Tuesday, February 9, 2016

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.

Citations –

1. Deng, X, et Al. “Posttranslational modifications of CXCR4: implications in cancer metastasis.” Receptors and Clinical Investigation. 2014. 1-6:e63.

2. Zlotnik, A. “Chemokines and cancer.” Int. J. Cancer. 2006. 119:2026-2029.

3. Muller, A, et Al. “Involvement of chemokine receptors in breast cancer metastasis.” Nature. 2001. 410:50-56.

4. Furusato, B, et Al. “CXCR4 and cancer.” Pathol. Int. 2010. 60:497-505.

5. Liekens, S, Schols, D, and Hatse, S. “CXCL12-CXCR4 axis in angiogenesis, metastasis and stem cell mobilization.” Curr. Pharm. Des. 2010. 16:3903-3920.

6. Oh, Y, et Al. “Hypoxia induces CXCR expression and biological activity in gastric cancer cells through activation of hypoxia-inducible factor-1alpha.” Oncol. Rep. 2012. 28:2239-2246.

7. Lee, H, et Al. “CXC chemokines and chemokine receptors in gastric cancer: from basic findings towards therapeutic targeting.” World. J. Gastroenterol. 2014. 20(7):1681-1693.

8. Rodriguez-F, J, et Al. “Blocking HIV-1 infection via CCR5 and CXCR4 receptors by acting in trans or the CCR2 chemokine receptor. EMBO. J. 2004. 23:66-76.

9. Sohy, D, et Al. “Hetero-oligomerization of CCR2, CCR5, and CXCR4 and the protean effects of “selective” antagonists.” J. Biol Chem. 2009. 284:31270-31279.

10. Levoye, A, et Al. “CXCR7 heterodimerizes with CXCR4 and regulates CXCL12-mediated G protein signaling.” Blood. 2009. 113:6085-6093.

11. Basmaciogullari, S, et Al. “Specific interaction of CXCR4 with CD4 and CD8alpha: functional analysis of the CD4/CXCR4 interaction in the context of HIV-1envelope glycoprotein-mediated membrane fusion.” Virology. 2006. 353:52-67.

12. Woerner, B, et Al. “Widespread CXCR4 activation in astrocytomas revealed by phospho-CXCR4-specific antibodies.” Cancer Res. 2005. 65:11392-11399.

13. Marchese, A, Benovic, J. “Agonist-promoted ubiquitnation of the G protein-coupled receptor CXCR4 mediates lysosomal sorting.” J. Biol. Chem. 2001. 276:45509-45512.

14. Wang, J, et Al. “Dimerization of CXCR4 in living malignant cells: control of cell migration by a synthetic peptide that reduces homologous CXCR4 interactions.” Mol. Cancer. Ther. 2006. 5(10):2474-2483.

15. Veldkamp, C, et Al. “Recognition of a CXCR4 sulfotyrosine by the chemokine stromal cell-derived factor-1 alpha (SDF-1alpha)/CXCL12). J. Mol. Biol. 2006. 359:1400-1409.

16. Xu, J, et Al. “Tyrosylprotein sulfotransferase-1 and tyrosine sulfation of chemokine receptor 4 are induced by Epstein-barr virus encoded latent membrane protein 1 and associated with the metastatic potential of human nasopharyngeal carcinoma.” PLoS ONE. 2013. 8(3):e56114.

17. Hu, J, et Al. “The expression of functional chemokine receptor CXCR4 is associated with the metastatic potential of human nasopharyngeal carcinoma.” Clin. Cancer Res. 2005. 11:4658-4665.

18. Fanelli, M, et Al. “The influence of transforming growth factor-alpha, cyclooxygenase-2, matrix metalloproteinase (MMP)-7, MMP-9, and CXCR4 proteins involved in epithelial-mesenchymal transition on overall survival of patients with gastric cancer.” Histopathology. 2012. 61:153-161.

