen-independence, but that resistance may be tied to CXCR4 interaction.64

With CXCR4 having its “fingerprints” over a number of pro-cancer processes, fortunately an additional element that makes it an attractive therapeutic target is its natural role in the body. In non-cancerous tissue CXCR4 is expressed on hematopoietic cells like CD34+ HSC, B-lymphocytes, neutrophils, monocytes, macrophages, and microglia, etc.65 CXCR4 or CXCL12 knockouts in mice result in impaired hematopoiesis through reduced hematopoietic stem cell (HSC) trafficking, which results in heart and brain defects as well as vascularization commonly producing embryonic death;66 in adults CXCR4 is important in HSC homing for the bone marrow microenvironment and lymphocyte trafficking.65 However, most of the time CXCR4 expression in normal cells is low, unless the body has been recently injured. Therefore, treatments that limit CXCL12/CXCR4 pathway activation should result in limited negative side effects for healthy non-injured individuals.

Another potential benefit from CXCR4 inhibition could involve reducing the probability of chemotherapy agent resistance, including Docetaxel (DTX) resistance. Some research suggests that the CXCL12-CXCR4 pathway interacts with p21-activated kinase 4 (PAK4)-induced LIM domain kinase 1 (LIMK1) via phosphorylation to reduce the ability of DTX to destabilize microtubules, which typically results in cell cycle arrest during the G2/M phase.67 Basically CXCR4 activation provides additional protection against cell death for tumors when exposed to DTX. This result suggests that LIMK1 could have a role similar to microtubule-associated protein (MAP) depending on whether or not it is phosphorylated. Therefore, this chemotherapy resistant pathway has two principal inhibition targets in CXCR4 or PAK4 to negatively influence prospective chemo resistance.

Existing potential therapies involving CXCR4 have focused largely on inhibiting the binding capacity of CXCR4 most notably either through the use of AMD3100, a specific CXCR4 antagonist, or synthetic peptide TM4.14,68,69 AD3100 (a.k.a. (Plerixafor) is a small molecule with two cyclam rings connected by a phenylene linker that have nitrogens on each ring that have charge-charge interactions with carboxylate groups on CXCR4, which inhibits CXCL12 binding.70-72

Plerixafor is most commonly used as a pre-treatment element for chemotherapy where, as mentioned, CXCR4 disruption reduces the probability of hematopoietic stem cells homing to bone marrow, thus it increases their circulation in the blood stream allowing for their collection for transplantation after chemotherapy regimens.73,74 Plerixafor has also proven promising as an anti-cancer treatment via its ability to reduce cancer cell chemotherapy resistance by either neutralizing the CXCL12-CXCR4 pathway or reducing the physical attachment of various micro-environment critical cells, similar to what is expected for a CXCR4 inhibitor.

An interesting side effect in plerixafor treatment is that surface expression of CXCR4 increases both in vitro and in vivo.65 One possible explanation for this outcome could be that principal signals that induce CXCR4 expression continue while plerixafor prevents CXCL12 from binding CXCR4; CXCL12 binding leads to internalization and activation of secondary pathways. When there is no CXCL12 binding there is no CXCR4 internalization, but the pathways governing CXCR4 expression towards the cell surface continue, thus explaining the overall increase in CXCR4 expression. If this tendency is accurate then long-term treatment with plerixafor alone may not be beneficial because the increased surface expression will substitute for the CXCR4 “removed” by plerixafor interaction. Basically plerixafor works well alone in the short-term, but may not work well alone in the long-term, which may be the same fate of all substrate based CXCR4 inhibitors.

Apart from preventing metastasis, treating the primary tumor is also an important task, especially when surgical options are unavailable. One promising potential therapeutic agent that can influence both primary tumors and metastasis is salinomycin (SAL), which has demonstrated effective ability to kill cancer via a perceived mixture of apoptotic and autophagic cell death in breast, prostrate, brain, blood, liver, pancreatic and lung cancers with no immediate lethal toxicity.75-77 Initially it was reported that SAL was toxic to certain neuronal cells (dorsal root ganglion in mice) at 1 uM, but this toxicity was neutralized when paired with factors that inhibited mitochondrial Na+/K+ exchangers with no resultant change in cancer cell cytotoxicity.78,79

One of the chief advantages of SAL in treating cancer is that it uses a different methodology apart from more common chemotherapy drugs like Doxorubicin, Cisplatin, Gemcitabine, Temozolamide, Tratsuzumab, Imatinib, etc.75,80-82 SAL has a preference for targeting cancer stem cells (CSCs), which reduces the probability of cancer reoccurrence after its primary removal.75 CSCs are important to address in treatment because they are commonly thought of as another element responsible for driving the core of cancer metastasis after responding to various signal triggers as well as driving cancer recurrence after the primary tumor is eliminated due to their ability to more frequently resist anti-cancer therapies. Thus, addressing CSCs, either directly or indirectly, is a critical part to addressing both cancer itself as well as its metastasis.

Another interesting behavioral aspect of SAL is amplified effectiveness under hypoxia or starvation conditions. This result makes sense on two different levels: first, it is thought that a means in which SAL triggers cell death is through damage to the mitochondria, in part to being a potassium ionophore, promoting hyperpolarization in the mitochondria, which decreases ATP availability and triggers caspase-3, 8 and 9, a consequence worsened by starvation conditions.76,83 Second, its effectiveness against CSCs is enhanced by the reaction of the tumor to hypoxia. In hypoxia the primary tumor will begin to focus an effort to metastasize due to the negative environment that currently exists; one step in this process requires the recruitment and creation of CSCs which reduce resource availability for the primary tumor, yet those CSCs are more effectively eliminated by SAL versus the cells that comprise the primary tumor.

The ability of SAL to “cooperative” with other anti-cancer drugs directly is questionable for most of the benefit from co-therapy between SAL and given drug x appears indirect.84 This result is somewhat interesting because there is some evidence to suggest that SAL can also function as an efflux pump inhibitor, which is commonly operated by a p-glycoprotein;85-87 efflux pumps increase the ability of cancer cells to remove chemotherapy agents before the inducement of cell death, so inhibiting them would increase tumor susceptibility to these chemotherapy agents. However, Metformin (METF), which is though to lower circulating insulin levels and stimulate AMPK-mediated suppression of mTOR, along with having some anti-cancer properties in thyroid, prostate, gastric, breast and glioblastoma,88-90 seems to have some form of direct enhancing cooperative relationship with SAL.84

This combination activity results in the “unspecific” inhibition of EGFR and HER2/HER3 leading to reduced concentrations of AKT and ERK1/2 via an unknown mechanism.84 However, it seems appropriate to suggest that based on SAL activity when acting alone that this “inhibition” is born from a reduction in available receptors due to cancer cell or associated cell death.

Another mechanism for inducing cancer death that includes SAL interaction is autophagy. In its most basic form autophagy involves the “self-digestion” of intracellular elements via the vacuolar lysosomal degradation pathway to recycle cytoplasmic constituents.91 Autophagy is typically used to prevent the accumulation of damaged proteins and organelles, largely born from cell damage due to outside agents; for cancer it would be anti-cancer drugs. This process also reduces the production potential of reactive oxygen species (ROS) that negatively impact cell survival.

