CXCR4/CXCL12 axis in non small cell lung cancer (NSCLC) pathologic roles and therapeutic potential.

Lung cancer is the second most common malignancy and the leading cause of cancer-related death in the western world. Moreover, despite advances in surgery, chemotherapy and radiotherapy, the death rate from lung cancer remains high and the reported overall five-year survival rate is only 15%. Thus, novel treatments for this devastating disease are urgently needed. Chemokines, a family of 48 chemotactic cytokines interacts with their 7 transmembrane G-protein-coupled receptors, to guide immune cell trafficking in the body under both physiologic and pathologic conditions. Tumor cells, which express a relatively restricted repertoire of chemokine and chemokine receptors, utilize and manipulate the chemokine system in a manner that benefits both local tumor growth and distant dissemination. Among the 19 chemokine receptors, CXCR4 is the receptor most widely expressed by malignant tumors and whose role in tumor biology is most thoroughly studied. The chemokine CXCL12, which is the sole ligand of CXCR4, is highly expressed in primary lung cancer as well as in the bone marrow, liver, adrenal glands and brain, which are all sites for lung cancer metastasis. This review focuses on the pathologic role of the CXCR4/CXCL12 axis in NSCLC and on the potential therapeutic implication of targeting this axis for the treatment of NSCLC.

Introduction - Lung Cancer

Lung cancer is the second most common malignancy and continues to be the leading cause of cancer-related death in both men and women in the United States and throughout the world 1. Lung cancer causes more deaths than the next four common cancers combined - colon, breast, pancreas and prostate 1, 2. Epidemiologic evidence and the complementary biological understanding of respiratory carcinogenesis support the conclusion that cigarette smoking causes lung cancer 3-6. Other occupational and environmental causes are being increasingly associated with lung cancer include exposure to radon, asbestos, outdoor air pollution and many other chemicals 3-6. It has been estimated that over 220,000 new cases of lung cancer would be diagnosed in the United States in 2012 7. Despite advances over the last decade in diagnostic, staging and surgical techniques as well as new chemotherapy and radiotherapy protocols, the death rate from lung cancer remains high 8. Lung cancer-related mortality is anticipated to peak above 160,000 cases/year and the reported overall five-year survival rate is only 15% 7. The anticipated implementation of nation-wide lung cancer screening programs will increase the proportion of patients diagnosed in early disease stages permitting such patients to undergo surgical resection, which is considered the best therapeutic approach 9. Nonetheless, the five years survival rates of these surgically treated patient, with or without the addition of adjuvant chemotherapy or radiotherapy is approximately 50% 10, 11. Following primary tumor development the disease tends to progress along three distinct, yet often concomitantly occurring pathways: 1. Local invasion of the tumor to adjacent structures in the mediastinum and the chest wall. 2. Lymphatic spread to regional lymph nodes 3. Hematogenous spreading of distant metastases, most commonly to the liver, adrenal glands, bone marrow and the brain 10-13. Each of these three manifestations of disease is only partially addressed by current medical and surgical treatments protocols, and novel therapeutic approaches that tackle each of these clinical manifestations are urgently needed 10-13.

Similar to many other solid epithelial malignancies, staging of lung cancer is based on the TNM classification system 14. The anatomical extent of disease is based on the assessment of three components: T - the extent of the primary tumor, N- the absence or presence of regional lymph nodes metastases, M- the absence or presence of distant metastases. This clinical staging is essential to select and evaluate therapy, and such evidence arises from physical examination, imaging, endoscopy, biopsy and surgical exploration 15. Pathologically lung cancer is classified into non-small cell lung cancer (NSCLC) accounting for 80% of cases, and small cell lung cancer (SCLC) accounting for the remaining 20% 16, 17. NSCLC originates from respiratory epithelial cells and adenocarcinoma is the most common histologic subtype in most countries, accounting for almost half of all lung cancers 18. The clinical, radiologic, molecular and pathologic spectrum is widely divergent within lung adenocarcinoma, and recently a new classification has been revised 18. The second most common type of lung cancer is squamous cell carcinoma and the least common type is large cell carcinoma 16-18. SCLC stem from cells with neuroendocrine differentiation that are located at distinct anatomic sites along the bronchial tree 19. The majority of data reviewed in this manuscript relates to NSCLC adenocarcinomas.

