Antioxidant Systems, lncRNAs, and Tunneling Nanotubes in Cell Death Rescue from Cigarette Smoke Exposure.

Cigarette smoke is a rich source of carcinogens and reactive oxygen species (ROS) that can damage macromolecules including DNA. Repair systems can restore DNA integrity. Depending on the duration or intensity of stress signals, cells may utilize various survival and adaptive mechanisms. ROS levels are kept in check through redundant detoxification processes controlled largely by antioxidant systems. This review covers and expands on the mechanisms available to cigarette smoke-exposed cancer cells for restoring the redox balance. These include multiple layers of transcriptional control, each of which is posited to be activated upon reaching a particular stress threshold, among them the NRF2 pathway, the AP-1 and NF-kB pathways, and, finally, TP53, which triggers apoptosis if extreme toxicity is reached. The review also discusses long noncoding RNAs, which have been implicated recently in regulating oxidative stress-with roles in ROS detoxification, the inflammatory response, oxidative stress-induced apoptosis, and mitochondrial oxidative phosphorylation. Lastly, the emerging roles of tunneling nanotubes in providing additional mechanisms for metabolic rescue and the regulation of redox imbalance are considered, further highlighting the expanded redox reset arsenal available to cells.

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.

Concomitant and decoupled effects of cigarette smoke and SCAL1 upregulation on oncogenic phenotypes and ROS detoxification in lung adenocarcinoma cells.

Lung cancer is the leading cause of cancer deaths worldwide, with smoking as its primary predisposing factor. Although carcinogens in cigarettes are known to cause oncogenic DNA alterations, analyses of patient cohorts revealed heterogeneous genetic aberrations with no clear driver mutations. The contribution of noncoding RNAs (ncRNAs) in the pathogenesis of lung cancer has since been demonstrated. Their dysregulation has been linked to cancer initiation and progression. A novel long noncoding RNA (lncRNA) called smoke and cancer-associated lncRNA 1 (SCAL1) was recently found upregulated in smoke-exposed adenocarcinomic alveolar epithelial cells. The present study characterized the phenotypic consequences of SCAL1 overexpression and knockdown using A549 cells as model system, with or without prior exposure to cigarette smoke extract (CSE). Increase in SCAL1 levels either by CSE treatment or SCAL1 overexpression led to increased cell migration, extensive cytoskeletal remodeling, and resistance to apoptosis. Further, SCAL1 levels were negatively correlated with intracellular levels of reactive oxygen species (ROS). In contrast, SCAL1 knockdown showed converse results for these assays. These results confirm the oncogenic function of SCAL1 and its role as a CSE-activated lncRNA that mediates ROS detoxification in A549 cells, thereby allowing them to develop resistance to and survive smoke-induced toxicity.

CircPVT1 attenuates negative regulation of NRAS by let-7 and drives cancer cells towards oncogenicity.

Circular RNAs have emerged as functional regulatory molecules whose aberrant expression has been linked to diverse pathophysiological processes. Here, we report that circPVT1 interferes with let-7 binding to NRAS, confirming this axis as one route by which circPVT1 can instigate an oncogenic program in A549 lung cancer cells and HCT116 colorectal cancer cells. CircPVT1 knockdown significantly reduced NRAS levels and attenuated cancer hallmark phenotypes such as proliferation, migration, resistance to apoptosis, cytoskeletal disorganization, and epithelial-mesenchymal transition. The effects of circPVT1 knockdown were at least partially rescued by blocking binding of let-7 to NRAS 3'UTR with a target protector, suggesting that a circPVT1/let-7/NRAS axis exists and acts in cells to reverse NRAS downregulation and favor oncogenicity. While the phenotypic effects of circPVT1 knockdown may be attributable to the global action of circPVT1, the target protection assays resolved the relative contribution of the circPVT1/let-7/NRAS axis specifically.

Exosomal microRNAs in colorectal cancer: Overcoming barriers of the metastatic cascade (Review).

