CXCL12/CXCR4 Axis- Mediated Epithelial-Mesenchymal Transition Facilitates Renal Cell Carcinoma Metastasis
Date of Award
Campus Access Dissertation
Doctor of Philosophy (PhD)
Biology/Molecular, Cellular, and Organismal Biology
Jill A. Macoska
Renal cell carcinoma (RCC) is one of the most commonly diagnosed cancers among adults. Annually, more than 70000 new cases are identified, and death from the disease transpires in most of these patients, making RCC a leading cause of cancer-related death. The high mortality of RCC is directly correlated to the high recurrence of the disease and limited therapeutic options. Metastasis to a distant organ occurs in more than 30% of patients, and most patients are diagnosed at a late stage after the tumor has already metastasized. RCC is also known to be highly heterogenic and resistant to conventional treatments like systemic chemotherapy and radiation. Nephrectomy is considered the most effective treatment so far for patients with localized disease. A better understanding of factors contributing to RCC metastasis might improve therapeutic development and patient outcomes.
One of the steps required for tumor cell progression from localized disease to metastatic disease is the acquisition of invasive abilities through epithelial to mesenchymal transition (EMT). EMT promotes loss of epithelial markers and gain of mesenchymal markers by downregulation of E-cadherin via the inhibitory transcription factors SNAIL and SLUG. The tumor cells acquire a mesenchymal phenotype that enables them to leave the primary site and intravasate into the blood or lymphatic systems and thereby colonize distant sites. Metastasis and colonization are highly directed mechanisms in which cancer cells migrate toward specific preferred organs that express specific protein receptors. The CXCL12/CXCR4 axis is a known chemoattractant pathway, it is highly active in advanced cancer, including RCC, and its activation was implicated in metastasis and poor patient survival.
This study explored the hypothesis that CXCL12/CXCR4 signaling promotes migratory and invasive ability in RCC by inducing EMT signaling. We demonstrate that RCC cell lines activate the CXCL12/CXCR4 axis to induce a migratory and invasive response, dependent on EMT cytoskeletal rearrangement by Rho GTPases and CXCR4 signaling. Moreover, CXCL12/CXCR4 induces a classic EMT signaling cascade, decreasing epithelial markers like E-cadherin and increasing the mesenchymal signature by up-regulating Vimentin, N-cadherin, and EMT transcription factors SLUG/SNAIL. Interestingly, this axis transactivates EGFR that stimulates the EMT signaling signature and promotes the phosphorylation of AKT and ERK, suggesting that these signaling pathways are coupled to CXCL12/CXCR4-mediated RCC progression.
Inhibition of the CXCL12/CXCR4 signaling axis represses the ability of RCC cell lines to activate EMT signaling and attenuates their ability to migrate and invade. We demonstrate that the role of CXCR4 is crucial in transmitting the EMT signaling signature induced by CXCL12 and not CXCR7, as genetic ablation of CXCR4 in RCC reduces their ability to up-regulate EMT signature and inhibits their ability to invade, even when CXCL12 is present as a chemoattractant. Lastly, we explore the expression pattern of CXCR4 and CXCR7 in different subtypes and stages of RCC disease. Using patient tumor samples and bioinformatics analysis of available transcription data, we found that CXCR4 is up-regulated in RCC patient tumors, and its overexpression is correlated with disease progression. We also associate that more aggressive RCC subtypes like ccRCC have higher CXCR4 and CXCR7 expression, and both RCC subtypes favor overexpressing CXCR4 over CXCR7, which might make CXCR4 expression level a suitable prognostic factor for RCC progression and metastasis. Finally, we implemented an in-vivo mouse model approach to further our understanding of the contribution of CXCR4 and CXCR7 to RCC aggressiveness. Using engineered CXCR4 and CXCR7 Knockdown (KD) clones, we demonstrate that RCC cells with intact CXCR4 have higher metastatic and colonization abilities than the CXCR4 KD clones. Interestingly, the CXCL12/CXCR4/CXCR7 axis does not seem to modulate the primary tumor size. These results are consistent with the hypothesis that the CXCL12/CXCR4 axis has a critical role in promoting lung metastatic lesions, suggesting that CXCR4 and not CXCR7 is the major driver of RCC aggressive malignancy.
In conclusion, our findings suggest that CXCL12/CXCR4 activation in RCC has a pivotal role in EMT induction and the acquisition of a migratory and invasive phenotype. The CXCL12/CXCR4 axis inhibition can repress the CXCL12 induced EMT induction and repress the acquisition of a migratory and invasive phenotype. Our results also demonstrate that the CXCL12/CXCR4 axis has a critical role in promoting malignant aggressive disease both in-vitro and in-vivo, which corresponds with the clinical outcome and tumor samples.
Understanding how CXCL12/CXCR4/CXCR7 axis signaling might define the cellular mechanisms involved in metastatic disease facilitates our further understanding of such mechanisms that underlay advanced metastatic RCC and provides new prospects for drug development.
Zhilin-Roth, Alisa, "CXCL12/CXCR4 Axis- Mediated Epithelial-Mesenchymal Transition Facilitates Renal Cell Carcinoma Metastasis" (2021). Graduate Doctoral Dissertations. 711.
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