19. Hashimoto, I, et Al. “Blocking on the CXCR4/mTOR signalling pathway induces the anti-metastatic properties and autophagic cell death in peritoneal disseminated gastric cancer cells.” Eur. J. Cancer. 2008. 44:1022-1029.

20. Cui, K, et Al. “The CXCR4-CXCL12 pathway facilitates the progression of pancreatic cancer via induction of angiogenesis and lymphagiogenesis.” Journal of Surgical Research. 2011. 171(1):143-150.

21. Kaplan, R, et Al. “VEGFR1-positive haematopoietic bone marrow progenitors initiate the pre-metastatic niche.” Nature. 2005. 438(7069):820-827.

22. Taichman, R, et Al. “Use of the stromal cell-derived factor-1/CXCR4 pathway in prostate cancer metastasis to bone.” Cancer Research. 2002. 62(6):1832-1837.

23. Wang, Q, et Al. “SUMO-specific protease 1 promotes prostate cancer progression and metastasis.” Oncogene. 2013. 32(19):2493-2498.

24. McCawley, L and Matrisian, L. “Matrix metalloproteinases: multifunctional contributors to tumor progression.” Mol. Med. Today. 2000. 6: 149–156.

25. Sternlicht, M and Werb, Z. “How matrix metalloproteinases regulate cell behavior.” Annu. Rev. Cell Dev. Biol. 2001. 17: 463–516.

26. Egeblad, M and Werb, Z. “New functions for the matrix metalloproteinases in cancer progression.” Nat. Rev. Cancer. 2002. 2: 161–174.

27. Leeman, M, Curran, S, and Murray, G. “New insights into the roles of matrix metalloproteinases in colorectal cancer development and progression.” J. Pathol. 2003. 201:528-534.

28. Yang, B, et Al. “Expression and prognostic value of matrix metalloproteinase-7 in colorectal cancer.” Asian Pacific J. Cancer Prev. 2012. 13:1049-1052.

29. Woessner, J, Jr. and Taplin, C. “Purification and properties of a small latent matrix metalloproteinase of the rat uterus.” J. Biol. Chem. 1988. 263:16918-16925.

30. Ito, Y, et Al. “Inverse relationships between the expression of MMP-7 and MMP-11 and predictors of poor prognosis of papillary thyroid carcinoma.” Pathology. 2006. 38:421-425.

31. de Vicente, J, et Al. “Expression of MMP-7 and MT1-MMP in oral squamous cell carcinoma as predictive indictor for tumor invasion and prognosis.” J. Oral. Pathol. Med. 2007. 36:415-424.

32. Liu, H, et Al. “Predictive value of MMP-7 expression for response to chemotherapy and survival in patients with non-small cell lung cancer.” 2008. Cancer Sci. 99:2185-2192.

33. Koskensalo, S et Al. “MMP-7 overexpression is an independent prognostic marker in gastric cancer.” Tumour. Biol. 2010. 31:149-155.

34. Davies, G, Jiang, W, and Mason, M. “Matrilysin mediates extracellular cleavage of E-cadherin from prostate cancer cells: a key mechanism in hepatocyte growth factor/scatter factor-induced cell-cell dissociation and in vitro invasion.” Clin. Cancer Res. 2001. 7:3289-3297.

35. Mitsiades, N, et Al. “Matrix metalloproteinase-7-mediated cleavage of Fas ligand protects tumor cells from chemotherapeutic drug cytotoxicity.” Cancer Res. 2001. 61:577-581.

36. Li, Q, et Al. “Matrilysin shedding of syndecan-1 regulates chemokine mobilization and transepithelial efflux of neutrophils in acute lung injury.” Cell. 2002. 111:635-646.

37. Noe, V, et Al. “Release of an invasion promoter E-cadherin fragment by matrilysin and stromelysin-1.” J. Cell. Sci. 2001. 114:111-118.

38. Lynch, C, et Al. “MMP-7 promotes prostate cancer-induced osteolysis via the solubilization of RANKL.” Cancer Cell. 2005. 7:485-496

39. Imai, K, et Al. “Degradation of decorin by matrix metalloproteinases: identification of the cleavage sites, kinetic analyses and transforming growth factor-beta1 release.” Biochem. J. 1997. 322: 809–814.