CXCR4 Expression, Regulation and Function in Solid Malignancies

Chemokines, a family of 48 chemotactic cytokines interact with their 7 transmembrane G-protein-coupled receptors to guide immune cell trafficking in the body under both physiologic and pathologic conditions 20, 21. Tumor cells, which express a relatively restricted repertoire of chemokine and chemokine receptors, utilize and manipulate the chemokine system in a manner that benefits both local tumor growth and distant dissemination 20, 22, 23. In the tumor microenvironment autocrine and paracrine chamokine/chemokine receptor loops interact to promote tumor cell survival and growth, and also to enhance tumor neo-angiogenesis 20, 22, 23. At distant sites, it is the tissue-produced chemokine which guide/attracts the metastasis of chemokine receptor expressing tumor cells 20.

Among the 19 chemokine receptors, CXCR4 is the receptor most widely expressed by malignant tumors and whose role in tumor biology is most thoroughly studied 20. The chemokine CXCL12 is the sole ligand of CXCR4 and the majority of research that focus on the role of CXCR4 in cancer relates to this chemokine/chemokine receptor pair 24, 25. Nevertheless, in 2006 another receptor for CXCL12 was identified and named CXCR7 26. CXCR7 is expressed during embriogenesis, angiogenesis and in various malignant tissues including NSCLC. CXCR7 is thought to act in part as a scavenger of CXCL12 however additional functions for this receptor have also been reported 26-28. In distinct form CXCR4, CXCR7 binds not only CXCL12 but also the chemokine CXCL11 26, 27. Moreover, the signaling cascades that are generated upon binding of CXCL12 to CXCR4 or CXCR7 vary at least partly, depending on which of the receptors is engaged 26, 27. This review focuses mainly on data collected regarding the expression and function of CXCR4 in NSCLC, nevertheless it is important to keep in mind that whenever CXCL12 is mentioned the effects related to its expression may be attributed in part to CXCR7 expression and function.

Relative to normal cells in the tumor's tissue of origin, malignant cells often over express CXCR4, this phenotype can be induced by multiple oncogenic alternations and appears to promote tumor cell survival, proliferation, invasion and metastasis 20, 29-35. For example, the RET/PTC rearrangement was shown to enhance CXCR4 expression in papillary thyroid carcinomas and this was associated with increased responsiveness of the malignant cells to CXCL12, leading to entry into S-phase and to enhanced survival of the tumor cells 36. Furthermore in the same study the researchers found that invasion through extracellular matrix was also supported by CXCL12 stimulation and inhibited by CXCR4 blocking antibodies 36. Along similar lines, Yan M. Li et al found that aberrant activation of the human epithelial growth factor receptor HER-2 in breast cancer cells results in PI-3K/Akt/mTOR pathway dependant up-regulation of CXCR4, that in turn enhances tumor cell invasion in vitro and lung metastasis in vivo 37. In addition to oncogenic alternations, the tumor microenvironment may also greatly affect the tumor's CXCR4 expression levels 38. For example, low tissue oxygen levels result in Hypoxia Inducible Factor 1 (HIF-1) expression that in turn up-regulates CXCR4 leading to enhanced NSCLC tumor cell invasion and metastasis 38. Likewise, in glioblastoma tumors, both HIF-1 and vascular endothelial growth factor (VEGF) were found to enhance CXCR4-dependant angiogenesis in the tumors 39. Thus it appears that, regardless of the reasons behind CXCR4 expression, the over-expression of this receptor renders tumor cells responsive to CXCL12-induced signaling, which in turn promotes their survival, proliferation, angiogenesis and metastasis 40, 41.

Pathologic Role For CXCR4/CXCL12 in Non-Small Cell Lung Cancer

Two line of evidence provide insight into the role of CXCR4/CXCL12 in NSCLC malignant propagation. The first comes from retrospective studies of human metastatic cancers, in which high CXCR4/CXCL12 expression levels are associated with a more advanced diseases stage and with worse prognosis 12, 25, 42-44. The second evidence comes from experimental in vitro and in vivo studies, which indicate that CXCR4/CXCL12 interactions in the tumor microenvironment may foster local tumor growth and that high CXCR4 expressing tumor cells, have a greater invasive and metastatic potential 12, 25, 42-44.

