The alternatively-included 11a sequence modifies the effects of Mena on actin cytoskeletal organization and cell behavior

During tumor progression, alternative splicing gives rise to different Mena protein isoforms. We analyzed how Mena11a, an isoform enriched in epithelia and epithelial-like cells, affects Mena-dependent regulation of actin dynamics and cell behavior. While other Mena isoforms promote actin polymerization and drive membrane protrusion, we find that Mena11a decreases actin polymerization and growth factor-stimulated membrane protrusion at lamellipodia. Ectopic Mena11a expression slows mesenchymal-like cell motility, while isoform-specific depletion of endogenous Mena11a in epithelial-like tumor cells perturbs cell:cell junctions and increases membrane protrusion and overall cell motility. Mena11a can dampen membrane protrusion and reduce actin polymerization in the absence of other Mena isoforms, indicating that it is not simply an inactive Mena isoform. We identify a phosphorylation site within 11a that is required for some Mena11a-specific functions. RNA-seq data analysis from patient cohorts demonstrates that the difference between mRNAs encoding constitutive Mena sequences and those containing the 11a exon correlates with metastasis in colorectal cancer, suggesting that 11a exon exclusion contributes to invasive phenotypes and leads to poor clinical outcomes.Virginia and D.K. Ludwig Fund for Cancer Research (Graduate Student Fellowship)National Institutes of Health (U.S.) (GM58801)Massachusetts Institute of Technology. Ludwig Center for Molecular OncologyDavid H. Koch Institute for Integrative Cancer Research at MIT (NCI Core Grant P30-CA14051

Colorectal Cancers from Distinct Ancestral Populations Show Variations in BRAF Mutation Frequency

It has been demonstrated for some cancers that the frequency of somatic oncogenic mutations may vary in ancestral populations. To determine whether key driver alterations might occur at different frequencies in colorectal cancer, we applied a high-throughput genotyping platform (OncoMap) to query 385 mutations across 33 known cancer genes in colorectal cancer DNA from 83 Asian, 149 Black and 195 White patients. We found that Asian patients had fewer canonical oncogenic mutations in the genes tested (60% vs Black 79% (P = 0.011) and White 77% (P = 0.015)), and that BRAF mutations occurred at a higher frequency in White patients (17% vs Asian 4% (P = 0.004) and Black 7% (P = 0.014)). These results suggest that the use of genomic approaches to elucidate the different ancestral determinants harbored by patient populations may help to more precisely and effectively treat colorectal cancer

Profiling Critical Cancer Gene Mutations in Clinical Tumor Samples

Background: Detection of critical cancer gene mutations in clinical tumor specimens may predict patient outcomes and inform treatment options; however, high-throughput mutation profiling remains underdeveloped as a diagnostic approach. We report the implementation of a genotyping and validation algorithm that enables robust tumor mutation profiling in the clinical setting. Methodology: We developed and implemented an optimized mutation profiling platform (“OncoMap”) to interrogate ∼400 mutations in 33 known oncogenes and tumor suppressors, many of which are known to predict response or resistance to targeted therapies. The performance of OncoMap was analyzed using DNA derived from both frozen and FFPE clinical material in a diverse set of cancer types. A subsequent in-depth analysis was conducted on histologically and clinically annotated pediatric gliomas. The sensitivity and specificity of OncoMap were 93.8% and 100% in fresh frozen tissue; and 89.3% and 99.4% in FFPE-derived DNA. We detected known mutations at the expected frequencies in common cancers, as well as novel mutations in adult and pediatric cancers that are likely to predict heightened response or resistance to existing or developmental cancer therapies. OncoMap profiles also support a new molecular stratification of pediatric low-grade gliomas based on BRAF mutations that may have immediate clinical impact. Conclusions: Our results demonstrate the clinical feasibility of high-throughput mutation profiling to query a large panel of “actionable” cancer gene mutations. In the future, this type of approach may be incorporated into both cancer epidemiologic studies and clinical decision making to specify the use of many targeted anticancer agents