There is reason to believe that SAL can interfere with autophagy in cancer cells by inhibiting lysosomal activity driven by cathepsins.85 Interestingly enough this activity occurs without impacting the lysosomal compartment.85 This aspect of SAL interaction is not surprising due to structural similarities with both nigericin and monensin, which have similar behavior as anti-porters themselves, but the lack of change in pH of the lysosomes from SAL treatment belies a different pathway.

Also the behavior of SAL runs contrast to ATG7 expression where ATG7 acts as a protectorate of sorts ensuring the proper functionality of autophagy. For breast cancer cells and more than likely other forms of cancer, aldehyde dehydrogenase 1 positive cells (ALDH+) promote autophagy.85,92 Thus, SAL directly works against ALDH as well as competes to “thwart” ATG7 autophagy protection. Therefore, ATG7 in some instances is able to neutralize the dual ability of SAL to kill cancer cells via induction of apoptosis while inhibiting autophagy inhibition. Therefore, inhibiting the activity of ATG7 may provide a useful co-therapy with SAL to significantly neutralize the ability of cancer cells to build resistance to SAL-related apoptotic activity.

Another popular method of action for SAL against cancerous agents is thought to be its interaction with Wnt signaling and its relation to b-catenin. The interaction between Wnt and b-catenin begins when Wnt binds to frizzled (Fzd) receptor and then that complex binds to lipoprotein receptor-related protein 5 or 6 (LRP5/6) co-receptors leading to a ternary complex that typically exists at the cell surface.93 The presence of this complex can trigger phosphorylation of either LRP leading to the recruitment of axin, which then undergoes endocytosis.93 The end result of this entire process is the breakdown or inactivation of the Adenomatous polyposis coli (APC)-Axin complex, which is responsible for b-catenin elimination.

This processes is important because b-catenin accumulation leads to its nuclear translocation and can even increase expression of Wnt genes via those tumors located at the invasive front, which have more interaction with growth factors and cytokines including hepatocyte growth factor.94 This interaction may even create a positive feedback loop of sorts.94,95 This nuclear translocation of b-catenin is thought to play some role in tumor cells experiencing cell-cycle arrest and EMTs via the loss of E-cadherin expression creating some form of cancer stem cell, with increased migration/metastasis potential.96,97

SAL interferes with the Wnt pathway by degrading the LRP6 protein and possibly LRP5 protein, which obviously reduces the probability that they form a complex with Wnt and are later phosphorylated activating the complex.85 LRP protein importance is further supported by a level of suppression in breast cancer tumor growth after treatment with a LRP antagonist, Mesoderm development (Mesd).98,99

However, SAL is not a cure-all when it comes to this supposed pro-cancer pathway, for Wnt and its associated complex is not the only significant destruction inhibition interaction experienced by b-catenin. Expression of platelet-derived growth factors (PDGF) can induce the tyrosine phosphorylation of p68 via c-Abl kinase.100 After phosphorylation p68 can bind b-catenin and inhibit GSK3b mediated phosphorylation reducing the probability that it is eliminated, thereby increasing the probability of b-catenin nuclear location.100 It is also thought that EGF and TGF-b can induce p68 phosphorylation via receptor tyrosine kinases.100,101

Another possible strategy to deal with LRP protein complex interaction involves the inhibition of vacuolar H+-adenosine triphosphatase using an agent like archazolid.102 LRP6 phosphorylation and internalization appears to require V-ATPase. The general role of V-ATPase is transport of both intracellular and extracellular organelles near the plasma membrane. Not surprisingly it also pumps protons leading to the acidification of vesicles, which promotes endocytosis.103

Another element in cancer development that has garnered attention for metastasis is the role of carcinoma-associated fibroblasts (CAFs). Tumor invasion is heavily influenced by the tumor microenvironment, especially the types of non-tumor cells. Various types of fibroblast recruitment lead to the production of soluble factors and extra-cellular matrix (ECM) remodeling usually through actin changes and cell migration born from MMPs, Rho targeted via ubiquitination and SUMO pathways104 as well as global DNA hypomethylation and recruitment of mesenchymal stromal cells; these changes increase the viability of future invasion.105-108

The cancer stroma is typically populated by various concentrations of fibroblastic cell groups that make up CAFs and are commonly divided into myofibroblast (MFs) and non-myofibroblast populations (non-MFs).105 MF populations have received much more attention than non-MF populations more than likely due to the diversity of the non-MF populations. It is thought that CAFs differ significantly from normal fibroblasts and myoblasts, but there is little information regarding the extent of these differences.105,109

CAFs are heavily involved in various pro-cancer pathways like tumor necrosis factor alpha (TNFa), IL-1 and IL-6,105,110,111 which lead to promoting invasion, immune suppression and angiogenesis through the secretion of SDF-1, TGF-b, hepatocyte growth factor (HGF), PDGFs, or vascular endothelial growth factors (VEGFs) principally driven by FSP-1- or PDGF receptor alpha-positive stromal fibroblasts.112-116

Not surprisingly if CAFs are thought to play a role in all of these pro-cancer processes, targeting them would prove useful for developing effective therapies. A number of proposals have been made regarding PDGF receptor inhibitors, SUMO inhibitors, Met receptor inhibitors or HGF inhibitors; however, on its face it is difficult to envision how to effectively target the “right” CAFs due to the widely diverse population of cells within the stroma. Interestingly enough some possibly contradictory research could prove some insight.

As mentioned above CAFs in the stroma are typically defined as either MF or non-MF, but both of these groups can be activated and/or transformed as well. One particular group to question is activated non-transformed MFs, which express alpha-smooth muscle actin (a-SMA). There are some that believe that these cells have “anti-cancer” activity instead of “pro-cancer” activity. Support for this mindset comes from studies of early and late stages of pancreatic cancer outcomes, clinical correlation between high a-SMA levels and improved survival on a general level, and studies of resected tumors.117-120 Also there is some question to whether or not a-SMA positive MF cells increase hyaluronic acid concentration.121 The anti-cancer attributes of MFs appear to stem from aiding both innate and adaptive immune response via increased fibrosis.117

Whether or not these MFs are pro-cancer or anti-cancer elements is important to deduce because anti-cancer therapies tend to kill indiscriminately around the principal tumor and its microenvironment, including a-SMA positive MFs. If these particular MFs are anti-cancer then these drugs are inherently less effective because while they are killing cancerous elements they are also killing anti-cancer elements.

The final possible important element to CAFs and their role in cancer is their ability to produce exosomes. For example in breast cancer CAFs secrete Cd81+ exosomes that can induce the planar cell polarity (PCP) signaling pathway targeting Wnt and influencing the polarity of carcinoma cells.122 Internalization of these exosomes also promotes Wnt11-PCP induction via autocrine through Frizzled receptor signaling leading to increased probability of pulmonary metastases.112 Targeting these exosomes may be a valid therapeutic strategy for reducing cancer potency.

