Faculty Bibliography

The outcome for pediatric ALL patients that relapse is dismal. A hallmark of relapsed disease is acquired resistance to multiple chemotherapeutic agents, particularly glucocorticoids. In this study, we performed a genome-scale shRNA screen to identify mediators of prednisolone sensitivity in ALL cell lines. The incorporation of this data with an integrated analysis of relapse-specific genetic and epigenetic changes allowed us to identify the MAPK pathway as a mediator of prednisolone resistance in pediatric ALL. We show that knockdown of the specific MAPK pathway members MEK2 and MEK4 increased sensitivity to prednisolone through distinct mechanisms. MEK4 knockdown increased sensitivity specifically to prednisolone by increasing the levels of the glucocorticoid receptor. MEK2 knockdown increased sensitivity to all chemotherapy agents tested by increasing the levels of p53. Furthermore, we demonstrated that inhibition of MEK1/2 with trametinib increased sensitivity of ALL cells and primary samples to chemotherapy in vitro and in vivo. To confirm a role for MAPK signaling in patients with relapsed ALL, we measured the activation of MEK1/2 target ERK in matched diagnosis and relapse primary samples and observed increased pERK levels at relapse. Furthermore, relapse samples have an enhanced response to MEK inhibition compared to matched diagnosis samples in xenograft models. Altogether, our data indicate that inhibition of the MAPK pathway increases chemosensitivity to glucocorticoids and possibly other agents, and is an attractive target for prevention and/or treatment of relapsed disease.

The microRNA-34a (miR-34a), a tumor suppressive microRNA (miRNA), is implicated in epithelial-mesenchymal transition (EMT) and cancer stem cells. Lymphoid enhancer-binding factor-1 (LEF1) is a key transcription factor in the Wnt signaling pathway, and has been suggested to be involved in regulation of cell proliferation and invasion. Here, the molecular mechanism of miR-34a and LEF1 in cooperatively regulating prostate cancer (PCa) cell invasion is described. Molecular profiling analysis of miRNA levels in PCa cells revealed a negative correlation between miR-34a and LEF1 expression, and the downregulation of LEF1 by miR-34a was confirmed by luciferase assays. Further, miR-34a specifically repressed LEF1 expression through direct binding to its 3'-untranslated (3'-UTR) regions. miR-34a modulated the levels of LEF1 to regulate EMT in PCa cells. Functionally, miR-34a negatively correlated with the migration and invasion of PCa cells through LEF1. An analysis of miR-34a expression levels in matched human tumor and benign tissues demonstrated consistent and statistically significant downregulation of miR-34a in primary prostate cancer specimens. These data strongly suggest that miR-34a/LEF1 regulation of EMT plays an important role in PCa migration and invasion. Implications: The miR-34a/LEF1 axis represents a potential molecular target for novel therapeutic strategies in PCa.

The two major melanoma histologic subtypes, superficial spreading and nodular melanomas, differ in their speed of dermal invasion but converge biologically once they invade and metastasize. Herein, we tested the hypothesis that distinct molecular alterations arising in primary melanoma cells might persist as these tumors progress to invasion and metastasis. Ribosomal protein S6 kinase, 90 kDa, polypeptide 1 (RSK1; official name RPS6KA1) was significantly hyperactivated in human melanoma lines and metastatic tissues derived from nodular compared with superficial spreading melanoma. RSK1 was constitutively phosphorylated at Ser-380 in nodular but not superficial spreading melanoma and did not directly correlate with BRAF or MEK activation. Nodular melanoma cells were more sensitive to RSK1 inhibition using siRNA and the pharmacological inhibitor BI-D1870 compared with superficial spreading cells. Gene expression microarray analyses revealed that RSK1 orchestrated a program of gene expression that promoted cell motility and invasion. Differential overexpression of the prometastatic matrix metalloproteinase 8 and tissue inhibitor of metalloproteinases 1 in metastatic nodular compared with metastatic superficial spreading melanoma was observed. Finally, using an in vivo zebrafish model, constitutive RSK1 activation increased melanoma invasion. Together, these data reveal a novel role for activated RSK1 in the progression of nodular melanoma and suggest that melanoma originating from different histologic subtypes may be biologically distinct and that these differences are maintained as the tumors invade and metastasize.

BACKGROUND: Identification of primary melanoma patients at the highest risk of recurrence remains a critical challenge, and monitoring for recurrent disease is limited to costly imaging studies. We recently reported our array-based discovery of prognostic serum miRNAs in melanoma. In the current study, we examined the clinical utility of these serum-based miRNAs for prognosis as well as detection of melanoma recurrence. METHODS: Serum levels of 12 miRNAs were tested using qRT-PCR at diagnosis in 283 melanoma patients (training cohort, n = 201; independent validation, n = 82; median follow-up, 68.8 months). A refined miRNA signature was chosen and evaluated. We also tested the potential clinical utility of the miRNAs in early detection and monitoring of recurrence using multiple longitudinal samples (pre- and postrecurrence) in a subset of 82 patients (n = 225). In addition, we integrated our miRNA signature with publicly available Cancer Genome Atlas data to examine the relevance of these miRNAs to melanoma biology. RESULTS: Four miRNAs (miR-150, miR-30d, miR-15b, and miR-425) in combination with stage separated patients by recurrence-free survival (RFS) and overall survival (OS) and improved prediction of recurrence over stage alone in both the training and validation cohorts (training RFS and OS, P < .001; validation RFS, P < .001; OS, P = .005). Serum miR-15b levels significantly increased over time in recurrent patients (P < .001), adjusting for endogenous controls as well as age, sex, and initial stage. In nonrecurrent patients, miR-15b levels were not significantly changed with time (P =.17). CONCLUSIONS: Data demonstrate that serum miRNAs can improve melanoma patient stratification over stage and support further testing of miR-15b to guide patient surveillance. Cancer 2015;121:51-59. (c) 2014 American Cancer Society.

Lymphoid enhancer-binding factor-1 (LEF1) is a key transcription factor mediating Wnt signaling pathway. Our previous studies indicate that LEF1 is highly expressed in androgen-independent prostate cancer (PCa) and enhances invasion ability in androgen-independent PCa cells. However, the molecular mechanism of LEF1 effect on invasion remains largely unknown. Using microRNA profiling analysis comparing androgen-independent LNCaP-AI PCa cells with high levels of endogenous LEF1 to LNCaP-AI cells with LEF1 knockdown by LEF1shRNA, we found miR-181a to be increased 12.3-fold in LNCaP-AI cells. We confirmed a positive correlation between LEF1 and miR-181a expression across multiple PCa cell lines. Additionally, we showed that in PCa cells, overexpression of LEF1 increased miR-181a expression and subsequently induced EMT associated migration and invasion, whereas LEF1 knockdown decreased miR-181a expression and subsequently resulted in inhibition of EMT, migration and invasion. Mechanistically, we demonstrated by chromatin immunoprecipitation assays that LEF1 could enhance miR-181a expression via its binding to the promoter regions of hsa-miR-181a. Overall, this study identified a novel LEF1-miR-181a-EMT axis in regulation of PCa migration and invasion.