Inactivation of the tumor suppressor gene RASSF1A by promoter hypermethylation represents a key event underlying the initiation and progression of lung cancer. RASSF1A inactivation is also associated with poor prognosis and may promote metastatic spread. In this study, we investigated how RASSF1A inactivation conferred invasive phenotypes to human bronchial cells. RNAi-mediated silencing of RASSF1A induced epithelialto-mesenchymal transition (EMT), fomenting a motile and invasive cellular phenotype in vitro and increased metastatic prowess in vivo. Mechanistic investigations revealed that RASSF1A blocked tumor growth by stimulating cofilin/PP2A-mediated dephosphorylation of the guanine nucleotide exchange factor GEF-H1, thereby stimulating its ability to activate the antimetastatic small GTPase RhoB. Furthermore, RASSF1A reduced nuclear accumulation of the Hippo pathway transcriptional cofactor Yes-associated protein (YAP), which was reinforced by RhoB activation. Collectively, our results indicated that RASSF1 acts to restrict EMT and invasion by indirectly controlling YAP nuclear shuttling and activation through a RhoB-regulated cytoskeletal remodeling process, with potential implications to delay the progression of RASSF1-hypermethylated lung tumors. Cancer Res; 76(6);

Aberrant promoter DNA hypermethylation is a hallmark of cancer; however, whether this is sufficient to drive cellular transformation is not clear. To investigate this question, we use a CRISPR-dCas9 epigenetic editing tool, where an inactive form of Cas9 is fused to DNA methyltransferase effectors. Using this system, here we show simultaneous de novo DNA methylation of genes commonly methylated in cancer, CDKN2A, RASSF1, HIC1 and PTEN in primary breast cells isolated from healthy human breast tissue. We find that promoter methylation is maintained in this system, even in the absence of the fusion construct, and this prevents cells from engaging senescence arrest. Our data show that the key driver of this phenotype is repression of CDKN2A transcript p16 where myoepithelial cells harbour cancer-like gene expression but do not exhibit anchorage-independent growth. This work demonstrates that hit-and-run epigenetic events can prevent senescence entry, which may facilitate tumour initiation.

SummaryAutophagy plays key roles in development, oncogenesis, cardiovascular, metabolic, and neurodegenerative diseases. Hence, understanding how autophagy is regulated can reveal opportunities to modify autophagy in a disease-relevant manner. Ideally, one would want to functionally define autophagy regulators whose enzymatic activity can potentially be modulated. Here, we describe the STK38 protein kinase (also termed NDR1) as a conserved regulator of autophagy. Using STK38 as bait in yeast-two-hybrid screens, we discovered STK38 as a novel binding partner of Beclin1, a key regulator of autophagy. By combining molecular, cell biological, and genetic approaches, we show that STK38 promotes autophagosome formation in human cells and in Drosophila. Upon autophagy induction, STK38-depleted cells display impaired LC3B-II conversion; reduced ATG14L, ATG12, and WIPI-1 puncta formation; and significantly decreased Vps34 activity, as judged by PI3P formation. Furthermore, we observed that STK38 supports the interaction of the exocyst component Exo84 with Beclin1 and RalB, which is required to initiate autophagosome formation. Upon studying the activation of STK38 during autophagy induction, we found that STK38 is stimulated in a MOB1- and exocyst-dependent manner. In contrast, RalB depletion triggers hyperactivation of STK38, resulting in STK38-dependent apoptosis under prolonged autophagy conditions. Together, our data establish STK38 as a conserved regulator of autophagy in human cells and flies. We also provide evidence demonstrating that STK38 and RalB assist the coordination between autophagic and apoptotic events upon autophagy induction, hence further proposing a role for STK38 in determining cellular fate in response to autophagic conditions.

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