As mentioned above directly targeting CAFs via therapies may be difficult, but one potential candidate could be TNF receptor associated factor 6 (TRAF6). At least for squamous cell carcinoma (SCC), TRAF6 plays a role in enabling nuclear factor kappa beta (NF-kB) signaling to activate a number of downstream pathways for CAFs, like Akt, Src-family kinases, IKK, IL-1beta and p38 and can regulate the formation of Cdc42-dependent F-actin microspikes.105,107,123 While the role of Cdc-42 is exactly unclear, TNFa plays a large role in promoting invasion in SCC and TRAF6 plays a significant role in producing sufficient TNFa concentration. The reduction of either one of these pathways significantly reduces cancer invasion, possibly due to the K63 ubiquitin ligase activation associated with TRAF6.124

The final issue when addressing cancer metastasis is developing a strategy to promote delivery of the anti-cancer agents to increase the probability of positive action, especially through ensuring proximity action. A reason behind this strategy is that some agents, like the rather useful SAL, demonstrate low quality aqueous solubility, which restricts their ability to be injected through a more standard IV strategy.125 Not surprisingly nanoparticles have become the most attractive vessel for transporting anti-cancer agents to cancer sites.

Overall nanoparticles are advantageous due to their low to non-immunogenic activity reducing complications and increasing their lifespan in the bloodstream, their natural and generally safe biodegradability and biocompatibility and their general design flexibility for producing the right type of particle for the given job. For example some nanoparticle structures use polypeptides with elastin and hydrophilic properties in effort to produce immune-tolerant elements. These elements are commonly referenced with the acronym, iTEP.126 However, nanoparticles need a form of “navigation” system to reach the appropriate target. The two most common targeting strategies are the use of antibodies or the use of aptamers.

Antibody targeting in some respects is the “old reliable” while aptamer targeting is somewhat new. Aptamers are comprised either of oligonucleic acid (DNA or RNA) or a peptide that are able to bind a specific target molecule. The major advantages of aptamers are their molecular specificity, their lack of immunogenicity, and their low molecular weights. These two latter advantages along with ease of production have further increased the popularity of aptamers versus antibodies regarding therapeutic targeting strategies including those dealing with cancer. A number of aptamers have already been developed for use in cancer treatment.127,128

Regardless of navigation methodology, the drug delivery vessel must have the right navigation point. One molecule that has drawn interest for potential cancer targeting ability is hyaluronic acid (HA). HA typically binds to CD44, a receptor commonly over-expressed on numerous types of tumors.129 Furthermore HA is frequently broken down by tumor cells by hyaluronidases (Hyals), which is widely thought to experience concentration increases in various cancers including prostate, bladder, colorectal, brain and breast due to the presence of increased low weight HA fragments found in these tumors versus normal cells.130-133

The general process of HA catabolism involves binding to CD44 resulting in its breakdown into smaller elements by Hyal-2 while still on the cell surface forming what is known as a caveolae. This caveolae eventually becomes an endosome that fuses with lysosomes resulting in the further degradation of HA fragments into tetrasaccharides by Hyal-1.134,135 Based on this process one could theorize that a self-assembled nanoparticle comprised of HA would serve as an effective means of drug delivery to the tumor site both tracking and through its degradation, a belief that has been supported with early empirical results.129

In addition to HA, it is widely thought that cluster of differentiation 133 (CD133) is a positive stem cell marker for both normal and cancerous tissue and is thought to be a critical agent in identifying CSCs. For example it is common for CD133+ cancer cells to form mammospheres that can initiate tumor growth in non-tumor cells. Due to the importance of CD133 expressing cells, an RNA aptamer (A15) has already been developed that binds to CD133 for use as a “tracking” marker of sorts and some groups have already explored the idea of using A15 as a drug delivery targeting agent.136

However, it must be noted that while CD133 expressing cells appear to be the most important in the CSC pool, tumors do produce CSCs that express other surface receptors while not expressing CD133. For example in osteosarcoma, CD133, CD117 and Stro-1 are all considered to be legitimate CSC markers.48 Additionally there is some evidence to support the idea that CSCs can convert to non-CSCs and back again.137 Therefore, while targeting CD133 is clearly an appropriate strategy for treating CSCs, it may not be the only targeting strategy necessary to eliminate CSCs.

The type of nanoparticle is only part of the issue involving drug delivery. Another important element is whether or not the principal drug should have other elements encapsulated with it to increase efficacy. For example SAL delivery involves a charged hydrophobic drug trapped inside a hydrophobic core of micelle-like nanoparticle; these interacting charges can increase destabilization potential, leading to ineffective drug application, which has been seen in past studies.125,126 Therefore, this charge interaction needs to be neutralized.

An early candidate for cooperation with SAL stability was N,N-dimethyloctadecylamine (DMOA) due to its similar hydrophobic nature, yet positive charge which is obviously counter to the negative charge of SAL. Unfortunately DMOA proved too toxic for this role.126 Fortunately it has a less toxic analogue in N,N-dimethylhexylamine. However, this reduced toxicity comes at the price of reduced hydrophobic strength due to a shorter hydrocarbon chain.126 Thus, researchers have added alpha-tocopherol as a second hydrophobic agent to enhance internal hydrophobicity to increase stability, which appears to work well.126

In the end while metastasis is still a process with a number of question marks associated with its occurrence and action there does appear to be certain elements that have important roles in its successful occurrence and function regardless of these question marks. First, it is quite clear that any therapy will have to involve some form of drug cocktail to cover multiple metastasis pathways including treatment of the principal tumor via either drugs or surgery. With this strategy in mind one interesting combination would involve the use of SAL in HA nanoparticles, some form of CXCR4 inhibitor, something like Plerixafor should be sufficient when not applied by itself, and a standard chemotherapy drug like Docetaxel.

Another possible addition to this cocktail could be an anti-angiogenesis drug. In recent treatment history anti-angiogenesis drugs have had a negative history of being useful anti-cancer agents despite the sound theoretical reasoning that reducing growth resources should reduce cancer growth potential. The failure of anti-angiogenesis drugs more than likely occurred due to the induced hypoxia environment increasing rates of metastasis. However, this increased metastasis may be a benefit when the anti-angiogenesis drug is used in combination with a CXCR4 inhibitor and/or SAL, which could speed cancer death by eliminating the metastatic elements versus attempting to eliminate the principal tumor. Of course this combination and the possible positive outcome is only theoretical, without appropriate empirical evidence the addition of an anti-angiogenesis agent may not provide a benefit, similar to how it functions currently.

While promising gains have been made in recent years in immunotherapy-based techniques to combat cancer, it is important to acknowledge that overall there is no magic bullet, but the above potential cocktail should be able to overlap the important negative cancerous element of both primary tumor elimination through multiple destruction pathways and metastasis neutralization via elimination of CSCs as well as the major pathways the drive the preparation and activation of metastasis itself. This method alone or in combination with a proven effective immunotherapy technique could provide a legitimate anti-cancer therapy for various stages of cancer development.