Multiple retrospective studies of human NSCLC implemented immunohistochemical staining to demonstrate that all major subtypes of NSCLC tumors express CXCR4 and more specifically to show that CXCR4 expression is detected in either the cytomembranous compartment or the nuclear compartment or both compartments of the malignant cells 42-44. When examining the correlation of such CXCR4 staining with patient-specific clinical parameters of disease, Su and colleagues found that tumors with high cytomembranous expression of CXCR4 were more prone to locally invade neighboring anatomical structures and to form distant metastasis than were low CXCR4-expressing tumors 43. Similarly, Chen et al have found that high-level of cytomembranous CXCR4 expression correlated with brain-specific metastasis in single station M1 NSCLC patients 45. Some insight into the processes that may promote this highly metastatic phenotype of CXCR4 expressing tumor cells comes from the recent work of Franco et al. In this report the authors found that high cytomembranous expression of CXCR4 in NSCLC tumor cells was associated with a significantly increased in density of micro-vascular structures in the tumors and that this was associated with increased micro-vessel invasion by tumor cells 46. Further support for involvement of CXCR4 in NSCLC spread and disease outcome comes from the two additional works. First, Oonakahara and associates found that pleural mesothelial cell produced CXCL12 and that high level of CXCL12 in the pleural fluid were associated with seeding of the pleural space with CXCR4 expressing tumor cells 47. Second, a work by Otsuka et al has recently shown that CXCR4 is expressed in the cytomembranous compartment of majority of NSCLC tumors, and that its over-expression is associated with significantly poorer survival in stage IV NSCLC patients. Moreover, this poor outcome was disproportionately represented in the female population thus suggesting a gender-dependent difference in clinical outcome based on CXCR4 over-expression 48. Somewhat contradictory to these observations, Spano and associates have reported that strong CXCR4-positive nuclear staining was associated with improved outcomes in early stage NSCLC 42. In a larger study, Wagner and colleagues have confirmed the above-mentioned findings and concluded that cytomembranous expression of the CXCR4 in lung adenocarcinoma is an independent risk factor for decreased disease-free survival, whereas nuclear expression of CXCR4 confers a survival benefit 44. The prognostic usefulness of determining CXCL12 expression levels in NSCLC has also been tested 44. For instance, we found that more than 80% of NSCLC tumors expressed CXCL12 in the cytomembranous compartment and that NSCLC recurrence rates were higher (7/21) among patients with intense CXCL12 staining than among those who had weak staining (0/8) 49. Using tumor tissue arrays we further found a correlation between high CXCL12 expression levels and an advanced pathologic stage of disease 50. Other reports have corroborated these findings, also nicely demonstrating a linkage between high tumoral CXCL12 expression levels and both a higher T score and an increased tendency to form lymph node metastasis 44. Taken together, although yet preliminary and requiring further validation, the above-mentioned studies certainly advocate in favor of a pro-malignant role for the CXCL12/CXCR4 axis in NSCLC disease progression.

An additional method to examine the potential correlation between CXCR4/CXCL12 expression and cancer patient outcomes is by performing genetic polymorphism studies that correlate distinct genetic alternations to patient-specific clinical and pathologic parameters 51. In this regard, two distinct genetics alternations in the CXCR4/CXCL12 axis have been characterized in the past. The first is the single nucleotide polymorphisms (SNP) in position 801 of the CXCL12 gene, (guanine to adenine (G→A), un-translated region at position 801) and the second is the SNP at codon 138 of the CXCR4 gene (the silent mutation cytosine to thymine (C→T), at codon 138) 52, 53. Looking at these SNPs, Hirata et al have found that prostate cancer patients that carry the CXCL12 AA trait tend to express higher levels of CXCL12 and also to up-regulate CXCR4 expression in their tumors 51. Furthermore, they found that carriers of this trait had a worse prognosis relative to GA and GG traits 51. The effects of the CXCR4 TT trait on CXCR4 expression levels are not well defined, nevertheless, the CXCR4 TT trait has been associated with more advanced disease stages in several malignancies, among them is head and neck cancer 53. Recent research by Lee and colleagues followed the same approach in order to examine the correlation of these two SNP mutations and the outcomes of NSCLC patients 52. The researchers found that individuals with the CXCL12 AA and CXCR4 TT genotypes had higher odds ratios (an OR of 1.95, 95% CI 1.08-3.50, p = 0.018 and OR of 4.71, 95% CI 1.99-11.2, p < 0.0001 respectively) for lung cancer development and that NSCLC patients with the CXCL12 AA and CXCR4 TT genotypes more frequently had an advanced disease stage and a worse prognosis 52. This study thus further provides evidence for the pro-malignant role for CXCL12/CXCR4 in NSCLC.

