Evaluating the Impact of a Medical School Student-Run Research Organization on Scholarly Activity.

Introduction The United States Medical Licensing Examination Step 1 change to Pass/Fail scoring has motivated medical students to pursue more research opportunities. To support students, a student-led organization was created at an allopathic medical school, offering initiatives such as workshops, mentorship, and research projects. Here, we evaluate its impact on medical student research. Methods An observational survey study was conducted to assess students' research involvement and productivity and their sense of support, confidence, and comfort in pursuing research at an institution during the first two years of medical school. These variables were compared between three contiguous classes of students and between club members and non-members. Analyses included t-tests, Chi-square tests, and ANOVA, among others. Results Findings revealed that organization membership was associated with an increased number of research projects. Club members (M= 4.49) reported a significantly greater number of projects compared to non-members (M= 4.49) (p= 0.002). Students who had access to the organization during their preclinical years (M= 4.38) reported significantly more projects compared to students whose preclinical years were before the organization's conception (M= 2.21) (p= 0.041). However, research productivity and feelings of support and confidence in research did not differ by class or club membership.  Conclusions Club members engaged in a greater number of research projects as compared to non-members and students who had access to the organization during their preclinical years. The implementation of similar organizations at every medical school can allow more students to engage in scholarly work.

Loss of Gap Junction Factor Connexin 43 Results in an Increase of Exosomal Tetraspanins in Human Lung Cancer Cell Line A549.

Gap junctions (GJs) and small extracellular vesicles such as exosomes are two fundamental intercellular communication (IC) mechanisms. We tested the hypothesis that the two IC mechanisms are connected by gene editing to inactivate a ubiquitously expression GJ factor (i.e., Cx43) in the human lung cancer cell line A549. Surprisingly, we observed that loss of Cx43 led to a buildup of exosomal tetraspanin proteins such as CD63 and CD9. Given the known activities of tetraspanins in cell-cell adhesion and vesicle uptake, our observation establishes an impetus to investigate further how these two IC mechanisms are intertwined.

CRISPR-Induced Loss of Connexin 43 Expression Sensitizes KRAS Mutant Cells to Cisplatin.

Gap Junction intercellular communication (GJIC) is often dysregulated in cancers, and this dysregulation has been shown to have pro-tumorigenic effects. Connexins (Cxs) are transmembrane proteins that make up gap junctions. Previous studies have indicated that RNA interference (RNAi)-based suppression of Cx43 increases cellular resistance to the chemotherapeutic agent cisplatin. Interestingly, we found that the loss of Cx43 expression induced by the CRISPR-Cas9 technology sensitizes cells to cisplatin in a KRAS mutant-dependent manner.

Connecting Cholesterol Efflux Factors to Lung Cancer Biology and Therapeutics.

Cholesterol is a foundational molecule of biology. There is a long-standing interest in understanding how cholesterol metabolism is intertwined with cancer biology. In this review, we focus on the known connections between lung cancer and molecules mediating cholesterol efflux. A major take-home lesson is that the roles of many cholesterol efflux factors remain underexplored. It is our hope that this article would motivate others to investigate how cholesterol efflux factors contribute to lung cancer biology.

Mechanistic Study of TTF-1 Modulation of Cellular Sensitivity to Cisplatin.

The lung lineage master regulator gene, Thyroid Transcription Factor-1 (TTF-1, also known as NKX2-1), is used as a marker by pathologists to identify lung adenocarcinomas since TTF-1 is expressed in 60 ~ 70% of lung ADs. Much research has been conducted to investigate roles of TTF-1 in lung cancer biology. But, how it modulates cellular chemosensitivity remains poorly characterized. Our study shows that TTF-1 sensitizes the KRAS-mutated A549 and NCI-H460 lung cancer cells to cisplatin, a common chemotherapy used to treat lung cancer. This chemosensitization activity does not appear to be mediated by a TTF-1-imposed alteration on nucleotide excision repair. Mechanistically, TTF-1 induced a reduction in p-AKT (S473), which in turn activated glycogen synthase kinase 3 (GSK3) and reduced ß-catenin. Intriguingly, in the EGFR-mutated NCI-H1975 and HCC827 cells, TTF-1 desensitized these cells to cisplatin; concomitantly, TTF-1 conferred an increase in p-AKT. Finally, the conditioned media of TTF-1-transefected cells sensitized TTF-1- cells to cisplatin, implicating that the TTF-1-driven chemosensitization activity may be dually pronged in both intracellular and extracellular compartments. In short, this study highlights the enigmatic activities of TTF-1 in lung cancer, and calls for future research to optimally manage chemotherapy of patients with TTF-1+ lung ADs.

Thyroid transcription factor 1 enhances cellular statin sensitivity via perturbing cholesterol metabolism.