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Is Curing Cancer the Appropriate Step? (Preparing for a World without Cancer)

Since 1971 when then President Nixon declared war on cancer society have awaited the breakthrough that would render the elimination of cancer as a societal and psychological boogie man. Interestingly the pursuit of this accomplishment has developed a certain type of viewpoint of how the future would operate after success with few individuals discussing how the world will change in both a positive and negative way. As counter-intuitive as it may seem would identifying a completely effective cancer treatment leave the world worse off than not identifying one at all?

At first thought almost everyone would find the above question preposterous citing both qualitative and quantitative rationalities for its ridiculousness. For example the National Institutes of Health (NIH) in 2008 estimated the annual costs of cancer to society were 201.5 billion (77.4 billion in direct medical costs and 124 billion in indirect mortality costs);1 those costs increased further in 2010 to $290 billion (154 billion in direct medical costs and 146 billion in indirect mortality costs).2 In 2007 cancer was responsible for approximately 13% of all human deaths globally (7.9 million)3 and in 2008 cancer killed another 7.6 million4 with overall occurrence rates increasing due to general increases in global life expectancies, changes in diets and lifestyles in the developing world, and changes to the environment. In addition there is the emotional damage that cancer afflicts against individuals who do not even have the condition (friends and family) that is not calculated in the mortality costs, regardless of whether or not the afflicted dies, raising economical and societal damage further.

Initially one may assume that the enormity of these numbers should dispel any individual arguing that a cure for cancer would be a bad thing, but the problem is that those numbers are only viewed through a positive lens not through a realistic lens. For example modeling economic costs associated with any condition or disease is currently simplistic and heavily dependent on certain assumptions. There are three common methods for estimating costs associated with various health conditions: Cost-of-illness (COI), Value of Lost Output (VLO) and value of statistical life (VSL).2

COI is the most common analysis method to calculate economic impact of an illness and assigned costs based on the sum of direct and indirect medical costs associated with the illness. Direct medical costs are sub-categorized as: diagnosis, drugs, hospital stays, surgeries, etc. Indirect mortality costs are sub-categorized as: transportation, income losses, pain and suffering reduced productivity, education, etc.5 Note that productivity losses (associated with indirect mortality costs) are typically modeled through the human capital approach, which calculates the potential production by an individual based on average wages adjusted for household productivity.5

The human capital approach is not the only method for calculating productivity, some analysis also use the friction cost method instead. Friction cost estimates the indirect costs of an illness relative to the amount of time it takes the employer/society to substitute the production lost from the individual who has taken ill.6 Basically costs are estimated by multiplying the number of days required for recovery by income and including an elasticity element for annual labor time versus labor productivity.6 Normally there is some form of time boundary (typically some number of days) associated with friction cost pertaining to the average expected recovery time for the illness. If recovery exceeds this boundary condition the excess costs are not counted towards the estimated production costs/losses. For example suppose person x contracts disease y that has an average recovery time of 30 days. If person x is not available to work for 17 days the productivity losses from all 17 days are counted. However, if person x takes 43 days only the first 30 are counted. This condition frequently results in lower estimated costs for a given disease because it typically does not encapsulate the more severe and costly cases of the disease.

COI also has two analysis “viewpoints”: prevalence or incidence. Prevalence estimates costs over a specific time period normally averaged to an annual cost, but does not focus on costs over the duration of the illness.5 Incidence estimates costs over the expected lifetime of an illness. Savings born from prevention strategies are derived from incidence-based analysis because of their timeline estimates over cross-sectional costs.5 While incidence estimates provide more information, they also require more assumptions and information, which can increase the probability for more inaccuracy in the results.

VLO estimates costs based on the impact on GDP with regards to lost capital, production, efficiency and other factors that affect commerce.2 VSL is categorized by two difference analysis methods: the human capital method and the mortality risk method.7,8 The human capital method calculates the present value of future income forgone due to death (similar to the method for COI). However, this method does not include intangible psychological elements on the family and friends of the deceased and ignores the non-working members of society who do not significantly act as human capital in the labor market.8 The mortality risk method calculates what costs members of society are willing to incur to change mortality risks from given illnesses. For example the World Bank classifies VSL as how people’s preferences affect the measurement of change (increase or decrease) in human well-being relative to the change in mortality risk based on the amount of money they would pay.9 Basically how much money would a person pay to reduce the chance of dying from disease A from 40% to 30%?

The reason these three methods exist is that they all examine economic costs relative to illness from different perspectives [public versus private (COI) or individual versus social (VOL) or some aspects of both (VSL) over different time periods]. However, because of these different analysis perspectives, along with complications due to co-morbidities, it is difficult to compare these methods directly.

Regardless of method calculating the possible savings from indirect costs is tricky. First of all productivity losses are more than likely overestimated because unlike past instances the current labor market functions on a shortage of jobs, not a shortage of labor. Of course some may argue against this point citing some of the higher paying jobs that have been vacant for years, but these jobs are highly specific demanding high education and experience levels, which are not available to a vast majority of the population, thus these jobs are radical outliers. Most modern vacant jobs are lower paying, but have high levels of competition including many individuals who are overqualified. Thus, most of the production from labor that is lost to cancer is more easily replaced now than in the past making the savings from curing cancer less than expected.

Another question is how the overall economy will be affected by the redirection of indirect cancer costs. For example current indirect cancer costs are high scale with a narrow focus (i.e. large amounts of money applied to a small section of the economy). If cancer is cured the indirect costs will be diverted more than likely at a smaller scale due to greater sector spread. Will this change in fund distribution result in a net gain or net loss for the economy? The loss of large-scale funds in the cancer treatment industry will result in job loss and contraction in this industry. The concern is that will the redirection of the funds be too scattered to significantly promote job growth in other fields, thus resulting in a negative economic impact? One counter-argument is that the new survivors will spend their money bolstering economic activity. While possible the real validity of this counter-argument is contingent on what happens to the assets owned by these individuals in probate and raises the question of whether government or private control of funds provides better stimulation to the economy.

Another concern is the increasing intracountry global inequality gap. While globalization has decreased the inequality gap between developed and developing countries, it has increased the inequality gap between rich and poor individuals within both developed and developing countries. Assuming that the general death total associated with cancer remains similar over the next 10 years as cited from 2007 figures above, then if a cure for cancer is discovered in 2015 over the next nine years 71.1 million people who should have died will live (and that does not include any offspring these individuals may have).

Note that the above estimate is probably on the low side because the World Health Organization (WHO) estimates that by 2030 at least 13.1 million worldwide will die from cancer.4 Based on the existing inequality level most of these survivors will not have vast amounts of wealth (this assumption makes sense because 70% of all cancer deaths in 2008 occurred in low- and middle-income countries),4 but instead will compete with other individuals for a pool of resources that is at best static and worst declining. This competition could place a greater strain on a vast majority of parties including those who developed cancer, but did not die, lowering the quality of life of a greater number of individuals.

