Hypoxia Stabilizes GAS6/Axl Signaling in Metastatic Prostate Cancer

Abstract

The receptor tyrosine kinase Axl is over-expressed in a variety of cancers and is known to play a role in proliferation and invasion. Previous data from our lab indicates that Axl and its ligand GAS6 may play a role in establishing metastatic dormancy in the bone marrow microenvironment. In the current study, we found that Axl is highly expressed in metastatic prostate cancer (PCa) cell lines PC3 and DU145 and has negligible levels of expression in a non-metastatic cancer cell line LNCaP. Knockdown of Axl in PC3 and DU145 cells resulted in decreased expression of several mesenchymal markers including Snail, Slug, and N-cadherin, and enhanced expression of the epithelial marker E-cadherin, suggesting that Axl is involved in the epithelial to mesenchymal transition in PCa cells. The Axl-knockdown PC3 and DU145 cells also displayed decreased in vitro migration and invasion. Interestingly, when PC3 and DU145 cells were treated with GAS6, Axl protein levels were down-regulated. Moreover, CoCl2, a hypoxia mimicking agent, prevented GAS6 mediated down-regulation of Axl in these cell lines. Immunochemical staining of human PCa tissue microarrays demonstrated that Axl, GAS6 and Hif1-α (indicator of hypoxia) were all co-expressed in PCa and in bone metastases, compared to normal tissues. Together, our studies indicate that Axl plays a crucial role in PCa metastasis, and that GAS6 regulates the expression of Axl. Importantly, in a hypoxic tumor microenvironment Axl expression maintained leading to enhanced signaling.

Interplay between receptor tyrosine kinases and hypoxia signaling in cancer

“…Hence, in a hypoxic tumor microenvironment stabilization of AXL expression may underlie driving of metastasis progression [66]. The finding that both AXL and MET are direct targets of HIF-1α/HIF-2α reveals a potent mechanism of hypoxic tumors to sustain malignant phenotypes [62].…”

“…Transcriptional activation by HIF-1α [49,55] SN12L1 clear cell renal cell carcinoma (ccRCC) cells MET AXL signalling mediated lateral activation by SRC [62] RCC4 ccRCC cells, 786-0 ccRCC cells, HepG2 hepatocellular carcinoma cells, Hep3B hepatocellular carcinoma cells AXL Transcriptional activation by HIF-1α and HIF-2α [62] HLEC lens epithelial cells, PC3 and Du-145 prostate cancer cells AXL Inhibition of GAS6-mediated AXL downregulation [65,66] MCF7, ZR-75, MDA-MB-231 and MDA-MB-468 breast cancer cells RON Transcriptional activation by HIF-1α [69] Numerous cancer types VEGF-A Transcriptional activation by HIF-1α [13,18,23] Rat pulmonary microvascular smooth muscle cells…”

Development of a Novel Method to Detect Prostate Cancer Circulating Tumor Cells (CTCs) Based on Epithelial-Mesenchymal Transition Biology

“…The Axl/GAS6 pathway is important for metastasis of several carcinomas [129][130][131][132][133], and is adversely prognostic [134][135][136][137][138]. Axl is necessary for EMT, as evidenced by reduction in mesenchymal biomarkers and increased migration and invasion upon knockdown of Axl in PC cells [139]. GAS6 downregulates expression of its receptor, Axl, and hypoxia is sufficient to prevent GAS6-mediated downregulation of Axl.…”

Identification of Novel HIF1A Target Genes That Regulate Tumor Progression and Metastasis

“…Analysis of the array data revealed that HIF-1α regulates multiple genes involved in stem cell maintenance, as discussed above and in Chapter 5. Many other genes we initially identified have since been implicated as being HIF-1α dependent genes such as Angptl4 [72], Jarid1b and the Jumanji proteins [246], several pro-collagens [291] and Axl [245]. However, we also discovered several genes that still had not been previously explored as hypoxiadependent or in the context of breast cancer.…”

“…Some of the genes identified have long been known to be HIF-1α target genes, such as Vegf, Glut1, Phd3/Egln3, Aldoc, Ldha, Gapdh and many more [24]. Furthermore, genes were identified that have recently been shown to be direct HIF-1α-target genes, including Axl [245], Angptl4 [72], and multiple jumonji-domain histone demethylases [246]. Additionally, analysis of the gene lists revealed enrichment of several genes in WT cells that are either involved in stem cell maintenance, or are commonly used stem cell surface markers including CD133/Prom1 [117], which has been shown to a HIF-1α target gene, Gata3 [247], Wnt signaling pathway genes such as Wnt10a, Wnt5b, Lrp5, and Fzd7 [248], Notch pathway genes such as Dll1 and Hey1 [164], and Itga6/CD49f.…”