40. Vandooren, J, Van den Steen, P, and Opdenakker, G. “Biochemistry and molecular biology of gelatinase B or matrix metalloproteinase-9 (MMP-9): the next decade.” Crit Rev Biochem Mol Biol. 2013. 48: 222–272.

41. Radisky, D, et Al. “Rac1b and reactive oxygen species mediate MMP-3-induced EMT and genomic instability.” Nature. 2005. 436:123-127.

42. Kheradmand, F, et Al. “Role of Rac1 and oxygen radicals in collegenase-1 expression induced by cell shape change.” Science. 1998. 280:898-902.

43. Khamis, Z, et Al. “Evidence for a pro-apoptotic role of matrix metalloproteinase-26 in human prostate cancer cells and tissues.” Journal of Cancer. 2016. 7:80-87.

44. Martin, M, and Matrisian, L. “The other side of MMPs: protective roles in tumor progression.” Cancer metastasis Rev. 2007. 26(3-4):717-724.

45. McCawley, L, et Al. “A protective role for matrix metalloproteinase-3 in squamous cell carcinoma.” Cancer Res. 2004. 64(19):6965–6972.

46. Kerkelä, E, et Al. “Metalloelastase (MMP-12) expression by tumour cells in squamous cell carcinoma of the vulva correlates with invasiveness, while that by macrophages predicts better outcome.” J. Pathol. 2002. 198(2):258–269.

47. Vilen, Suvi-Tuuli, et Al. “Fluctuating roles of matrix metalloproteinase-9 in oral squamous cell carcinoma.”

48. Balkwill, F. “The chemokine system and cancer.” J. Pathol. 2012. 226:148-157.

49. Burger, J. “Chemokines and chemokine receptors in chronic lymphocytic leukemia (CLL): from understanding the basics towards therapeutic targeting.” Semin Cancer Biol. 2010. 20:424-430.

50. Chen, G, et Al. “Inhibition of chemokine (CXC motif) ligand 12/chemokine (CXC motif) receptor 4 axis (CXCL12/CXCR4)-mediated cell migration by targeting mammalian target of rapamycin (mTOR) pathway in human gastric carcinoma cells. J. Biol. Chem. 2012. 287:12132-12141.

51. Luker, K, and Luker, G. “Functions of CXCL12 and CXCR4 in breast cancer.” Cancer Lett. 2008. 238:30-41.

52. Lee, H, et Al. “Chemokine (C-X-C motif) ligand 12 is associated with gallbladder carcinoma progression and is a novel independent poor prognostic factor.” Clin Cancer Res. 2012. 18:3270-3280.

53. Choi, Y, et Al. “CXCR4, but not CXCR7, discriminates metastatic behavior in non-small cell lung cancer cells.” Mol. Cancer Res. 2014. 12(1):38-47.

54. Carbajal, K, et Al.”Migration of engrafted neural stem cells is mediated by CXCL12 signaling through CXCR4 in a viral model of multiple sclerosis.” PNAS 2010. 107(24):11068-11073.

55. Sanchez-Alcaniz, J, et Al. “CXCR7 controls neuronal migration by regulating chemokine responsiveness.” Neuron. 2011. 69(1):77-90.

56. Tawadros, T, et Al. “Release of macrophage migration inhibitory factor by neuroendocrine-differentiated LNCaP cells sustains the proliferation and survival of prostate cancer cells.” Endocrine-Related Cancer. 2013. 20:137-149.

57. Calandra, T, and Roger, T “Macrophage migration inhibitory factor: a regulator of innate immunity.” Nature Reviews Immunology. 2003. 3:791–800.

58. Bucala, R, and Donnelly, S. “Macrophage migration inhibitory factor: a probable link between inflammation and cancer.” Immunity. 2007. 26:281–285.