Some may argue that while the savings born from indirect costs may be inaccurate and possibly non-existent, the savings from direct costs are certainly real and substantial. Direct cost savings are real, but more than likely will not be useful to society. The reason for this judgment is the commerce arrow that governs cancer treatment. When individual x develops cancer and receives treatment (note that second part) most of the time he/she has health insurance where after paying a deductible the insurance company will pay for a vast majority of the costs associated with the cancer treatment (80-100%, depending on the specifics of the plan). This payment will typically go to a hospital (most doctors do not own their own treatment centers, etc.). The hospital uses some of those funds to purchase elements of the treatment from various companies. Overall most of the costs associated with cancer treatment are born by the insurance companies, whether or not those costs are passed on to consumers is unclear, but is definitely a possibility.

If a cancer cure is developed, the direct costs of treating cancer will significantly drop reducing the amount of money that insurance companies pay to hospitals. Therefore, insurance companies will gain most of the savings from a cancer cure. However, the benefit to society from a standpoint of direct costs depends on what the insurance company does with these saved funds. There are ten common actions that a company can take with their earned profits:

1) Keep it on their balance sheet to pay for future expenses (i.e. suppose next year is not profitable);
2) Pay off existing debt;
3) Invest in new equipment or other modernization upgrades to increase production and/or lower costs;
4) Issue a dividend or increase the dividend to stockholders;
5) Hire more employees;
6) Increase wages and/or pay bonuses to various employees;
7) Buyback stock;
8) Invest in other companies either as a passive investment or a takeover;
9) Provide some benefit for the consumer (in the case of insurance this would most likely be reducing premiums);
10) Donate the money to a charitable cause;

There is little reason to suspect that insurance companies would pledge profit neutrality where insurance premiums would be lowered to correspond to the savings garnered by the cancer cure versus current cancer treatment. Whether legitimate or not the failure to lower premiums would be justified through arguing the prevalence of other chronic conditions like high blood pressure, obesity and high cholesterol. Anything else that could be done with the saved money that would benefit society directly or even indirectly like donating the money to charity, increasing stock dividends or hiring more employees seems even more far-fetched than lowering premiums.

With the most likely use of the saved funds going into the coffers of the insurance company to be used for CEO bonuses and other frivolous actions to bolster the already wealthy (modern reality dictates that this will happen) the newly saved direct cancer costs will not be reinvested into society at any real magnitude. Any rational person realizes that with decades of empirical evidence against it, supply side economics (a.k.a. trickle down) does not work thus increasing CEO bonuses is rather irrelevant to societal economic benefit. In addition other economic changes would lead various hospitals to lose millions of dollars (it stands to reason that they charge more for cancer treatments over the elements used in those treatments) and the medical companies that provide current cancer treatments will more than likely lose money as well because longer-term treatments yield more revenue than shorter-term treatments (which is what a cancer cure is assumed to be). Whether or not these losses will result in further expanded economic damage to society through things like a cutback in the existing workforce is unclear.

It is also important to address the fate of money devoted to researching a cancer cure. A vast majority of the money that funds cancer research comes from the government through the National Institute of Health (NIH), somewhere in the neighborhood of at least 5.6 billion.10 Therefore, it is reasonable to suggest that this money could be directed elsewhere from the NIH general fund to other worthy grant applicants. However, whether or not this will occur is unknown because of possible future cutbacks in NIH funds. For example some members of Congress may simply decide to eliminate a large amount of past cancer funding from the NIH in an inaccurate and foolish attempt to “cut government pork/waste”. While it is difficult for a legislator to cut funds for cancer research from a public relations standpoint, the public would not be so resistant to a cut in cancer funds that would have been redirected to say Chronic Fatigue Syndrome or something else.

The money derived from charitable organizations like the National Cancer Society is less likely to be recovered. Clearly the development of a cancer cure would heavily limit the continued functionality of such organizations. Most people who donate to cancer charities do so because of a personal connection to cancer, either they had it or know someone who has/had it. Therefore, it is unlikely that these individuals will donate to other charities if such a personal connection does not exist, they do not have a “charity donation quota” that needs to be met each year. Overall it stands to reason that most of the government funds for future cancer research will be directed to other medical research (unless Congress gets involved) and most of the private funds that would have gone to cancer research will not go towards other medical research. Some of these private funds could be invested in the economy yielding positive results, but the extent of benefit for such investment is unclear.

Part of the economic analysis associated with the possible societal influence of a cure for cancer is based on the assumptions that the cure would be widely available and will treat most, if not all, of the major forms of cancer. Initially it could be argued that the widely available assumption is not appropriate. However, the reasoning behind including this assumption is as followed. In the developed world it stands to reason that major insurance companies would cover the cost of the cure. This reasoning makes sense on two fronts: first, it is highly probable that a cure for cancer will cost less than the existing standard cancer treatment, thus it makes direct business sense for insurance companies that cover existing cancer treatments to shift coverage plans to cover the cancer cure.

Second, from a public perception standpoint any insurance company not providing coverage for a cancer cure would more than likely be basically ostracized from the marketplace with consumers avoiding that company in favor of one that does provide cancer cure coverage (recall that the assumption for this discussion is a cancer cure not a cancer vaccine thus people will still develop cancer). For the developing world it seems likely that various NGOs and charities will cover a majority of cancer cases with significant cost parameters.

So the general summary of change to society with the development of a cancer cure like treatment is as followed:

- Approximately 7.6 - 7.9 million people per year no longer die (assuming that the cure is available to all and that estimate may be on the lower end if cancer rates and deaths increase in the future which they are expected to);
- A vast majority of the direct costs associated with cancer treatment becomes increased profit for various insurance companies that will not result in an increased benefit to society;
- The growth/loss transfer to the general economy associated with indirect costs is unclear due to the simplistic level of modeling associated with cost replacement in the models themselves;
- The increased population will increase strain on existing resources, especially in developing countries, which have the majority of cancer deaths, as well as increase competition between individuals more than likely decreasing the quality of life of a larger number of individuals;
- There may be a contraction in employment at hospitals and other medical service companies, especially in high healthcare cost countries like the United States;
- Redirection of government grants and other monetary awards, both public and private, from cancer research to another medical condition/treatment should occur for government funds (again depending on how Congress acts), but not for private funds;

Understand that this blog post is not suggesting that the medical research community stop pursuing better cancer treatments, including one that may result in a cure, but instead is raising the important discussion point regarding how society may change in response to a cure. These changes are important to consider because of the momentous influence cancer as a physical disease and a psychological condition has on modern society. Cancer is in a rather unique position as a disease because while technically increasing age increases probability of development, no other disease permeates human health along all age groups at such a level of scale. Most other major diseases only afflict the elderly population. Therefore, eliminating this influence of cancer will cause significant change that must be accounted for and properly addressed to maximize the efficiency of curing cancer.