The above-mentioned studies point toward two distinct mechanisms by which CXCL12/CXCR4 interactions may promote NSCLC progression. First the association of high CXCR4 expression levels in the primary tumor with distant metastasis suggests that this receptor is involved in NSCLC disease spread. Second, the high expression of CXCL12 in the tumor microenvironment suggests that CXCR4/CXCL12 interaction may act locally in autocrine and paracrine manners to enhance primary tumor growth and to alter its inflammatory milieu. Experimental evidence in support of the role of CXCR4 in NSCLC metastasis comes from the work of Phillips and colleagues who demonstrated that administration of anti-CXCL12 neutralizing antibodies to immunodeficient mice harboring human NSCLC tumors abrogated organ specific metastasis 54, 55. Recent research also suggests that CXCR4/CXCL12 interactions can advance NSCLC local tumor growth 50. For examples, we have shown in a series of studies that CXCL12 stimulation enhances the colony forming capacity of NSCLC cells and that drugs such as AMD3100 and BKT140, which specifically block the CXCL12/CXCR4 axis, inhibit NSCLC growth in vitro and in vivo 50, 56-58. Finally, the inflammatory milieu of NSCLC tumors may also be affected by the CXCL12/CXCR4 axis 49, 59. In this regard we have shown that CXCL12 protein levels in NSCLC tumors are significantly higher relative to CXCL12 levels in the serum and that stimulation of NSCLC cells with CXCL12 induces the production of additional pro-inflammatory (CCL20) and pro-angiogenic (CXCL1 - IL-8) chemokines 49, 50. Furthermore, we found an increased inflammatory load in NSCLC tumors that expressed high levels of CXCL12 relative to tumors that expressed low levels of CXCL12 49. Thus, CXCL12 may participate in shaping the tumor microenvironment by affecting the recruitment/retention of immune and bone marrow derived cell in the tumor. Also noteworthy are the works that report the expression of CXCR4 in the nucleus 43, 44. The role of CXCR4 in this sub-cellular compartment is yet unknown and if indeed validated, it may lay direction for intriguing future research.

Role for CXCR4 in NSCLC Tumor initiating cells (cancer stem cells) biology

Cancer initiating cells (CIC), also referred to as cancer stem cells, are functionally defined by their high self-renew capacities and by their potential to recapitulate tumors in ectopic setting 60-63. In addition, CIC are considered to possess drug-resistance properties indicating an important role for these cells in disease relapse following treatment 60-63. In recent years, several groups have reported the identification of NSCLC cells with CIC-like properties, and argued that patients with tumors harboring increased number of these CIC have a worse prognosis 64. Various membranous expressed proteins (CD133, CD166, CD24, CD44 and CD117), intracellular enzymes (saldehyde dehydrogenase 1 (ALDH 1) and glycine decarboxylase (GLDC)) and additional proteins (OCT-4 SOX2 and ABCG2) were used to isolate and characterize the NSCLC CIC population, therefore suggesting that great heterogeneity may actually exist in this unique subpopulation of tumor cells 12, 61-66. Interestingly, recent reports argue that CXCR4 is expressed by NSCLC CIC and is functionally important for maintenance of their unique characteristics 65, 67. For example, Bertolini et al has found that NSCLC cells with high self-renewal capacity and with increased chemotherapeutic resistant expressed high levels of CXCR4 relative to NSCLC that lack these characteristics 67. Similarly, Jung et al have shown that pharmacological inhibition and/or siRNA targeting of CXCR4 and of the downstream action of STAT3 significantly suppressed the self renewal capacity of various NSCLC cell lines and concluded that CXCR4 positive NSCLC cells are strong candidates for tumorigenic stem-like cancer cells that maintain stemness through a CXCR4-mediated STAT3 pathway 65. In addition, Damelin and colleagues suggested that CXCR4 might act downstream to the transcription factor 5T4 in epithelialto-mesenchymal transition (EMT) and in migration of NSCLC CIC 68. Taken together, these studies provide evidence in support of the existence of subpopulations of NSCLC cells with stem-like properties and also mark CXCR4 as an important player in their unique biology. Considering the resistance of NSCLC CIC to conventional chemotherapeutic drugs it is reasonable to speculate that in the clinical setting such CICs may be responsible for treatment failure and disease recurrence following therapy 69.

