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Mutational loss of CDKN2A (encoding p16INK4A) tumor-suppressor function is a key genetic step that complements activation of KRAS in promoting the development and malignant growth of pancreatic ductal adenocarcinoma (PDAC). However, pharmacologic restoration of p16INK4A function with inhibitors of CDK4 and CDK6 (CDK4/6) has shown limited clinical efficacy in PDAC. Here, we found that concurrent treatment with both a CDK4/6 inhibitor (CDK4/6i) and an ERK-MAPK inhibitor (ERKi) synergistically suppresses the growth of PDAC cell lines and organoids by cooperatively blocking CDK4/6i-induced compensatory upregulation of ERK, PI3K, antiapoptotic signaling, and MYC expression. On the basis of these findings, a Phase I clinical trial was initiated to evaluate the ERKi ulixertinib in combination with the CDK4/6i palbociclib in patients with advanced PDAC (NCT03454035). As inhibition of other proteins might also counter CDK4/6i-mediated signaling changes to increase cellular CDK4/6i sensitivity, a CRISPR-Cas9 loss-of-function screen was conducted that revealed a spectrum of functionally diverse genes whose loss enhanced CDK4/6i growth inhibitory activity. These genes were enriched around diverse signaling nodes, including cell-cycle regulatory proteins centered on CDK2 activation, PI3K-AKT-mTOR signaling, SRC family kinases, HDAC proteins, autophagy-activating pathways, chromosome regulation and maintenance, and DNA damage and repair pathways. Novel therapeutic combinations were validated using siRNA and small-molecule inhibitor-based approaches. In addition, genes whose loss imparts a survival advantage were identified (e.g., RB1, PTEN, FBXW7), suggesting possible resistance mechanisms to CDK4/6 inhibition. In summary, this study has identified novel combinations with CDK4/6i that may have clinical benefit to patients with PDAC., Significance: CRISPR-Cas9 screening and protein activity mapping reveal combinations that increase potency of CDK4/6 inhibitors and overcome drug-induced compensations in pancreatic cancer., (©2022 American Association for Cancer Research.)

ECT2 is an activator of RHO GTPases that is essential for cytokinesis. In addition, ECT2 was identified as an oncoprotein when expressed ectopically in NIH/3T3 fibroblasts. However, oncogenic activation of ECT2 resulted from N-terminal truncation, and such truncated ECT2 proteins have not been found in patients with cancer. In this study, we observed elevated expression of full-length ECT2 protein in preneoplastic colon adenomas, driven by increased ECT2 mRNA abundance and associated with APC tumor-suppressor loss. Elevated ECT2 levels were detected in the cytoplasm and nucleus of colorectal cancer tissue, suggesting cytoplasmic mislocalization as one mechanism of early oncogenic ECT2 activation. Importantly, elevated nuclear ECT2 correlated with poorly differentiated tumors, and a low cytoplasmic:nuclear ratio of ECT2 protein correlated with poor patient survival, suggesting that nuclear and cytoplasmic ECT2 play distinct roles in colorectal cancer. Depletion of ECT2 reduced anchorage-independent cancer cell growth and invasion independent of its function in cytokinesis, and loss of Ect2 extended survival in a Kras G12D Apc -null colon cancer mouse model. Expression of ECT2 variants with impaired nuclear localization or guanine nucleotide exchange catalytic activity failed to restore cancer cell growth or invasion, indicating that active, nuclear ECT2 is required to support tumor progression. Nuclear ECT2 promoted ribosomal DNA transcription and ribosome biogenesis in colorectal cancer. These results support a driver role for both cytoplasmic and nuclear ECT2 overexpression in colorectal cancer and emphasize the critical role of precise subcellular localization in dictating ECT2 function in neoplastic cells. SIGNIFICANCE: ECT2 overexpression and mislocalization support its role as a driver in colon cancer that is independent from its function in normal cell cytokinesis., (©2021 American Association for Cancer Research.)