Profiling Critical Cancer Gene Mutations in Clinical Tumor Samples

BACKGROUND: Detection of critical cancer gene mutations in clinical tumor specimens may predict patient outcomes and inform treatment options; however, high-throughput mutation profiling remains underdeveloped as a diagnostic approach. We report the implementation of a genotyping and validation algorithm that enables robust tumor mutation profiling in the clinical setting. METHODOLOGY: We developed and implemented an optimized mutation profiling platform ("OncoMap") to interrogate approximately 400 mutations in 33 known oncogenes and tumor suppressors, many of which are known to predict response or resistance to targeted therapies. The performance of OncoMap was analyzed using DNA derived from both frozen and FFPE clinical material in a diverse set of cancer types. A subsequent in-depth analysis was conducted on histologically and clinically annotated pediatric gliomas. The sensitivity and specificity of OncoMap were 93.8% and 100% in fresh frozen tissue; and 89.3% and 99.4% in FFPE-derived DNA. We detected known mutations at the expected frequencies in common cancers, as well as novel mutations in adult and pediatric cancers that are likely to predict heightened response or resistance to existing or developmental cancer therapies. OncoMap profiles also support a new molecular stratification of pediatric low-grade gliomas based on BRAF mutations that may have immediate clinical impact. CONCLUSIONS: Our results demonstrate the clinical feasibility of high-throughput mutation profiling to query a large panel of "actionable" cancer gene mutations. In the future, this type of approach may be incorporated into both cancer epidemiologic studies and clinical decision making to specify the use of many targeted anticancer agents

Mena 11a-isoform specific regulation of actin cytoskeleton organization and cell behavior

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biology, 2016.Cataloged from PDF version of thesis.Includes bibliographical references.EnaNASP proteins are a conserved family of actin regulatory proteins that modulate cell motility and morphology by altering actin dynamics. Mena, an EnaNASP protein, is alternatively spliced, producing protein isoforms with distinct functions. Here, we characterize the expression, function, and regulation of the Mena11a splice isoform at the molecular and cellular level. We find that Menal 1 a is enriched in epithelia and muscle in both embryonic and adult tissues. Recent evidence demonstrates Mena upregulation in breast cancer; Mena promotes cell motility, invasiveness, and metastasis in a growth factor dependent manner. We demonstrate that Mena11a depletion in epithelial-like breast cancer cells disrupts cell-cell junctions and increases cell motility. Mena11a dampens growth factor-mediated membrane protrusion and attenuates G-actin incorporation to barbed ends of actin filaments. In addition, Menal Ia alters the actin cytoskeleton and decreases Arp2/3 recruitment to the leading edge of lamellipodia. Mass spectrometry analysis demonstrates that Mena11a is phosphorylated. The expression of a nonphosphorylatable mutant of Mena 1Ia does not retain the ability of Mena11a to dampen membrane protrusion, decrease G-actin incorporation at barbed ends, and reduce Arp2/3 recruitment at the lamellipodial edge; thus, our results suggest that phosphorylation of Mena11a is coupled to its activity.by Chandrani Mondal.Ph. D

Hymenolepis diminuta Reduce Lactic Acid Bacterial Load and Induce Dysbiosis in the Early Infection of the Probiotic Colonization of Swiss Albino Rat

Tapeworm infection continues to be an important cause of morbidity worldwide. Recent metagenomics studies have established a link between gut microbiota and parasite infection. The identification of gut probiotics is of foremost importance to explore its relationship and function with the parasite in the host. In this study, the gut content of hosts infected with tapeworm Hymenolepis diminuta and non-infected host gut were disected out to determine their Lactic acid bacterial (LAB) population in MRS agar and microbial community was analysed by metagenomics. The bacterial count was calculated on a bacterial counting chamber and their morphology was determined microscopically and biochemically. Further, to determine the safety profile antibiotic resistance test, antimicrobial, hemolytic activity, and adhesion capability were calculated. We found six dominant probiotic strains and a decrease in LAB load from 1.7–2.3 × 107 CFU/mL in the uninfected group to a range of 8.4 × 105 CFU/mL to 3.2 × 105 CFU/mL in the infected groups with respect to an increase in the parasite number from 10–18. In addition, we found a depletion in the probiotic relative abundance of Lactobacillus and an enrichment in potentially pathogenic Proteobacteria, Fusobacteria, and Streptococcus. Phylogenetic analysis of the six probiotics revealed a close similarity with different strains of L. brevis, L. johnsonii, L. taiwansis, L. reuteri, L. plantarum, and L. pentosus. Thus, this study suggests that the parasite inhibits probiotic colonization in the gut during its early establishment of infection inside the host