We have discovered an unexpected connection between a critical lung development and cancer gene termed thyroid transcription factor 1 (TTF-1 also known as NKX2-1) and cholesterol metabolism. Our published work implicates that TTF-1 positively regulates miR-33a which is known to repress ATP-binding cassette transporter 1 (ABCA1) and thus its cholesterol efflux activity. We set out to demonstrate that a higher TTF-1 expression would presumably inhibit cholesterol efflux and consequently raise intracellular cholesterol level. Surprisingly, raising TTF-1 expression actually lowers intracellular cholesterol level, which, we believe, is attributed to a direct transactivation of ABCA1 by TTF-1. Subsequently, we show that lung cancer cells primed with a TTF-1-driven decrease of cholesterol were more vulnerable to simvastatin, a frequently prescribed cholesterol biosynthesis inhibitor. In view of the fact that pathologists routinely interrogate human lung cancers for TTF-1 immunopositivity to guide diagnosis and the prevalent use of statins, TTF-1 should be further investigated as a putative biomarker of lung cancer vulnerability to statins.

Roles of Thyroid Transcription Factor 1 in Lung Cancer Biology.

Thyroid transcription factor 1 (TTF-1 or NKX2-1) is a transcription factor of fundamental importance in driving lung maturation and morphogenesis. In the last decade, scientists began to appreciate the functional roles of TTF-1 in lung tumorigenesis. This movement was triggered by the discoveries of genetic alterations of TTF-1 in the form of gene amplification in lung cancer. Many downstream target genes of TTF-1 relevant to the lung cancer biology of TTF-1 have been documented. One of the most surprising findings was that TTF-1 may exhibit either pro- or antitumorigenic activities, an outcome with the complexity exceeding the original anticipation purely based on the fact that TTF-1 undergoes gene amplification in lung cancer. In the coming decade, we believe, we will witness additional surprises as the research exploring the cancer roles of TTF-1 progresses.

Thyroid Transcription Factor 1 Reprograms Angiogenic Activities of Secretome.

Through both gain- and loss-of-TTF-1 expression strategies, we show that TTF-1 positively regulates vascular endothelial growth factor (VEGF) and that the VEGF promoter element contains multiple TTF-1-responsive sequences. The major signaling receptor for VEGF, i.e VEGFR2, also appears to be under a direct and positive regulation of TTF-1. The TTF-1-dependent upregulation of VEGF was moderately sensitive to rapamycin, implicating a partial involvement of mammalian target of rapamycin (mTOR). However, hypoxia did not further increase the secreted VEGF level of the TTF-1(+) lung cancer cells. The TTF-1-induced VEGF upregulation occurs in both compartments (exosomes and exosome-depleted media (EDM)) of the conditioned media. Surprisingly, the EDM of TTF-1(+) lung cancer cells (designated EDM-TTF-1(+)) displayed an anti-angiogenic activity in the endothelial cell tube formation assay. Mechanistic studies suggest that the increased granulocyte-macrophage colony-stimulating factor (GM-CSF) level in the EDM-TTF-1(+) conferred the antiangiogenic activities. In human lung cancer, the expression of TTF-1 and GM-CSF exhibits a statistically significant and positive correlation. In summary, this study provides evidence that TTF-1 may reprogram lung cancer secreted proteome into an antiangiogenic state, offering a novel basis to account for the long-standing observation of favorable prognosis associated with TTF-1(+) lung adenocarcinomas.

Mnk2 alternative splicing modulates the p38-MAPK pathway and impacts Ras-induced transformation.

The kinase Mnk2 is a substrate of the MAPK pathway and phosphorylates the translation initiation factor eIF4E. In humans, MKNK2, the gene encoding for Mnk2, is alternatively spliced yielding two splicing isoforms with differing last exons: Mnk2a, which contains a MAPK-binding domain, and Mnk2b, which lacks it. We found that the Mnk2a isoform is downregulated in breast, lung, and colon tumors and is tumor suppressive. Mnk2a directly interacts with, phosphorylates, activates, and translocates p38α-MAPK into the nucleus, leading to activation of its target genes, increasing cell death and suppression of Ras-induced transformation. Alternatively, Mnk2b is pro-oncogenic and does not activate p38-MAPK, while still enhancing eIF4E phosphorylation. We further show that Mnk2a colocalization with p38α-MAPK in the nucleus is both required and sufficient for its tumor-suppressive activity. Thus, Mnk2a downregulation by alternative splicing is a tumor suppressor mechanism that is lost in some breast, lung, and colon tumors.

The complexity of thyroid transcription factor 1 with both pro- and anti-oncogenic activities.

After the original identification of thyroid transcription factor 1 (TTF-1 or NKX2-1) biochemical activity as a transcriptional regulator of thyroglobulin in 1989, the bulk of the ensuing research has concentrated on elucidating the roles of NKX2-1 in the development of lung and thyroid tissues. Motivated by its specific expression pattern, pathologists adopted the NKX2-1 immunoreactivity to distinguish pulmonary from nonpulmonary nonthyroid adenocarcinomas. Interestingly, the concept of NKX2-1 as an active participant in lung tumorigenesis did not take hold until 2007. This minireview contrasts the recent advancements of NKX2-1-related observations primarily in the realm of pulmonary malignancies.

Tight junction proteins: from barrier to tumorigenesis.

