The Novel Phosphatase Domain Mutations Q171R and Y65S Switch PTEN from Tumor Suppressor to Oncogene.

Phosphatase and tensin homolog deleted on chromosome 10, or PTEN, is a well-characterized tumor suppressor with both lipid and protein phosphatase activities. PTEN is often downregulated by epigenetic mechanisms such as hypermethylation, which leads to constitutive activation of the PI3K-Akt pathway. Large datasets from next-generation sequencing, however, revealed that mutations in PTEN may not only hamper protein function but may also affect interactions with downstream effectors, leading to variable oncogenic readouts. Here, two novel PTEN mutations, Q171R and Y65S, identified in Filipino colorectal cancer patients, were phenotypically characterized in NIH3T3 and HCT116 cells, alongside the C124S canonical mutant and wild-type controls. The novel mutants increased cellular proliferation, resistance to apoptosis and migratory capacity. They induced gross morphological changes including cytoplasmic shrinkage, increased cellular protrusions and extensive cytoskeletal reorganization. The mutants also induced a modest increase in Akt phosphorylation. Further mechanistic studies will help determine the differential oncogenic potencies of these mutants, and resolve whether the structural constraints imposed by the mutations may have altered associations with downstream effectors.

Identification and validation of mRNA 3'untranslated regions of DNMT3B and TET3 as novel competing endogenous RNAs of the tumor suppressor PTEN.

PTEN inactivation is a frequent event in oncogenesis. Multiple regulatory mechanisms such as promoter hypermethylation, antisense regulation, histone modifications, targeting by microRNAs (miRNAs/miRs) and regulation by transcription factors have all been shown to affect the tumor suppressor functions of PTEN. More recently, the functional involvement of competing endogenous RNAs (ceRNAs) in miRNA­dependent and coding­independent regulation of genes shed light on the highly nuanced control of PTEN expression. The present study has identified and validated DNA methyltransferase 3ß (DNMT3B) and TET methylcytosine dioxygenase 3 (TET3) as novel ceRNAs of PTEN, with which they share multiple miRNAs, in HCT116 colorectal cancer cells. miR­4465 was identified and characterized as a miRNA that directly targets and regulates all 3 transcripts via their 3'untranslated regions (3'UTRs) through a combination of luciferase reporter assays, abrogation of miRNA response elements (MREs) via site­directed mutagenesis, target protection of MREs with locked nucleic acids, RT­qPCR assays and western blot analysis. Competitive miRNA sequestration was demonstrated upon reciprocal 3'UTR overexpression and siRNA­mediated knockdown of their respective transcripts. Overexpression of DNMT3B or TET3 3'UTR promoted apoptosis and decreased migratory capacity, potentially because of shared miRNA sequestration and subsequent activation of PTEN expression. Knockdown of TET3 and DNMT3B decoupled their protein­coding from miRNA­dependent, coding­independent functions. Furthermore, the findings suggested that the phenotypic outcome of ceRNAs is dictated largely by the number of shared miRNAs, and predictably, by the existence of other ceRNA networks in which they participate. Taken together, the findings of the present study identified DNMT3B and TET3 as novel ceRNAs of PTEN that may impact its dose­sensitive tumor suppressive function.

Non-Redundant and Overlapping Oncogenic Readouts of Non-Canonical and Novel Colorectal Cancer KRAS and NRAS Mutants.

RAS oncogene family members are molecular switches of signaling pathways that control cell growth, proliferation, differentiation, and survival. In colorectal cancer, Kirsten-RAS (KRAS) and neuroblastoma-RAS (NRAS) are the commonly mutated isoforms. Activating mutations in RAS result in cellular transformation independent of upregulated epidermal growth factor receptor (EGFR)-initiated signaling. The present study characterized the functional consequences of non-canonical/novel KRAS and NRAS mutants identified in a targeted next-generation sequencing study of colorectal cancer specimens from Filipino patients. In vitro assays in NIH3T3 cells showed that similar to the canonical KRAS G12D mutant, overexpression of KRAS G12S, A59T, and Y137C, but not NRAS G12D and NRAS A11V, confer higher proliferation and migration rates. HCT116 cells transfected with the novel NRAS A11V and the canonical NRAS G12D, but not the KRAS mutants, display enhanced resistance to apoptosis. All four non-canonical/novel KRAS and NRAS mutants induce gross changes in F-actin cytoskeletal organization and cellular morphology of NIH3T3 cells. Only KRAS G12S and KRAS A59T appear to deregulate extracellular signal-regulated kinase (ERK) and its downstream target ETS transcription factor ELK1 (ELK1). Elucidation of differential effector engagement responsible for the variable phenotypic readouts of the mutants is warranted. If validated by mouse studies and clinical correlates, these can have wider implications in choosing treatment options.

MicroRNA­92a promotes cell proliferation, migration and survival by directly targeting the tumor suppressor gene NF2 in colorectal and lung cancer cells.

Inactivation of the tumor suppressor protein Merlin leads to the development of benign nervous system tumors in neurofibromatosis type2 (NF2). Documented causes of Merlin inactivation include deleterious mutations in the encoding neurofibromin2 gene (NF2) and aberrant Merlin phosphorylation leading to proteasomal degradation. Rare somatic NF2 mutations have also been detected in common human malignancies not associated with NF2, including colorectal and lung cancer. Furthermore, tumors without NF2 mutations and with unaltered NF2 transcript levels, but with low Merlin expression, have been reported. The present study demonstrated that NF2 is also regulated by microRNAs (miRNAs) through direct interaction with evolutionarily conserved miRNA response elements (MREs) within its 3'­untranslated region (3'UTR). Dual­Luciferase assays in human colorectal carcinoma (HCT116) and lung adenocarcinoma (A549) cells revealed downregulation of NF2 by miR­92a­3p via its wild­type 3'UTR, but not NF2­3'UTR with mutated miR­92a­3p MRE. HCT116 cells overexpressing miR­92a­3p exhibited significant downregulation of endogenous NF2 mRNA and protein levels, which was rescued by co­transfection of a target protector oligonucleotide specific for the miR­92a­3p binding site within NF2­3'UTR. miR­92a­3p overexpression in HCT116 and A549 cells promoted migration, proliferation and resistance to apoptosis, as well as altered F­actin organization compared with controls. Knockdown of NF2 by siRNA phenocopied the oncogenic effects of miR­92a overexpression on HCT116 and A549 cells. Collectively, the findings of the present study provide functional proof of the unappreciated role of miRNAs in NF2 regulation and tumor progression, leading to enhanced oncogenicity.