The journey of cancer cells from a primary tumor to distant sites is a multi­step process that involves cellular reprogramming, the breaking or breaching of physical barriers and the preparation of a pre­metastatic niche for colonization. The loss of adhesion between cells, cytoskeletal remodeling, the reduction in size and change in cell shape, the destruction of the extracellular matrix, and the modification of the tumor microenvironment facilitate migration and invasion into surrounding tissues. The promotion of vascular leakiness enables intra­ and extravasation, while angiogenesis and immune suppression help metastasizing cells become established in the new site. Tumor­derived exosomes have long been known to harbor microRNAs (miRNAs or miRs) that help prepare secondary sites for metastasis; however, their roles in the early and intermediate steps of the metastatic cascade are only beginning to be characterized. The present review article presents a summary and discussion of the miRNAs that form part of colorectal cancer (CRC)­derived exosomal cargoes and which play distinct roles in epithelial to mesenchymal plasticity and metastatic organotropism. First, an overview of epithelial­to­mesenchymal transition (EMT), metastatic organotropism, as well as exosome biogenesis, cargo sorting and uptake by recipient cells is presented. Lastly, the potential of these exosomal miRNAs as prognostic biomarkers for metastatic CRC, and the blocking of these as a possible therapeutic intervention is discussed.

Clinicopathologic Factors Associated with Mismatch Repair Status Among Filipino Patients with Young-Onset Colorectal Cancer.

INTRODUCTION: Young-onset colorectal cancer is recognized as a distinct disease that may be sporadic or hereditary in nature. Microsatellite instability testing is recommended as a routine procedure in evaluating colorectal cancer specimens, especially in young-onset disease, because of implications in management. Immunohistochemistry of mismatch repair proteins serves as an inexpensive alternative to microsatellite instability testing with the added advantage of monitoring protein expression levels that may suggest underlying genetic or epigenetic alterations. This descriptive study aimed to determine the frequencies of proficient and deficient mismatch repair status among Filipino young-onset colorectal cancer patients, and to investigate their clinicopathologic profile. METHODS: Tumor tissues were prospectively collected from patients from two tertiary hospitals in the Philippines. Patients of age ≤45 years with resected adenocarcinoma of the colon or rectum were recruited. RESULTS: Seventy-seven out of 124 patients had tumor samples sent for immunohistochemistry. Of these, 61 samples (79%) were found to have proficient status while 16 samples (21%) had deficient status. Mismatch repair protein deficiencies, when present, more commonly involved MSH2 and MSH6 (9%) rather than MLH1 and PMS2 (5%). The deficient group had a mean age of 37.1 years and a female preponderance (56.25%), presenting as locally advanced ascending or descending colon tumors with mucinous histology in half of the population. The mismatch repair proficient group presented as locally advanced rectal and sigmoid tumors but with fewer mucinous adenocarcinomas (26.2%) compared to the deficient group. In both the mismatch repair proficient and deficient patients with family history reports, most did not have any known relative with cancer (75.4% and 68.75%, respectively). CONCLUSION: This is the first attempt to perform mismatch repair testing among young-onset colorectal cancer patients in the Philippines and to gather data on their clinicopathologic characteristics. However, the limited sample size precludes conclusive results for the associations of mismatch repair with clinicopathologic features.

Laboratory classes in biochemistry and molecular biology: A parallel session at the IUBMB/PSBMB 2019 "Harnessing Interdisciplinary Education in Biochemistry and Molecular Biology" conference.

Laboratory classes are a central element of all biochemistry and molecular biology programs. These play a role in developing students' hands-on and technical skills and also offer much more. The design of laboratory classes depends on many factors including the programs the students are enrolled in, the level they are at, employment destinations, and learning outcomes. This conference session considered the design and outcomes of laboratory experiences for undergraduate students.

NRAS mutant E132K identified in young-onset sporadic colorectal cancer and the canonical mutants G12D and Q61K affect distinct oncogenic phenotypes.