59. Meyer-Siegler, K, Leifheit, E, and Vera, P. “Inhibition of macrophage migration inhibitory factor decreases proliferation and cytokine expression in bladder cancer cells.” BMC Cancer. 2004. 4:34.

60. Muramaki, M, et Al. “Clinical utility of serum macrophage migration inhibitory factor in men with prostate cancer as a novel biomarker of detection and disease progression.” Oncology Reports. 2006. 15:253–257.

61. Schwartz, V, et Al. “A functional heteromeric MIF receptor formed by CD74 and CXCR4.” FEBS Letters. 2009. 583:2749–2757.

62. Keller, M, et Al. “Active caspase-1 is a regulator of unconventional protein secretion.” Cell. 2008. 132:818–831.

63. Merk, M, et Al. “The Golgi-associated protein p115 mediates the secretion of macrophage migration inhibitory factor.” Journal of Immunology. 2009. 182:6896–6906.

64. MIF induces cell proliferation via sustained activation of ERK1/2 MAPKs and promotes cell survival through the inhibition of p53 and the activation of PI3K/AKT signaling.

65. Teicher, B, and Fricker, S. “CXCL12 (SDF-1)/CXCR4 pathway in cancer.” Clin. Cancer Res. 2010. 16(11):2927-2931.

66. Ratajczak, M, et Al. “The plieotropic effects of the SDF-1 CXCR4 axis in organogenesis, regeneration and temorigenesis.” Leukemia. 2006. 20:1915-1924.

67. Bhardwaj, A, et Al. “CXCL12/CXCR4 signaling counteracts docetaxel-induced microtubule stabilization via p21-activated kinase 4-depednent activation of LIM domain kinase 1.” Oncotarget. 2014. 5(22):11490-11500.

68. Yasumoto, K, et Al. “Role of the CXCL12/CXCR4 axis in peritoneal carcinomatosis of gastric cancer.” Cancer Res. 2006. 66:2181-2187.

69. Burger, J, and Peled, A. “CXCR4 antagonists: targeting the microenvironment in leukemia and other cancers.” Leukemia. 2009. 23:43–52.

70. Rosenkilde, M, et Al. “Molecular mechanism of AMD3100 antagonism in the CXCR4 receptor.” J. Biol. Chem. 2004. 279:3033–3041.

71. Hatse, S, et Al. “Chemokine receptor inhibition by AMD3100 is strictly confined to CXCR4.” FEBS Lett. 2002. 527:255–62.

72. Fricker, S, et Al. “Characterization of the molecular pharmacology of the G-protein coupled chemokine receptor, CXCR4.” Biochem Pharmacol. 2006. 72:588–96.

73. Flomenberg, N, et Al. “The use of AMD3100 plus G-CSF for autologous hematopoietic progenitor cell mobilization is superior to G-CSF alone.” Blood. 2005. 106:1867-1874.

74. DiPersio, J, et Al. “Plerixafor and G-CSF versus placebo and G-CSF to mobilize hematopoietic stem cells for autologous stem cell transplantation in patients with multiple myeloma.” Blood. 2009. 113:5720-5726.

75. Jaganmohan, R., et Al. “Glucose starvation-mediated inhibition of salinomycin induced autophagy amplifies cancer cell specific cell death.” 2015. Oncotarget. 6(12):10134-10146.

76. Jangamreddy, J, et Al. “Salinomycin induces activation of autophagy, mitophagy and affects mitochondrial polarity: differences between primary and cancer cells.” Biochimica et biophysica acta. 2013. 1833(9):2057-2069.

77. Ghavami, S, et Al. “Autophagy and apoptosis dysfunction in neurodegenerative disorders.” Progress in neurobiology. 2014. 112:24-49.

78. Boehmerle, W, and Endres, M. “Salinomycin induces calpain and cytochrome c-mediated neuronal cell death.” Cell death & disease. 2011. 2:e168.

79. Boehmerle, W, et Al. “Specific targeting of neurotoxic side effects and pharmacological profile of the novel cancer stem cell drug salinomycin in mice.” Journal of molecular medicine. 2014. 92(8):889-900.