Overall it is troubling that no one really discusses these potential changes. Perhaps society is functioning under the belief that a cure to cancer is still decades away so addressing potential problems stemming from a cure is not necessary or that there will be no problems stemming from a cure. Neither of these explanations makes sense because there will be problems and even if a cure is delayed by decades developing a thought methodology to analyze how society could change is beneficial. Therefore, there are no excuses for the lack of attention being paid to possible negative societal changes that could be brought on by the development of a cure for cancer. As a starting point the most pressing issue for study is how the increased population will increase competition and possibly negatively influence society as a whole more so than if cancer is not cured.

Citations –

1. American Cancer Society. Cancer Facts & Figures 2012. Atlanta: American Cancer Society; 2012, page 1.

2. Bloom, D, et Al. “The Global Economic Burden of Non-communicable Diseases.” Harvard School of Public Health. World Economic Forum. 2011.

3. Wikipedia Entry – Cancer. 2013.

4. World Health Organization Fact Sheet N-297. Cancer. January 2013. In conjunction with Globocan 2008, IARC, 2010. http://www.who.int/mediacentre/factsheets/fs297/en/index.html

5. Corso, P, Soyemi, A, Lane, R. “Part II: Economic Impact Analysis. Cost of Illness.” Center for Disease Control and Heart Disease and Stroke Prevention.

6. Hutubessy, R, et Al. “Indirect costs of back pain in the Netherlands: a comparison of human capital method with the friction cost method.” Pain. 1999. 80:201-207.

7. Johansson, P. O. (2001). Is there a meaningful definition of the value of a statistical life? J Health Econ. 20(1):131-139.

8. Viscusi, W, and Aldy, J. “The Value of a Statistical Life: A Critical Review of Market Estimates Throughout the World.” Journal of Risk and Uncertainty. 2003. 27(1):5-76.

9. World Bank. “The Effects of Pollution on Health: The Economic Toll, in Pollution Prevention and Abatement Handbook, World Bank, Washington, DC. 1998.

10. National Institute of Health. Estimates of Funding for Various Research, Condition and Disease Categories (RCDC) between 2009 and 2014. April 2013. http://report.nih.gov/categorical_spending.aspx

Sleep Drugs and Cancer

Sleep drugs (commonly referred to as hypnotic drugs in the pharmaceutical industry) are widely prescribed with an estimated 6-12% of U.S. adults using some form of sleep drug in 2010 and even higher estimates of use in Europe.1,2 Unfortunately one reason the overall level of consumption, both in unique use and repeat use of sleep drugs, is so high is because they do not cure insomnia instead simply reduce its effects. Basically they treat the symptoms of a chronic condition instead of treating the underlying cause. Despite not addressing the cause directly, reducing the influence of insomnia at any level is an important issue because chronic insomnia is thought to significantly increase the probability of developing psychiatric ailments and also reduces wakeful efficiency, productivity and health.2-5

Sleep drugs are commonly divided into pharmacological agents and non-pharmacological agents. While non-pharmacological agents, which range from stimulus control strategies to sleep pattern development with relaxation therapy, are viewed as an initial treatment, most research focuses on pharmacological agents, which are further sub-divided into two categories: benzodiazepines and non-benzodiazepines. The most common sleep drugs are zolpidem, temazepam, eszopiclone and zaleplon with zolpidem as the most prescribed sleep drug between 2002 and 2006 with temazepam in second place.1

Unfortunately meta-analysis has revealed some disturbing information regarding the consumption of sleep inducing drugs and overall mortality. When compared against placebo a number of trials involving commonly used sleep drugs demonstrate a significantly higher rate of cancer including pancreatic, non-melanoma skin, lymphoma, lung, colon or prostate cancer.1,6 The rate of death for those who consume these drugs increases more than three times over those who do not consume these drugs even at the smallest dosage (1-18 pills per year).1 Not surprisingly the probability of death increases as individuals increase the dosage. Also there was no significant difference between the different drugs in relation to how they increase the probability of death,1 thus it appears that these drugs operate over the same or at least a similar mechanism.

One of the more concerning issues with this increased rate of death is that the time variance is scatted; there are probability increases in both short-term and long-term rates of death. The reasoning behind the short-term death increases is currently unknown (peptic ulcers and esophageal damage due to regurgitation are leading theories), but most believe that long-term deaths increases are due to increased rates of cancer.1 In fact in one study the top third of sleep drug consumers (> 132 pills per year) had a 35% greater chance of developing cancer versus non-consumers.1 Another study monitoring 13,177 individuals taking zopiclone determined that 42% of the total deaths were due to cancer.7

The rationality behind the increased probability of cancer development has largely revolved around increasing infections and/or inflammation. The prevailing theory is that sleep drugs somehow suppress immune function.1,6 This suppression of immune function leads to reduced abnormal cell and pathogen destruction resulting in greater rates of cancer and other infections. However, this explanation may not be the only one that accurately describes the increased rates of cancer in individuals that consume sleep drugs.

Most sleep drugs are either benzodiazepines or operate with a similar mechanism of influence on GABAA receptors. Benzodiazepines are agnoists for most GABAA receptors, which increase frequency and duration of their activation. The mechanism of action increases the firing of GABAergic neurons,which reduces the firing rate of excitory neurons increasing the probability of initating sleep and its duration. Application of benzodiazepines result in sedative, anxiolytic, anti-convulsant and hypnotic characterization in the user. There are three types of benzodiazepines largely defined through their residance times: short, intermediate or long.8 Short and intermediate mechanisms are used in controlling insomnia and long mechanisms are used to control anxiety. However, because these sleep compounds are only GABA agnoists their effectiveness is still contingent on the total concentrations of GABA.

Gamma-amino butyric acid (GABA) plays three critical roles in the brain as a signaling molecule, neurotransmitter and metabolite. During development GABA guides neurite outgrowth and directionality.9 Once development of the Central Nervous System (CNS) is complete GABA then alters its function becoming the chief inhibitory neurotransmitter for both the CNS and Peripheral Nervous System (PNS). As the chief inhibitory neurotransmitter GABA plays a role in various neurodegenerative diseases most notably Temporal Lobe Epilepsy (TLE), Parkinson’s Disease (PD) and Huntington’s Disease (HD) stemming from a breakdown in critical components that govern GABA regulation.10-13 However, there is also evidence that GABA plays an important role in the development and progression of certain types of cancer.

GABA has three corresponding biological receptors, which are classified as GABAA, GABAB and GABAC (a.k.a. GABAA-rho). The ion largely associated with GABA receptors is chloride (Cl-), which drives the inhibitory action of GABA. GABAA exists in two activator based constructs, nicotinic and muscimol, and are oligomeric comprised of five different subunits from a pool of seven possible (alpha1-6, beta1-3, gamma1- 3, sigma, epsilon, pi and theta).14-16 GABAA receptors are ionotropic meaning that when an appropriate molecular agent binds the receptor forms a channel/pore in the membrane that allows an appropriate ion to pass through the membrane (direction of passage is largely governed by concentration and charge gradients).17

During development GABAA receptors are more commonly excitatory over inhibitory due to the absence of a chloride pump on the membrane that transfers chloride ions from inside the membrane to the extracellular space. Without this pump there is an excess amount of chloride ions in the cell, thus when the GABAA receptor activates chloride ions escape the neuron along the concentration gradient increasing membrane potential increasing probability of depolarization. Upon the incorporation of the chloride pump the chloride concentration gradient reverses so upon GABAA activation chloride ions flow from the extracellular space into the neuron increasing the probability of hyperpolarization. GABAB receptors are metabotropic activating a G-protein pathway. A majority of GABAB receptors are located on pre-synaptic cells and act as feedback mechanisms.