Recent data show a global increase in colorectal cancer (CRC) cases among younger demographics, which portends poorer prognosis. The cause of rising incidence is uncertain, and its mutational landscape remains largely unexplored, including those in genes of the epidermal growth factor receptor pathway. Among these are NRAS mutants where there is paucity of functional studies compared to KRAS. Here, the novel NRAS mutant E132K, identified in three tumor samples from Filipino young-onset, sporadic colorectal cancer patients, was investigated for its effects on different cancer hallmarks, alongside the NRAS canonical mutants G12D and Q61K which are yet poorly characterized in the context of CRC. The novel NRAS mutant E132K and the canonical G12D and Q61K mutants show resistance to apoptosis, cytoskeletal reorganization, and loss of adhesion. In contrast to activating KRAS mutations, including the analogous KRAS G12D and Q61K mutations, all three NRAS mutants have no apparent effect on cell proliferation and motility. The results highlight the need to characterize isoform- and mutation-specific oncogenic phenotypes which can have repercussions in disease management and choice of therapeutic intervention. Further analyses of young-onset versus late-onset CRC datasets are necessary to qualify NRAS E132K as a biomarker for the young-onset subtype.

Mutation-Associated Phenotypic Heterogeneity in Novel and Canonical PIK3CA Helical and Kinase Domain Mutants.

Phosphatidylinositol 3-kinase, catalytic subunit alpha (PIK3CA) is an oncogene often mutated in colorectal cancer (CRC). The contribution of PIK3CA mutations in acquired resistance to anti-epidermal growth factor receptor (EGFR) therapy is well documented, but their prognostic and predictive value remain unclear. Domain- and exon-specific mutations are implicated in either favorable or poor prognoses, but there is paucity in the number of mutations characterized outside of the mutational hotspots. Here, two novel non-hotspot mutants-Q661K in exon 13 and C901R in exon 19-were characterized alongside the canonical exon 9 E545K and exon 20 H1047R mutants in NIH3T3 and HCT116 cells. Q661K and E545K both map to the helical domain, whereas C901R and H1047R map to the kinase domain. Results showed variable effects of Q661K and C901R on morphology, cellular proliferation, apoptosis resistance, and cytoskeletal reorganization, with both not having any effect on cellular migration. In comparison, E545K markedly promoted proliferation, survival, cytoskeletal reorganization, migration, and spheroid formation, whereas H1047R only enhanced the first three. In silico docking suggested these mutations negatively affect binding of the p85 alpha regulatory subunit to PIK3CA, thereby relieving PIK3CA inhibition. Altogether, these findings support intra-domain and mutation-specific variability in oncogenic readouts, with implications in degree of aggressiveness.

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.

Phenotypic characterization of the novel, non-hotspot oncogenic KRAS mutants E31D and E63K.

KRAS proto-oncogene, GTPase (KRAS) functions as a molecular switch at the apex of multiple signaling pathways controlling cell proliferation, differentiation, migration, and survival. Canonical KRAS mutants, such as those in codons 12 and 13, produce constitutively active oncoproteins that short-circuit epidermal growth factor receptor (EGFR)-initiated signaling, resulting in dysregulated downstream effectors associated with cellular transformation. Therefore, anti-EGFR therapy provides little to no clinical benefit to patients with activating KRAS mutations. Current genotyping procedures based on canonical mutation detection only account for ~40% of non-responders, highlighting the need to identify additional predictive biomarkers. In the present study, two novel non-hotspot KRAS mutations were functionally characterized in vitro: KRAS E31D was identified from a genetic screen of colorectal cancer specimens at the UP-National Institutes of Health. KRAS E63K is curated in the Catalogue of Somatic Mutations in Cancer database. Similar to the canonical mutants KRAS G12D and KRAS G13D, NIH3T3 cells overexpressing KRAS E31D and KRAS E63K showed altered morphology and were characteristically smaller, rounder, and highly refractile compared with their non-transformed counterparts. Filamentous actin staining also indicated cytoplasmic shrinkage, membrane ruffling, and formation of pseudopod protrusions. Further, they displayed higher proliferative rates and higher migratory rates in scratch wound assays compared with negative controls. These empirical findings suggest the activating impact of the novel KRAS mutations, which may contribute to resistance to anti-EGFR therapy. Complementary studies to elucidate the molecular mechanisms underlying the transforming effect of the rare mutants are required. In parallel, their oncogenic capacity in vivo should also be investigated.

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.