80. Oak, P, et Al. “Combinatorial treatment of mammospheres with trastuzumab and salinomycin efficiently targets HER2-positive cancer cells and cancer stem cells.” International journal of cancer. 2012. 131(12):2808-2819.

81. Parajuli, B, et Al. “Salinomycin inhibits Akt/NF-kappaB and induces apoptosis in cisplatin resistant ovarian cancer cells.” Cancer epidemiology. 2013. 37(4):512-517.

82. Zhang, G, et Al. “Combination of salinomycin and gemcitabine eliminates pancreatic cancer cells.” Cancer letters. 2011. 313(2):137-144.

83. Jangamreddy, J and Los, M. “Mitoptosis, a novel mitochondrial death mechanism leading predominantly to activation of autophagy.” Hepatitis monthly. 2012. 12(8):e6159.

84. Xiao, Z, et Al. “Metformin and salinomycin as the best combination for the eradication of NSCLC monolayer cells and their alveospheres (cancer stem cells) irrespective of EGFR, KRAS, EML4/ALK and LKB1 status.” Oncotarget. 2014. 5(24):12877-12891.

85. Yue, W, et Al. “Inhibition of the autophagic flux by salinomycin in breast cancer stem-like/progenitor cells interferes with their maintenance.” Autophagy. 2013. 9(5):1-16.

86. Fuchs, D, et Al. “Salinomycin overcomes ABC transporter-mediated multidrug and apoptosis resistance in human leukemia stem cell-like KG-1a cells.” Biochem Biophys Res Commun. 2010. 394:1098-104.

87. Riccioni, R, et Al. “The cancer stem cell selective inhibitor salinomycin is a p-glycoprotein inhibitor.” Blood Cells Mol Dis. 2010. 45:86-92.

88. Chen, G, et Al. “Metformin inhibits growth of thyroid carcinoma cells, suppresses self-renewal of derived cancer stem cells, and potentiates the effect of chemotherapeutic agents.” Journal of Clinical Endocrinology & Metabolism. 2012. 97(4):E510-E520.

89. Brown, K, et Al. “Metformin inhibits aromatase expression in human breast adipose stromal cells via stimulation of AMP-activated protein kinase.” Breast cancer research and treatment. 2010. 123(2):591-596.

90. Isakovic, A, et Al. “Dual antiglioma action of metformin: cell cycle arrest and mitochondria-dependent apoptosis.” Cellular and molecular life sciences. 2007. 64(10):1290-1302.

91. Mizushima, N. “Autophagy: process and function.” Genes Dev. 2007. 21:2861-73;

92. Mortensen, M, et Al. “The autophagy protein Atg7 is essential for hematopoietic stem cell maintenance.” J Exp Med. 2011. 208:455-67.

93. Lu, D, et Al. “Salinomycin inhibits Wnt signaling and selectively induces apoptosis in chronic lymphocytic leukemia cells.” PNAS. 2011. 108(32):13253-13257.

94. Tamai, K, et Al. “A mechanism for Wnt coreceptor activation.” Mol Cell. 2004. 13:149–156.

95. Zeng, X, et Al. “A dual-kinase mechanism for Wnt co-receptor phosphorylation and activation.” Nature. 2005. 438:873–877.

96. Fodde, R, and Brabletz, T.”Wnt/b-catenin signaling in cancer stemness and malignant behavior.” Current Opinion in Cell Biology. 2007. 19:150-158.

97. Jung, A, et Al. “The invasion front of human colorectal adenocarcinomas shows co-localization of nuclear b-catenin, cyclin D1, and p16INK4A and is a region of low proliferation.” Am J Pathol. 2001. 159:1613-1617.

98. Liu, C, et Al. “LRP6 overexpression defines a class of breast cancer subtype and is a target for therapy.” PNAS. 2010. 107:5136–5141.