The third class of GABA receptors, GABAC, is somewhat controversial in whether or not it is uniquely different enough from GABAA to be considered a separate class.18 GABAC receptors are typically insensitive to GABAA receptor allosteric modulators like benzodiazepine and barbiturates because they are exclusively composed of a unique subunit (rho.18,19 This composition does not significantly change the functionality of GABAC receptors compared to GABAA receptors with respect to their interaction with GABA.

Each subunit in a GABAA receptor possesses four hydrophobic membrane-spanning domains.17 Studies have shown that functional GABAA receptors contain at least one alpha and one beta with one gammasubunit typically also involved; sigma, epsilon, pi and thetasubunits are thought to be assembled into GABAA receptors in place of subunits or complementary pairings.20 Overall it is rare to have a GABAA receptor comprised of subunits that lack an alpha or a beta and such a conformation will not be functional.

The importance of receptor composition is largely demonstrated in how individuals subunits are able to confer different sensitivities to GABA and its associated agonists and antagonists.21,22 For example pi subunits appear to highly sensitive to excitation by loreclezole (where non-pi subunit receptors are either unaffected or inhibited), inhibited by lanthanum and unaffected by benzodiazepine diazepam.19,23 Specificially the pi subunit has drawn interest with respects to the role of GABA in the development of cancer.24

Using cDNA libraries the pi subunit was isolated to multiple reproductive tissue (uterus, ovaries, etc.), digestive tissue (gall bladder, small intestine) and specific regions of the brain namely the hippocampus and temporal cortex; two of the major expression cell types in the brain are teratocarcinoma NT2 neuronal precursor and terminally differnetiated NT2-N cells.25,26 However, while NT2 neuronal cells express pi subunit mRNA there is some question to whether those pi subunits are actually incorported into NT2 neuronal GABA receptors.19 Unfortunately despite the apparent importance of the pi subunit, both the developmental expression of the epsilon and pi subunits have yet to be isolated, but both have been cloned.27 From cloning analysis the pi subunit has a 37% relation to the beta subunit, a 35% relation to the sigma subunit and a 33% relation to the rho subunit with very little relation on any of the other subunits.11 Most specifically the pi subunit appears to have similarities to alpha-5, beta-3 and gamma-3.19,27

This similarity of the pi subunits to these other subunits is not surprising in that the pi subunits are commonly incorporated into receptors with alpha-5beta-3 or alpha-5beta-3gamma-3 configurations.19 With respect to insomnia the amplification of benzodiazepine sensitivity is governed by the type of gamma subunit which determines extent of benzodiazepine influence with the requirement of a gamma subunit for a GABA receptor to have signiifcant affinity for benzodiazepines.16,19 Most GABA receptors in the brain have gamma-2 subunits, which demonstrate the highest gamma subunit sensivity to benzodiazepines.16 Receptors with pi subunits are thought to interfere with the ability of the gamma subunit to form the benzodiazepine binding site by either replacing the gamma subunit or blocking the interaction between the gamma subunit and alpha subunit.19,25,28 Receptor interaction with zinc is also influenced by the gamma subunit, but incorporation of the pi subunit into different receptors does not appear to interfear with zinc interaction.19

Receptors that incorporate the pi subunit have demonstrated higher GABA EC50 values, less outward rectification and larger single-channel conductance.19 There is also reason to believe that pi subunits flip activity from hyperpolarization to depolarization in cancer cells.19,24 Therefore, these changes imply longer duration firing over receptors without pi subunits. If this information is accurate then a depolarizing GABAA pi subunit receptor has an advantage over normal hyperpolarizing GABAA receptors demanding greater activity from non-pi subunit GABAA receptors for hyperpolarization and cancer limitation. The extent of pi subunit pentration in cancer cells versus non-pi subunit may also explain the somewhat contradicting results with whether or not GABA aids or hinders cancer development.

The differing action between pi subunit and non-pi subunit containing GABA receptors could offer one reason for why taking benzodiazepine based sleep aids increase cancer rates. Increasing benzodiazepine concentrations act on GABAA receptors, which lead to increased GABAA receptor expression. Increased expression rates would increase the number of mutations simply through volume changes alone (subunit mutation rates may not change, but because more subunits would be created there would be more subunit mutations). Among these subunit mutations could be gamma subunits mutating into pisubunits or pi subunits being incorporated over gamma subunits. Increasing the number of pi subunits would increase the number of GABAA receptors that depolarize instead of hyperpolarize, which would aid cancer development instead of hinder it.

Outside very specific subunit interactions like those involving the pi subunit, the relationship between cancer and GABA appears complex for GABA may influence different cancers in different ways. However, there does appear to be a general pattern of operation between GABA, cancer and the two major GABA receptors. To best understand this relationship temporal issues must be acknowledged between immature/developing cancer and mature cancer. Note that developing cancer refers to cells that have become cancerous and are starting to grow, not cells that are going through the initial mutation stages to become cancerous.

Typically the expression of GABA and its corresponding synthesizing enzyme GAD (both isoforms 65 and 67) significantly increase in concentration in the presence of neoplastic cells ((colorectal carcinoma, breast cancer, prostate cancer, glioma, pancreatic and gastric cancer).29-37 However, what does that increase mean relative to cancer growth? Based on existing evidence it appears that a reduction in GABAA receptor functionality leads to accelerated cancer growth.29,38 Such a result implies that GABA activity when binding to GABAA results in reduced cancer growth, which has been supported through reductions in membrane potentials of cancerous cells.39,40 If GABAA binding is detrimental to cancer growth then why do GABA and GAD concentrations increase in the presence of cancer versus non-cancerous cells? One explanation is that this increased expression may be a general ‘safety’ feedback mechanism designed to curtail excess (i.e. cancer) growth, especially if GABAA receptor expression decreases.38

GABAB interaction appears to play a similar role to GABAA with regards to reducing cancer growth. Activation of GABAB in a cancer cell does not kill the cancer cell, but instead arrests its growth between stages G0 and G1.41 In addition GABAB activation also reduces intra-cellular cAMP concentrations through the inhibition of adenylyl cyclase due to the activation of G-protein alpha-2.42,43 cAMP is important in cellular growth (both normal and cancerous) because it activates phosphokinase A (PKA) which among other things (i.e. ERK1/2 cascade) activates phospholamban.44 Phospholamban activation increases the rate of calcium release from the endoplasmic reticulum (ER). While some of this excess calcium is sequestered by the existing cAMP, in typical situations the calcium release from the ER exceeds the rate of sequestration by cAMP resulting in an increased level of cytosolic calcium.44 This increased cytosolic calcium concentration activates calcium dependent secondary messenger systems increasing the rate of cellular growth. Thus, the ability of GABAB activation to reduce cAMP concentrations reduces the rate of cancer growth through this particular mechanism.