Therapeutic Potential of CXCR4 Antagonists Against Non Small Cell Lung Cancer

Multiple solid malignant tumors such as breast, prostate, colorectal and pancreatic carcinomas, in addition to gliomas, neuroblastomas, osteosarcoma, and NSCLC express CXCR4 70. As reviewed above, CXCR4 plays a complex role in the evolution and progression of these malignancies, acting in multiple ways to increase both local tumor growth and metastatic spread 22, 23, 71. CXCR4 has additionally been found to be involved in regulating tumor vascularization and in facilitating the interaction between tumor cells and stromal cells in a manner that favors activation of adhesion-mediated drug and radiation-resistance pathways 25, 72, 73. Finally CXCR4 also appears to play a critical role in biology of cancer stem cells 65. Thus, it is unsurprising that the CXCR4/CXCL12 axis is considered as an exciting target for the development of novel anti-cancer chemokine-based therapeutics 25, 70, 74. Indeed, considering the accumulating data regarding the role of CXCR4 in NSCLC biology and the urgent need for novel therapeutics for this devastating disease it is tempting to speculate that CXCR4 antagonists will join in the near future the current armamentarium of drugs that are utilized against NSCLC.

Currently over 15 new drugs that target the CXCR4/CXCL12 axis are being developed with one drug, AMD3100 (also known as Plerixafor and Mozobil), already gaining FDA approval 70, 75-77. The indication for AMD3100 administration however is mobilization of hematopoietic stem cells for bone marrow transplantation rather than cancer treatment 76. Nevertheless AMD3100 and BKT140, an additional CXCR4 antagonist, have been reported to act in conjunction with conventional anticancer therapies against human NSCLC 50, 65. AMD3100 has been reported to render chemotherapeutic resistant NSCLC CIC to chemotherapy and was further shown to prevent these cells from maintaining their stem like properties 65. Similarly, BKT140 was shown to act in an additive manner with cisplatin, paclitaxel and radiation pretreatment to prevent NSCLC tumor cell proliferation 58. Although these findings are very encouraging, some key issues deserve proper consideration prior to designing clinical trials that utilize CXCR4 inhibitors for treatment of cancer 70. One intriguing issue to define is whether CXCR4 inhibitors should be administered in order to prevent disease spread or rather in order to enhance the effects of concomitantly administered drugs/radiation against preexisting tumors. The answer to this question will probably further guide the duration of drug administration: chronic administration versus pulse therapy in combination with chemo/radiotherapy. Another important topic to consider is the potential harmful effects of prolonged anti-CXCR4 therapy. While short administration of anti-CXCR4 drugs is considered to have a relatively tolerable profile of side effects, the prolonged effects of such drugs are less clear 70, 75-77. This issue is of great concern since CXCR4/CXCL12 interactions are involved in a number of essential homeostatic processes in the body such as regulating the recirculation of hematopoietic stem cells 78-80. More specifically, it is questionable whether prolonged mobilization of hematopoietic stem cells is tolerable and whether the effects of mobilized bone marrow cells on the tumor will be pro or anti metastatic. An intriguing possibility to overcome some of these concerns was recently introduced by the group of Liang et al who reported about the development of a novel small molecule, MSX-122, that is a partial CXCR4 antagonist without stem cell mobilizing properties 81. The researchers speculate that drugs such as MSX-122 may be safely administered for long-term blockade of metastasis avoiding the side effects of stem cell mobilization 81. Nonetheless, it seems that, when taking into account the characteristics of the CXCR4 antagonists that have already been approved for use in clinical trials (see ref no 62) and when considering the potential roles of CXCR4 in NSCLC, it might be more logical to first design clinical studies that will examine the short term effects of CXCR4 antagonist administration. More specifically, such studies should measure the potential additive effects of combining CXCR4 antagonists with current chemotherapeutic / radiation protocols in attempt to increase and prolong their effects. As mentioned, the alternative approach of attempting to prevent metastasis formation may require long-term administration of CXCR4 antagonist, which could possibly result in significant side effect. In addition studies that focus on metastasis inhibition might be more complex to design and will necessitate a longer follow up period prior to concluding their outcomes. Thus, the short-term approach seems to offer an ideal next step in the urgent need to develop novel therapeutics to NSCLC.

Concluding Remarks

In the last decade much has been learned about the role of CXCR4/CXCL12 axis in solid malignancies in general and in NSCLC in particular 12, 20, 21. Nowadays, this knowledge is on the edge of being translated into clinical trials that will examine the potential of CXCR4/CXCL12 antagonist to fight cancer 70. The urgent need for new therapeutic approaches for NSCLC patients make this disease an attractive target for such evolving trials.

Recommended Readings

For a recent overview of CXCR4 signaling, development of novel CXCR4-based therapeutics, and current clinical trials, the readers are referred to reference number 70.

For an overview about the identification of CXCR4 expression in tumor cells as a diagnostic and therapeutic tool, the readers are referred to the manuscript by Jacobson and Weiss et al in this issue of Theranostics Journal.