99. Zhang, J, et Al. “Wnt signaling activation and mammary gland hyperplasia in MMTV-LRP6 transgenic mice: Implication for breast cancer tumorigenesis.” Oncogene. 2010. 29:539–549.

100. Yang, L, Lin, C, and Liu, Z. “P68 RNA helicase mediates PDGF-induced epithelial mesenchymal transition by displacing axin from b-catenin.” Cell. 2006. 127:139-155.

101. He, X. “Unwinding a path to nuclear b-catenin.” Cell. 2006. 127:40-42.

102. Schempp, C, et Al. “V-ATPase inhibition regulates anoikis resistance and metastasis of cancer cells.” Mol. Cancer. Ther. 2014. 13(4):926-937.

103. Marshansky, V, and Futai, M. “The V-type H+ ATPase in vesicular trafficking: targeting regulation and function.” Curr. Opin. Cell Biol. 2008. 20:415-426.

104. Ridley, A. “Rho GTPases and actin dynamics in membrane protrusions and vesicle trafficking.” Trends Cell Biol. 2006. 16(10):522–529.

105. Chaudhry, S, et Al. “Autocrine IL-1beta-TRAF6 signalling promotes squamous cell carcinoma invasion through paracrine TNFalpha signaling to carcinoma-associated fibroblasts.” Oncogene. 2013. 32(6):747-758.

106. Gaggioli C, Hooper S, Hidalgo-Carcedo C, Grosse R, Marshall JF, Harrington K, et al. Fibroblast led collective invasion of carcinoma cells with differing roles for RhoGTPases in leading and following cells. Nat Cell Biol. Dec; 2007 9(12):1392–1400.

107. Wang K, Wara-Aswapati, N, et Al. “TRAF6 activation of PI 3-kinase-dependent cytoskeletal changes is cooperative with Ras and is mediated by an interaction with cytoplasmic Src.” J Cell Sci. 2006. 119(Pt 8):1579–1591.

108. Nystrom, M, et Al. “Development of a quantitative method to analyse tumour cell invasion in organotypic culture.” J Pathol. 2005. 205(4):468–475.

109. Kalluri, R, and Zeisberg, M. “Fibroblasts in cancer.” Nat Rev Cancer. 2006. 6(5):392–401.

110. Erez, N, et Al. “Cancer-Associated Fibroblasts Are Activated in Incipient Neoplasia to Orchestrate Tumor-Promoting Inflammation in an NF-kappa B-Dependent Manner.” Cancer Cell. 2010. 17(2):135–147.

111. Stuelten, C, et Al. “Breast cancer cells induce stromal fibroblasts to express MMP-9 via secretion of TNF-alpha and TGF-beta.” J Cell Sci. 2005. 118(Pt 10):2143–2153.

112. Polanska, U, and Orimo, A. “Carcinoma-associated fibroblasts: non-neoplastic tumor promoting mesenchymal cells.” J. Cell. Physiol. 2013. 228:1651-1657.

113. Guo, X, et Al. “Stromal fibroblasts activated by tumor cells promote angiogenesis in mouse gastric cancer.” J Biol Chem. 2008. 283:19864–19871.

114. Hanahan, D, and Coussens, L. “Accessories to the crime: Functions of cells recruited to the tumor microenvironment.” Cancer Cell. 2012. 21:309–322.

115. Polanska, U, Mellody, K, and Orimo, A. “Tumour-promoting stromal myofibroblasts in human carcinomas.” Cancer Drug Discov Dev (Springer Chapter). 2010. 16:325–349.

116. Togo, S, et Al. “Carcinoma-associated fibroblasts are a promising therapeutic target.” Cancers. 2013. 5:149–169.

117. Ozdemir, B, et Al. “Depletion of carcinoma-associated fibroblasts and fibrosis induces immunosuppression and accelerates pancreas cancer with reduced survival.” Cancer Cell. 2014. 25:1-16.

118. Armstrong, T, et Al. “Type I collagen promotes the malignant phenotype of pancreatic ductal adenocarcinoma.” Clin. Cancer Res. 2004. 10:7427–7437.