Activation of Aflatoxin Biosynthesis Alleviates Total ROS in Aspergillus parasiticus

An aspect of mycotoxin biosynthesis that remains unclear is its relationship with the cellular management of reactive oxygen species (ROS). Here we conduct a comparative study of the total ROS production in the wild-type strain (SU-1) of the plant pathogen and aflatoxin producer, Aspergillus parasiticus, and its mutant strain, AFS10, in which the aflatoxin biosynthesis pathway is blocked by disruption of its pathway regulator, aflR. We show that SU-1 demonstrates a significantly faster decrease in total ROS than AFS10 between 24 h to 48 h, a time window within which aflatoxin synthesis is activated and reaches peak levels in SU-1. The impact of aflatoxin synthesis in alleviation of ROS correlated well with the transcriptional activation of five superoxide dismutases (SOD), a group of enzymes that protect cells from elevated levels of a class of ROS, the superoxide radicals (O2−). Finally, we show that aflatoxin supplementation to AFS10 growth medium results in a significant reduction of total ROS only in 24 h cultures, without resulting in significant changes in SOD gene expression. Our findings show that the activation of aflatoxin biosynthesis in A. parasiticus alleviates ROS generation, which in turn, can be both aflR dependent and aflatoxin dependent

A tumor-derived type III collagen-rich ECM niche regulates tumor cell dormancy

Cancer cells disseminate and seed in distant organs, where they can remain dormant for many years before forming clinically detectable metastases. Here we studied how disseminated tumor cells sense and remodel the extracellular matrix (ECM) to sustain dormancy. ECM proteomics revealed that dormant cancer cells assemble a type III collagen-enriched ECM niche. Tumor-derived type III collagen is required to sustain tumor dormancy, as its disruption restores tumor cell proliferation through DDR1-mediated STAT1 signaling. Second-harmonic generation two-photon microscopy further revealed that the dormancy-to-reactivation transition is accompanied by changes in type III collagen architecture and abundance. Analysis of clinical samples revealed that type III collagen levels were increased in tumors from patients with lymph node-negative head and neck squamous cell carcinoma compared to patients who were positive for lymph node colonization. Our data support the idea that the manipulation of these mechanisms could serve as a barrier to metastasis through disseminated tumor cell dormancy induction

High-throughput mutation profiling of CTCL samples reveals KRAS and NRAS mutations sensitizing tumors toward inhibition of the RAS/RAF/MEK signaling cascade

Cutaneous T-cell lymphomas (CTCLs) are malignancies of skin-homing lymphoid cells, which have so far not been investigated thoroughly for common oncogenic mutations. We screened 90 biopsy specimens from CTCL patients (41 mycosis fungoides, 36 Sézary syndrome, and 13 non-mycosis fungoides/Sézary syndrome CTCL) for somatic mutations using OncoMap technology. We detected oncogenic mutations for the RAS pathway in 4 of 90 samples. One mycosis fungoides and one pleomorphic CTCL harbored a KRAS(G13D) mutation; one Sézary syndrome and one CD30(+) CTCL harbored a NRAS(Q61K) amino acid change. All mutations were found in stage IV patients (4 of 42) who showed significantly decreased overall survival compared with stage IV patients without mutations (P = .04). In addition, we detected a NRAS(Q61K) mutation in the CTCL cell line Hut78. Knockdown of NRAS by siRNA induced apoptosis in mutant Hut78 cells but not in CTCL cell lines lacking RAS mutations. The NRAS(Q61K) mutation sensitized Hut78 cells toward growth inhibition by the MEK inhibitors U0126, AZD6244, and PD0325901. Furthermore, we found that MEK inhibitors exclusively induce apoptosis in Hut78 cells. Taken together, we conclude that RAS mutations are rare events at a late stage of CTCL, and our preclinical results suggest that such late-stage patients profit from MEK inhibitors