We found that while inactivation of Hif1α or Hif2α did not affect the initiation or progression of Pten;Braf melanomas, it significantly reduced the incidence of lymphatic metastases (Figure F). Consistent with these findings, we show that HIF1α and HIF2α expression are increased in thick melanomas and metastases from patients and are both necessary and sufficient for the invasion and invadopodia formation of melanoma cell lines. Moreover, HIF1α and HIF2α activate distinct transcriptional programs that converge to activate SRC and coordinate ECM degradation via PDGFRα/MT1-MMP and FAK/MMP2, respectively. Finally, we demonstrate that hypoxia activates SRC and increases the invasiveness of cell lines derived from Pten;Braf melanomas and that hypoxia-induced invasion of these cells can be abrogated by knockdown of Hif1α or Hif2α. Collectively, these findings demonstrate that, while HIF1α and HIF2α do not have an impact on primary melanoma initiation or progression, they promote melanoma cell invasion, invadopodia formation, and metastases through distinct yet convergent transcriptional programs.
Previous work examining the angiogenic profile of malignant melanomas shows that a significant percentage of human melanomas express detectable levels of HIF1α and HIF2α by immunohistochemistry, but that only levels of HIF2α correlate with VEGF expression and a poor prognosis (14
). Other work has confirmed that both mouse and human skin is relatively hypoxic and that, at least in the context of Ink4a
-deficient mouse melanocytes, hypoxia and specifically stabilized HIF1α can cooperate with constitutively active AKT in transformation (15
). Whether HIF2α can mediate a similar effect was not determined. In our own studies, we stained a TMA from a large sample of patients with a spectrum of melanocytic lesions ranging from benign nevi to metastatic melanomas and showed that both HIF1α and HIF2α expression are increased in thick primary melanomas and metastases. In addition, we determined whether either loss of Hif1α or Hif2α affected the initiation of Pten;Braf
melanomas in GEM models. We found that inactivation of Hif1
α or Hif2
α did not affect either tumor formation or the rate of melanoma growth after their formation. While there are distinct differences in the systems utilized (i.e., soft agar assays and xenografts versus an in vivo autochthonous model) as well as the genetic background of the cells (i.e., constitutively activated Akt and Ink4a/Arf deletion versus Pten loss and mutant Braf activation), our results suggest that, at least in a GEM model of melanoma initiated by Pten
loss and Braf
activation, inactivation of Hif1α or Hif2α did not appreciably affect melanocyte transformation or melanoma progression.
Our in vitro studies in both human melanoma cell lines and the cell lines derived from Pten;Braf
murine melanomas suggest that HIF1α and HIF2α are critical nodes that mediate the hypoxia-induced motility and invasiveness of melanoma cells. This work delineates the pathways activated by HIF1α and HIF2α that mediate actin nucleation and ECM degradation at the forefront of invadopodia and shows for what we believe is the first time that HIF1α and HIF2α are necessary and sufficient for invadopodia formation. Notably, our data indicate what we believe to be a novel paradigm, that HIF1α and HIF2α act to promote a metastatic program through distinct pathways. Specifically, HIF1α induces SRC activity and ECM degradation via PDGFRα and MT1-MMP, while HIF2α signals to SRC via FAK and promotes ECM remodeling through MMP2. Therefore, the transcriptional suite of genes activated by HIF1α and HIF2α in response to hypoxia coordinates the 2 processes that define invadopodia: (a) actin nucleation at cell membrane protrusions and (b) expression of proteases involved in the degradation of the ECM (25
). Moreover, these HIF-dependent increases in invasion and invadopodia formation are accompanied by decreases in cell stiffness, a physical property of cells that has been previously shown to inversely correlate with the invasive capacity of cells (32
). Whether these HIF-induced alterations in cell compliance are a result of the same signaling pathways affecting HIF-induced invasion has yet to be determined.
There are striking clinical similarities between malignant melanoma and RCC, including their hypervascularity and propensity for hemorrhagic central nervous system metastases, relative resistance to radiation therapy, and, most intriguingly, their responsiveness to immunomodulatory agents such as IL-2, INF-α, and, most recently, antibodies directed at the T cell inhibitory ligand/receptor complex PD-L1 and PD1 (36
). On a molecular level, HIF activation in the setting of pVHL inactivation is both necessary and sufficient for RCC tumorigenesis, and HIF appears to mediate the majority of phenotypes seen in the setting of VHL deficiency in mice (6
). Interestingly, HIF activation is a consequence of several of the signature genetic events found in melanoma. For example, loss-of-function mutations of PTEN
result in an Akt-dependent increase in mTOR activity and the translation of HIFα subunits (15
). HIF is also transcriptionally upregulated in tumors with mutant RAS
as well as in tumors overexpressing MITF (16
). Intriguingly, a recent report describes a rare germline MITF mutation that predisposes to the development of melanoma, RCC, or both (40
). Perhaps most interestingly, this MITF mutation, Mi-E318K, prevents its SUMOylation, enhances its chromatin occupancy, and appears to upregulate expression of genes involved in melanomagenesis and HIF1α (potential effects on HIF2α have yet to be reported). Therefore, the germline MITF mutation, Mi-E318K, appears to link two seemingly similar cancers on a molecular level, possibly through activation of HIF.
Hypoxia is associated with metastasis in many solid tumors, including melanoma. The data presented here suggest that HIF1α and HIF2α contribute to hypoxia-mediated metastatic spread through the pathways presented. PDGFRα, FAK, and SRC are kinases and are targetable by inhibitors; indeed, inhibitors against PDGFRα (imatinib) and SRC (dasatinib) are already FDA approved and are in use for the treatment of multiple cancers. Our results imply that PDGFRα, FAK, or SRC inhibition in early stage melanoma patients may be beneficial, as these pathways are critical for hypoxia-induced melanoma cell intravasation. However, whether activation of these pathways remains necessary for the maintenance of established metastases remains to be determined.
In summary, our results establish that HIF plays an active role in the metastatic progression of melanoma and demonstrate that HIF1α and HIF2α activate independent transcriptional programs, which induce invadopodia formation and invasion and decrease cell stiffness. We demonstrate in an autochthonous mouse model of metastatic melanoma that inactivation of Hif1α or Hif2α results in a significant reduction in metastases to regional lymph nodes, suggesting that these in vitro observations are operational in vivo (Figure F). This work defines 2 HIF target genes, PDGFRA and FAK, and is the first examination, to our knowledge, of the role of HIF2α in melanomagenesis. Importantly, our studies directly link hypoxia, HIF1α, and HIF2α to enhanced invadopodia formation, adding another pathologic stimulus to the list of those currently known to modify invadopodia assembly and turnover. In aggregate, our studies define HIF1α and HIF2α as intimately involved in the process of metastasis in malignant melanoma and further enforce the notion that the similarities between melanoma and RCC extend beyond mere clinical observations and rare germline genetic events.