Epithelial-to-mesenchymal transition (EMT) is a dynamic process that relies on cellular plasticity. Recently, the process of an oncogenic EMT, followed by a reverse mesenchymal-to-epithelial transition (MET), has been implicated as critical in the metastatic colonization of carcinomas. Unlike governance of epithelial programming, regulation of mesenchymal programming is not well understood in EMT. Here, we describe and characterize the first microRNA that enhances exclusively mesenchymal programming. We demonstrate that miR-424 is upregulated early during a TWIST1 or SNAI1-induced EMT, and that it causes cells to express mesenchymal genes without affecting epithelial genes, resulting in a mixed/intermediate EMT. Furthermore, miR-424 increases motility, decreases adhesion, and induces a growth arrest, changes associated with a complete EMT that can be reversed when miR-424 expression is lowered, concomitant with an MET-like process. Breast cancer patient miR-424 levels positively associate with TWIST1/2 and EMT-like gene signatures, and miR-424 is increased in primary tumors versus matched normal breast. However, miR-424 is downregulated in patient metastases versus matched primary tumors. Correspondingly, miR-424 decreases tumor initiation and is posttranscriptionally downregulated in macrometastases in mice, suggesting the need for biphasic expression of miR-424 to transit the EMT–MET axis. Next-generation RNA sequencing revealed miR-424 regulates numerous EMT and cancer stemness-associated genes, including TGFBR3, whose downregulation promotes mesenchymal phenotypes, but not tumor-initiating phenotypes. Instead, we demonstrate that increased MAPK–ERK signaling is critical for miR-424–mediated decreases in tumor-initiating phenotypes. These findings suggest miR-424 plays distinct roles in tumor progression, potentially facilitating earlier, but repressing later, stages of metastasis by regulating an EMT–MET axis. Cancer Res; 75(9); 1–14. ©2015 AACR.

Epithelial-to-mesenchymal transition (EMT) is a dynamic process that relies on cellular plasticity. Recently, the process of an oncogenic EMT, followed by a reverse mesenchymal-to-epithelial transition (MET), has been implicated as critical in the metastatic colonization of carcinomas. Unlike governance of epithelial programming, regulation of mesenchymal programming is not well understood in EMT. Here, we describe and characterize the first microRNA that enhances exclusively mesenchymal programming. We demonstrate that microRNA-424 is upregulated early during a TWIST1 or SNAI1-induced EMT, and that it causes cells to express mesenchymal genes without affecting epithelial genes, resulting in a mixed/intermediate EMT. Furthermore, microRNA-424 increases motility, decreases adhesion and induces a growth arrest, changes associated with a complete EMT, that can be reversed when microRNA-424 expression is lowered, concomitant with an MET-like process. Breast cancer patient microRNA-424 levels positively associate with TWIST1/2 and EMT-like gene signatures, and miR-424 is increased in primary tumors versus matched normal breast. However, microRNA-424 is downregulated in patient metastases versus matched primary tumors. Correspondingly, microRNA-424 decreases tumor initiation and is post-transcriptionally downregulated in macrometastases in mice, suggesting the need for biphasic expression of miR-424 to transit the EMT-MET axis. Next-generation RNA sequencing revealed microRNA-424 regulates numerous EMT and cancer stemness-associated genes, including TGFBR3, whose downregulation promotes mesenchymal phenotypes, but not tumor-initiating phenotypes. Instead, we demonstrate that increased MAPK/ERK signaling is critical for miR-424-mediated decreases in tumor-initiating phenotypes. These findings suggest microRNA-424 plays distinct roles in tumor progression, potentially facilitating earlier, but repressing later, stages of metastasis by regulating an EMT-MET axis.

Melanoma development involves members of the AGC kinase family, including AKT, PKC, and, most recently, PDK1, as elucidated recently in studies of Braf::Pten mutant melanomas. Here, we report that PDK1 contributes functionally to skin pigmentation and to the development of melanomas harboring a wild-type PTEN genotype, which occurs in about 70% of human melanomas. The PDK1 substrate SGK3 was determined to be an important mediator of PDK1 activities in melanoma cells. Genetic or pharmacologic inhibition of PDK1 and SGK3 attenuated melanoma growth by inducing G1 phase cell-cycle arrest. In a synthetic lethal screen, pan-PI3K inhibition synergized with PDK1 inhibition to suppress melanoma growth, suggesting that focused blockade of PDK1/PI3K signaling might offer a new therapeutic modality for wild-type PTEN tumors. We also noted that responsiveness to PDK1 inhibition associated with decreased expression of pigmentation genes and increased expression of cytokines and inflammatory genes, suggesting a method to stratify patients with melanoma for PDK1-based therapies. Overall, our work highlights the potential significance of PDK1 as a therapeutic target to improve melanoma treatment. Cancer Res; 75(7); 1–14. ©2015 AACR.

Melanoma development involves members of the AGC kinase family including AKT, PKC and, most recently, PDK1, as elucidated recently in studies of Braf::Pten mutant melanomas. Here we report that PDK1 contributes functionally to skin pigmentation and to the development of melanomas harboring a wild-type PTEN genotype, which occurs in ~70% of human melanomas. The PDK1 substrate SGK3 was determined to be is an important mediator of PDK1 activities in melanoma cells. Genetic or pharmacological inhibition of PDK1 and SGK3 attenuated melanoma growth by inducing G1 phase cell cycle arrest. In a synthetic lethal screen, pan-PI3K inhibition synergized with PDK1 inhibition to suppress melanoma growth, suggesting that focused blockade of PDK1/PI3K signaling might offer a new therapeutic modality for wild-type PTEN tumors. We also noted that responsiveness to PDK1 inhibition associated with decreased expression of pigmentation genes and increased expression of cytokines and inflammatory genes, suggesting a method to stratify melanoma patients for PDK1-based therapies. Overall, our work highlights the potential significance of PDK1 as a therapeutic target to improve melanoma treatment.

Although metabolic defects have been investigated extensively in differentiated tumor cells, much less attention has been directed to the metabolic properties of stem-like cells that repopulate tumors [tumor-repopulating cells (TRC)]. Here, we show that melanoma TRCs cultured in three-dimensional soft fibrin gels reprogram glucose metabolism by hijacking the cytosolic enzyme phosphoenolpyruvate carboxykinase (PCK1), a key player in gluconeogenesis. Surprisingly, upregulated PCK1 in TRCs did not mediate gluconeogenesis but promoted glucose side-branch metabolism, including in the serine and glycerol-3-phosphate pathways. Moreover, this retrograde glucose carbon flow strengthened rather than antagonized glycolysis and glucose consumption. Silencing PCK1 or inhibiting its enzymatic activity slowed the growth of TRCs in vitro and impeded tumorigenesis in vivo. Overall, our work unveiled metabolic features of TRCs in melanoma that have implications for targeting a unique aspect of this disease. Cancer Res; 75(7); 1–6. ©2015 AACR.