While GABA does appear to influence cancer growth in a negative way regardless of which receptor it binds to it could play an even more important role in cancer migration/metastasis, albeit a slightly confusing one. The confusion in the issue of metastasis appears to come from somewhat conflicting evidence, but the confliction is not insurmountable. First, increased expression of GABA67 is seen in patients with higher Gleason scores30 (note Gleason scores are derived from examination of histological samples in an effort to track cancer progression). Initially such a result could be explained, as mentioned earlier, as a feedback mechanism from the body in effort to control cancer growth and as cancer growth increases (leading to a higher Gleason score) the body compensates further. However, what if there is another explanation, what if GABA actually assists in metastasis? This amplification of cancer metastasis seems to be in play at least for some forms of prostate and lymph node cancers where increasing GABA concentration resulted in an increase in matrix metalloproteinase (MMP) expression due to GABAB activation.30

Metastasis is a complex series of interactions leading from tumor development to detachment from its principle location and movement to a different more distant location in the body. The major events involve detachment of from the primary tumor, invasion of the stromal tissue, entrance to the bloodstream, extravasate, invasion of the new target organ and finally the formation of the metastatic colony.17,30 One of the key steps in this process is the proteolytic degradation of the extracellular matrix and in this step MMPs are critical agents.

Initially MMPs were thought to be degenerative proteases that were limited to cleaving matrix components, but that role has expanded to include the release of growth factors and other bioactive peptides localized at cleaved extracellular matrices.45-47 Most MMP action involves MMP-3 cleaving decorin which releases transforming growth factor-b leading to greater levels of angiogenesis in addition to cleaving TGF-alpha activating MAPK inducing cellular proliferation.48,49 MMPs also cleave matrix receptors that may inhibit metastasis inhibitors like E-cadherin and activate a semi-self-regulating pathway with MMP-3 activating MMP-7 and 9.50

However, there appears to be a problem with concluding that GABA increases metastasis probability through GABAB activation in that another study has demonstrated that GABA decreases metastasis probability through GABAB activation.44 In a study using SW480 colon carcinoma cells cellular locomotion (basically metastasis probability) was reduced due to reduction in cAMP concentration. One possible reason for this difference is the differing cell types, but another line of thought produces another possible solution.

First, the SW480 colon study focused on metastasis induced through norepinephrine, not MMPs. In norepinephrine induced metastasis norepinephrine activates beta-arrestin through the beta-2 adrenoceptor which activates Protein Tyrosine Kinase (PTK) which activates the key agent, protein kinase C gamma (PKC).44 Activation of PKC-gamma results in the dual activation of both inositol-1,4,5-trisphosphate and diacylglycerol.44,51 Inositol-1,4,5-triphosphate opens intracellular calcium channels increasing cAMP activation and diacylglycerol activates PKC-alpha, which has been shown to increase metastasis in cancer cells.52,53

Basically norepinephrine increases cancer proliferation by activating inositol-1,4,5-triphosphate and increases cancer metastasis probability through activating diacylglycerol. This dual activation is important because cAMP cannot activate diacylglycerol on its own, yet it does appear to augment diacylglycerol activity in that if cAMP concentration is reduced diacylglycerol does not aid metastasis. Thus GABAB is able to prevent this form of metastasis by reducing cAMP concentration through secondary messenger inhibition of adenylyl cyclase.

However, while GABAB prevents metastasis through PKC-gamma, why doesn’t the ability to increase MMP expression compensate for the PKC-gamma blocking resulting in greater metastasis versus controls? One explanation may be temporal in nature in that the colon cells were not in a high enough state of maturity to express and/or interact with the MMP that should have been generated from the GABAB activation. If this theory is accurate then GABAB may be a beneficial therapeutic agent in the early stages of cancer, but as cancer progresses its usefulness flips and it becomes more detrimental than beneficial.

Unfortunately the role of GABAA in metastasis may not be as simple. While a number of studies suggest that GABAA binding plays no role in metastasis30,41,44 other studies suggest that GABAA binding increases metastasis probability54 or decrease metastasis.55 Despite this contradiction based on currently understood GABAA behaviors and mechanisms it is hard to believe that GABAA positively influences metastasis if configured properly because of its hyperpolarizing nature. The difference between ‘neutrality’ and reducing metastasis for GABAA may also be temporal, similar to GABAB.

Early in cancer development GABAA has little influence, but has MMP concentrations increase, GABAA works to reduce those concentrations despite GABAB augmenting them.55 Another reason for this disparity may be that in some studies the tumors did not mature to the point where metastasis was at a reasonable probability of occurrence because GABAA activity arrested tumor growth, thus GABAA influenced growth, but not metastasis. Overall it appears that the theory to describe GABA receptor behavior with respect to cancer is that GABAA negatively affects cancer growth and has little influence on cancer metastasis whereas GABAB negative affects cancer growth and has a negative influence on early cancer metastasis and a positive influence on late cancer metastasis.

If the above characterization of GABA receptors with respect to cancer is taken as accurate, then it appears that ability of sleep drugs to increase cancer rates largely relies on receptor subunit configuration. Other than pi subunits47 there is reason to suspect that incorporation of theta or rho subunits also increases cancer proliferation probabilities.56,57 These receptor confirmations may be self-augmenting in that if cancer develops due to their depolarizing characteristics over hyperpolarizing the cancer mutates to produce more receptors with similar configurations explaining why rarer pi and theta subunit configurations, with even rho at times, are so prevalent in cancers.24,27,56,57

Another possibility may be that augmenting GABAA activation through agonists like benzodiazepine may cause greater GABAB activity as a result of feedback. While GABAB activation would be viewed as more cancer-preventative than cancer-aiding, recall that as cancer develops the benefit/detriment ratio for GABAB with respect to cancer prevention decreases. Thus one question to ask is if cancer rates increase with sleep drug administration or does cancer metastasis also increase relative to the increase in cancer rates?

Overall one of the chief concerns is that while rates of death are slightly lower with non-benzodiazepine sleep drugs there is little firm scientific evidence that these non-benzodiazepine treatments result in higher rates of increased sleep time or reduction in wake probability after sleep onset compared against placebo.1,58 Benzodiazepine treatments do decrease sleep latency and increase sleep duration8,58 (although there are some questions regarding the statistical significance of the latter effect despite concerns of overestimation of sleep latency and underestimation of total sleep time),58 thus taking non-benzodiazepine treatments which have less cancer promoting tendencies may not be a suitable alternative to addressing a lack of sleep. While GABA seems to prevent cancer more than aid in its development stimulation of the GABA system through artificial means could also increases the rate of mutation in the subunits which comprise GABA receptors and these mutations significantly increase the probability of generating cancer.

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Citations –
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