Keith S. Hoek

Systematic classification of melanoma cells by phenotype‐specific gene expression mapping

There is growing evidence that the metastatic spread of melanoma is driven not by a linear increase in tumorigenic aggressiveness, but rather by switching back and forth between two different phenotypes of metastatic potential. In vitro these phenotypes are respectively defined by the characteristics of strong proliferation/weak invasiveness and weak proliferation/strong invasiveness. Melanoma cell phenotype is tightly linked to gene expression. Taking advantage of this, we have developed a gene expression–based tool for predicting phenotype called Heuristic Online Phenotype Prediction. We demonstrate the predictive utility of this tool by comparing phenotype‐specific signatures with measurements of characteristics of melanoma phenotype‐specific biology in different melanoma cell lines and short‐term cultures. We further show that 86% of 536 tested melanoma lines and short‐term cultures are significantly associated with the phenotypes we describe. These findings reinforce the concept that a two‐state system, as described by the phenotype switching model, underlies melanoma progression.

Differential LEF1 and TCF4 expression is involved in melanoma cell phenotype switching

Recent observations suggest that melanoma cells drive disease progression by switching back and forth between phenotypic states of proliferation and invasion. Phenotype switching has been linked to changes in Wnt signalling, and we therefore looked for cell phenotype‐specific differences in the levels and activity of β‐catenin and its LEF/TCF co‐factors. We found that while cytosolic β‐catenin distribution is phenotype‐specific (membrane‐associated in proliferative cells and cytosolic in invasive cells), its nuclear distribution and activity is not. Instead, the expression patterns of two β‐catenin co‐factors, LEF1 and TCF4, are both phenotype‐specific and inversely correlated. LEF1 is preferentially expressed by differentiated/proliferative phenotype cells and TCF4 by dedifferentiated/invasive phenotype cells. Knock‐down experiments confirmed that these co‐factors are important for the phenotype‐specific expression of M‐MITF, WNT5A and other genes and that LEF1 suppresses TCF4 expression independently of β‐catenin. Our data show that melanoma cell phenotype switching behaviour is regulated by differential LEF1/TCF4 activity.

Heparan Sulfate Proteoglycan Modulation of Wnt5A Signal Transduction in Metastatic Melanoma Cells

Heparan sulfate proteoglycans (HSPGs) are important modulators for optimizing signal transduction of many pathways, including the Wnt pathways. We demonstrate that HSPG glycosaminoglycan levels increased with increasing metastatic potential of melanoma cells. Previous studies have demonstrated that Wnt5A increases the invasiveness of melanoma cells. We further demonstrate that HSPGs potentiate Wnt5A signaling, since enzymatic removal of the HSPG backbone resulted in a decrease in cellular Wnt5A levels, an increase in secreted Wnt5A in cell media, a decrease in downstream signaling, and ultimately, a decrease in invasiveness. Specifically, syndecan 1 and syndecan 4 expression correlated to Wnt5A expression and melanoma malignancy. Knockdown of syndecan 1 or 4 caused decreases in cell invasion, which could be restored by treating the cells with recombinant Wnt5A. These data indicate that syndecan 1 and 4 correlate to increased metastatic potential in melanoma patients and are an important component of the Wnt5A autocrine signaling loop, the activation of which leads to increased metastasis of melanoma.

The immunohistochemistry of invasive and proliferative phenotype switching in melanoma: a case report

To date there is no effective therapy for metastatic melanoma and at the molecular level the disease progression is poorly understood. A recent study by our group led to the development of a novel phenotype switching model for melanoma progression, wherein cells transition back-and-forth between states of proliferation and invasion to drive disease progression. To explore the models clinical relevance we interrogated phenotype-specific expression patterns in human melanoma patient material. A matched primary/metastasis pair from a human melanoma patient was obtained and immunohistochemically stained for proliferative and invasive phenotype markers. These were also stained for hypoxia and blood vessel markers. Proliferative phenotype markers Melan-A and Mitf showed consistent anti-correlation with invasive phenotype marker Wnt5A and hypoxia marker Glut-1. These also correlated with observed intra-tumoural vascularization patterns. Similar pattern distributions were present in both primary and metastasis samples. Strikingly, we observed that late phase metastatic melanoma cells adopt morphologies and behaviours identical to very early phase cells. The expression patterns observed closely matched expectations derived from previous in vitro and xenografting experiments. These results highlight the likelihood that disease progression involves melanoma cells retaining the capacity to regulate the expression of metastatic potential critical factors according to changing microenvironmental conditions.

E-cadherin determines Caveolin-1 tumor suppression or metastasis enhancing function in melanoma cells

The role of caveolin‐1 (CAV1) in cancer is highly controversial. CAV1 suppresses genes that favor tumor development, yet also promotes focal adhesion turnover and migration of metastatic cells. How these contrasting observations relate to CAV1 function in vivo is unclear. Our previous studies implicate E‐cadherin in CAV1‐dependent tumor suppression. Here, we use murine melanoma B16F10 cells, with low levels of endogenous CAV1 and E‐cadherin, to unravel how CAV1 affects tumor growth and metastasis and to assess how co‐expression of E‐cadherin modulates CAV1 function in vivo in C57BL/6 mice. We find that overexpression of CAV1 in B16F10 (cav‐1) cells reduces subcutaneous tumor formation, but enhances metastasis relative to control cells. Furthermore, E‐cadherin expression in B16F10 (E‐cad) cells reduces subcutaneous tumor formation and lung metastasis when intravenously injected. Importantly, co‐expression of CAV1 and E‐cadherin in B16F10 (cav‐1/E‐cad) cells abolishes tumor formation, lung metastasis, increased Rac‐1 activity, and cell migration observed with B16F10 (cav‐1) cells. Finally, consistent with the notion that CAV1 participates in switching human melanomas to a more malignant phenotype, elevated levels of CAV1 expression correlated with enhanced migration and Rac‐1 activation in these cells.