Insights into mechanisms of drug resistance could extend the efficacy of cancer therapy. To probe mechanisms in melanoma, we performed siRNA screening of genes that mediate the development of neural crest cells, from which melanocytes are derived. Here, we report the identification of ZIC5 as a mediator of melanoma drug resistance. ZIC5 is a transcriptional suppressor of E-cadherin expressed highly in human melanoma. ZIC5 enhanced melanoma cell proliferation, survival, drug resistance, in vivo growth and metastasis. Microarray analysis revealed that ZIC5 downstream signaling included PDGFD and FAK activation, which contributes to drug resistance by enhancing STAT3 activation. Silencing of ZIC5 or PDGFD enhanced the apoptotic effects of BRAF inhibition and blocked survival of melanoma cells resistant to BRAF inhibitors. Furthermore, inhibition of FAK or STAT3 suppressed expression of ZIC5, which was positively regulated by PDGFD, FAK, and STAT3 in a positive feedback loop. Taken together, our results identify ZIC5 and PDGFD as candidate therapeutic targets to overcome drug resistance in melanoma. Cancer Res; 77(2); 366-77. ©2016 AACR.

Colorectal cancer (CRC) is one of the most common causes of cancer-related deaths worldwide, and Kirsten rat sarcoma viral oncogene homolog (KRAS) mutations in CRC predict the ineffectiveness of EGF receptor-targeted therapy. Previous transcriptional microarray analysis suggests the association between phospholipase Cδ1 (PLCδ1) expression and KRAS mutation status in CRC. However, both the roles and the regulatory mechanisms of PLCδ1 in CRC are not known. Here, we found that the expression of PLCδ1, one of the most basal PLCs, is down-regulated in CRC specimens compared with normal colon epithelium by immunohistochemistry. Furthermore, we examined the roles of PLCδ1 in CRC cell lines that harbor an activating KRAS mutation. Ectopic expression of PLCδ1 in CRC cells induced the expression of E-cadherin, whereas knockdown of PLCδ1 repressed the expression of E-cadherin. Moreover, the overexpression of PLCδ1 suppressed the expression of several mesenchymal genes and reduced cell motility, invasiveness, and in vivo tumorigenicity of SW620 CRC cells. We also showed that PLCδ1 expression is repressed by the KRAS/mitogen-activated protein kinase kinase (MEK) pathway. Furthermore, PLCδ1 suppressed the phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 through E-cadherin induction in CRC cells, suggesting the presence of a negative regulatory loop between KRAS/MEK/ERK signaling and PLCδ1. These data indicate that PLCδ1 has tumor-suppressive functions in CRC through E-cadherin induction and KRAS/MEK/ERK signal attenuation.phospholipase C delta 1 | epithelial-to-mesenchymal transition | tumor suppressor C olorectal cancer (CRC) is one of the most common cancers and causes of cancer-related deaths worldwide. Although CRC patients with unresectable tumors and metastasis have been treated with chemotherapy, recent advances in molecular research about CRC have resulted in the development of molecular targeted therapies, such as cetuximab and panitumumab. Chemotherapy combined with these targeted therapies improves the prognosis of CRC patients with unresectable tumors to some extent (1, 2). However, some CRC patients with activating Kirsten rat sarcoma viral oncogene homolog (KRAS) mutations are unable to benefit from such drugs, because both cetuximab and panitumumab are epidermal growth factor (EGF) receptor-targeting agents and mutant KRAS constitutively activates the downstream signaling of EGF receptor (3). A more vigorous study using KRAS-mutant CRC is needed to identify novel molecular targets for drugs that will improve the prognosis of CRC patients with unresectable tumors, especially those with KRAS mutations.Mutations in KRAS are found in about 40% of CRC patients. Constitutively active KRAS mutations lead to the hyperactivation of mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) [mitogen-activated protein kinase (MAPK)] signaling and/or phosphatidylinositol-3 kinase (PI3K) pathways (4). Activation of MEK/ERK (MAPK) signaling results in increased phosphorylation...

<p>Table S1. List of siRNA Screening Library Sequences. Table S2. Identification of PDGFD as a ZIC5 Downstream Factor</p>

<p>Supplementary Materials and Methods of plasmids, siRNA, shRNA, transfections, western blot analysis, reagents, luciferase reporter assays, chromatin immunoprecipitation, immunocytochemistry, immunohistochemistry, scratch assays, cell proliferation assays, cell cycle analysis, quantitative real-time PCR, migration and invasion assays, animal experiments, microarray analysis, apoptosis assays, and generation of vemurafenib-resistant cell lines.</p>

<p>Table S1. List of siRNA Screening Library Sequences. Table S2. Identification of PDGFD as a ZIC5 Downstream Factor</p>