119. Wang, W, et Al. “Intratumoral a-SMA enhances the prognostic potency of CD34 associated with maintenance of microvessel integrity in hepatocellular carcinoma and pancreatic cancer.” PLoS ONE. 2013. 8:e71189.

120. Omary, M, et Al. “The pancreatic stellate cell: a star on the rise in pancreatic diseases.” J. Clin. Invest. 117:50–59.

121. Jacobetz, M, et Al. “Hyaluronan impairs vascular function and drug delivery in a mouse model of pancreatic cancer.” Gut. 2013. 62:112–120.

122. Luga, V, et Al. “Exosomes mediate stromal mobilization of autocrine Wnt-PCP signaling in breast cancer cell migration.” Cell. 2012. 151:1542–1556.

123. Yang, W, et Al. “The E3 ligase TRAF6 regulates Akt ubiquitination and activation.” Science. 2009. 325(5944):1134–1138.

124. Windheim, M, et Al. “Interleukin-1 (IL-1) induces the Lys63-linked polyubiquitination of IL-1 receptor-associated kinase 1 to facilitate NEMO binding and the activation of IkappaBalpha kinase.” Mol Cell Biol. 2008. 28(5):1783–1791.

125. Zhang, Y, et Al. “The eradica­tion of breast cancer and cancer stem cells using octreotide modified paclitaxel active targeting micelles and salinomycin passive targeting micelles.” Biomaterials. 2012. 33(2):679–691.

126. Zhao, P, et Al. “iTEP nanoparticle-delivered salinomycin displays an enhanced toxicity to cancer stem cells in orthotopic breasts tumors.” Mol. Pharmaceutics. 2014. 11:2703-2712.

127. Barbas, A, et Al. “Aptamer applications for targeted cancer therapy.” Future Oncol. 2010. 6(7):1117–1126.

128. Ni, M. “Poly(lactic-co-glycolic acid) nanoparticles conjugated with CD133 aptamers for targeted salinomycin delivery to CD133+ osteosarcoma cancer stem cells.” International Journal of Nanomedicine. 2015. 10:2537-2554.

129. Choi, K, et Al. “Smart nanocarrier based on PEGylated hyaluronic acid for cancer therapy.” American Chemical Society Nano. 2011. 5(11):8591-8599.

130. Lokeshwar, V, et Al. “Hyaluronidase in prostate cancer: a tumor promoter and suppressor.” Cancer Res. 2005. 65:7782–7789.

131. Chao, K, Muthukumar, L, and Herzberg, O. “Structure of Human Hyaluronidase-1, a Hyaluronan Hydrolyzing Enzyme Involved in Tumor Growth and Angiogenesis.” Biochemistry. 2007. 46:6911–6920.

132. Franzmann, E, et Al. “Expression of Tumor Markers Hyaluronic Acid and Hyaluronidase (Hyal1) in Head and Neck Tumors.” Int. J. Cancer. 2003. 106:438–445.

133. Bourguignon, L, et Al. “Cd44 Interaction with Naþ-Hþ Exchanger (Nhe1) Creates Acidic Microenvironments Leading to Hyaluronidase-2 and Cathepsin B Activation and Breast Tumor Cell Invasion.” J. Biol. Chem. 2004. 279:26991–27007.

134. Stern, R. “Hyaluronidases in Cancer Biology.” Semin. Cancer Biol. 2008, 18:275–280.

135. Coradini, D, Perbellini, A, and Hyaluronan, A. “Suitable Carrier for an Histone Deacetylase Inhibitor in the Treatment of Human Solid Tumors.” Cancer Ther. 2004. 2:201–216.

136. Shigdar, S, et Al. “RNA aptamers targeting cancer stem cell marker CD133.” Cancer Lett. 2013. 330(1):84–95.

137. Adhikari, A, et Al. “CD117 and Stro-1 identify osteosarcoma tumor-initiating cells associated with metastasis and drug resistance.” Cancer Res. 2010. 70(11):4602–4612.

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