Intracellular signaling governed by serine/threonine kinases comprises the molecular interface between cell surface receptors and the nuclear transcriptional machinery. The protein kinase C (PKC) family members are involved in the control of many signaling processes directing cell proliferation, motility, and survival. Here, we examined a role of different PKC isoenzymes in protein phosphatase 2A (PP2A) and HRSL3 tumor suppressor-dependent cell death induction in the ovarian carcinoma cell line OVCAR-3. Phosphorylation and activity of PKC isoenzymes were measured in response to PP2A or phosphoinositide 3-kinase inhibition or HRSL3 overexpression. These experiments indicated a regulation of PKC, epsilon, zeta, and iota through PP2A and/or HRSL3, but not of PKCalpha and beta. Using isoform-specific peptide inhibitors and overexpression approaches, we verified a contribution to PP2A- and HRLS3-dependent apoptosis only for PKCzeta, suggesting a proapoptotic function of this kinase. We observed a significant proportion of human ovarian carcinomas expressing high levels of PKCzeta, which correlated with poor prognosis. Primary ovarian carcinoma cells isolated from patients also responded to okadaic acid treatment with increased phosphorylation of PKCzeta and apoptosis induction. Thus, our data indicate a contribution of PKCzeta in survival control in ovarian carcinoma cells and suggest that upregulation or activation of tyrosine kinase receptors in this tumor might impinge onto apoptosis control through the negative regulation of the atypical PKCzeta.

MUC18/MCAM is a melanoma-associated cell adhesion molecule that is also occasionally found on carcinomas and other tumor types. On melanomas, MUC18 expression increases with tumor progression and is found on more than 70% of metastatic lesions. To investigate the regulation of MUC18 expression, cell lines of diverse tissue origin were exposed to cytokines, regulators of intracellular cyclic AMP (cAMP), and to phorbol ester. MUC18 expression could not be induced in negative cell lines and could only be modulated by changes in cAMP levels or by exposure to phorbol ester in positive cells. An increase in intracellular cAMP led to an up-regulation in cell surface MUC18 that was maximal at 48 h. Increased MUC18 mRNA levels were observed as soon as 4 h and were 3-fold higher than in control cells by 48 h. Exposure of the cells to phorbol ester reduced MUC18 surface expression to background levels by 24 h. This downregulation was associated with decreased mRNA levels that were apparent at 8 h. By 24 h, steady-state levels of MUC18 mRNA had been reduced by 58%. Whereas similar changes in MUC18 surface expression were observed in MUC18-expressing glioma and carcinoma cell lines, melanoma cells were more resistant to the MUC18-modulating effects of cAMP analogues and phorbol ester. These observations suggest that the strong MUC18 expression observed in advanced melanomas may reflect disturbances in the normal regulation of this molecule.

The MUC18 protein, a member of the immunoglobulin superfamily and related to several adhesion molecules, shows an expression pattern in human malignant melanoma which is closely associated with tumor progression and the onset of metastasis. To determine the expression pattern of MUC18 in normal human tissues, immunohistochemical analysis was performed on frozen sections of a variety of normal human tissues using monoclonal antibodies against three different epitopes. This analysis showed that expression of MUC18 is limited to smooth muscle cells and to vascular endothelium. No reactivity could be observed with epithelial cells or with quiescent or activated hemopoetic cells. Smooth muscle cells in lung, skin, and in the gastrointestinal tract express MUC18 as does vascular smooth muscle, whereas myocardium or skeletal muscle appeared negative. Comparison of MUC18 staining with that of the panendothelial marker CD31 showed that MUC18 is expressed on the endothelia of a subset of blood capillaries and in tumor vessels but is absent on the endothelium of arterial vessels and large veins. The regulation of MUC18 expression was investigated in vascular smooth muscle cells and endothelial cells cultured in vitro. These studies revealed induction of the gene in endothelial cells upon proliferation. The observation that the MUC18 protein is not only present on melanoma cells but also on the endothelia of blood vessels penetrating primary and metastatic melanomas suggests a complex involvement of this potential cell adhesion molecule in tumor angiogenesis and metastasis.