The tight junction is a multi-protein complex and is the apical most junctional complex in certain epithelial and endothelial cells. A great deal of attention has been devoted to the understanding of these proteins in contributing to the barrier function - that is, regulating the paracellular flux or permeability between adjacent cells. However, tight junction proteins are now recognized as having functions beyond the barrier. The focus of this review is to discuss the barrier function of the tight junction and to summarize the literature with a focus on the role of tight junction proteins in proliferation, transformation, and metastasis.

Two Distinct Categories of Focal Deletions in Cancer Genomes.

One of the key questions about genomic alterations in cancer is whether they are functional in the sense of contributing to the selective advantage of tumor cells. The frequency with which an alteration occurs might reflect its ability to increase cancer cell growth, or alternatively, enhanced instability of a locus may increase the frequency with which it is found to be aberrant in tumors, regardless of oncogenic impact. Here we've addressed this on a genome-wide scale for cancer-associated focal deletions, which are known to pinpoint both tumor suppressor genes (tumor suppressors) and unstable loci. Based on DNA copy number analysis of over one-thousand human cancers representing ten different tumor types, we observed five loci with focal deletion frequencies above 5%, including the A2BP1 gene at 16p13.3 and the MACROD2 gene at 20p12.1. However, neither RNA expression nor functional studies support a tumor suppressor role for either gene. Further analyses suggest instead that these are sites of increased genomic instability and that they resemble common fragile sites (CFS). Genome-wide analysis revealed properties of CFS-like recurrent deletions that distinguish them from deletions affecting tumor suppressor genes, including their isolation at specific loci away from other genomic deletion sites, a considerably smaller deletion size, and dispersal throughout the affected locus rather than assembly at a common site of overlap. Additionally, CFS-like deletions have less impact on gene expression and are enriched in cell lines compared to primary tumors. We show that loci affected by CFS-like deletions are often distinct from known common fragile sites. Indeed, we find that each tumor tissue type has its own spectrum of CFS-like deletions, and that colon cancers have many more CFS-like deletions than other tumor types. We present simple rules that can pinpoint focal deletions that are not CFS-like and more likely to affect functional tumor suppressors.

MicroRNA-33a mediates the regulation of high mobility group AT-hook 2 gene (HMGA2) by thyroid transcription factor 1 (TTF-1/NKX2-1).

In lung cancers, TTF-1 displays seemingly paradoxical activities. Although TTF-1 is amplified in primary human lung cancers, it inhibits primary lung tumors from metastasizing in a mouse model system. It was reported that the oncogenic proepithelial mesenchymal transition (EMT) high mobility group AT-hook 2 gene (HMGA2) mediates the antimetastatic function of TTF-1. To gain mechanistic insight into the metastasis-critical signaling axis of TTF-1 to HMGA2, we used both reverse and forward strategies and discovered that microRNA-33a (miR-33a) is under direct positive regulation of TTF-1. By chromatin immunoprecipitation, we determined that TTF-1 binds to the promoter of SREBF2, the host gene of miR-33a. The 3'-untranslated region (UTR) of HMGA2 contains three predicted binding sites of miR-33a. We showed that the first two highly conserved sites are conducive to HMGA2 repression by miR-33a, establishing HMGA2 as a genuine target of miR-33a. Functional studies revealed that enforced expression of miR-33a inhibits the motility of lung cancer cells, and this inhibition can be rescued by overexpression of the form of HMGA2 without the 3'-UTR, suggesting that TTF-1 keeps the prometastasis gene HMGA2 in check via up-regulating miR-33a. This study reports the first miRNAs directly regulated by TTF-1 and clarifies how TTF-1 controls HMGA2 expression. Moreover, the documented importance of SREBF2 and miR-33a in regulating cholesterol metabolism suggests that TTF-1 may be a modulator of cholesterol homeostasis in the lung. Future studies will be dedicated to understanding how miRNAs influence the oncogenic activity of TTF-1 and the role of TTF-1 in cholesterol metabolism.

The splicing factor SRSF6 is amplified and is an oncoprotein in lung and colon cancers.

An increasing body of evidence connects alterations in the process of alternative splicing with cancer development and progression. However, a direct role of splicing factors as drivers of cancer development is mostly unknown. We analysed the gene copy number of several splicing factors in colon and lung tumours, and found that the gene encoding for the splicing factor SRSF6 is amplified and over-expressed in these cancers. Moreover, over-expression of SRSF6 in immortal lung epithelial cells enhanced proliferation, protected them from chemotherapy-induced cell death and converted them to be tumourigenic in mice. In contrast, knock-down of SRSF6 in lung and colon cancer cell lines inhibited their tumourigenic abilities. SRSF6 up- or down-regulation altered the splicing of several tumour suppressors and oncogenes to generate the oncogenic isoforms and reduce the tumour-suppressive isoforms. Our data suggest that the splicing factor SRSF6 is an oncoprotein that regulates the proliferation and survival of lung and colon cancer cells.

Occludin is a direct target of thyroid transcription factor-1 (TTF-1/NKX2-1).

The thyroid transcription factor 1 gene (TTF-1 or NKX2-1) is essential to lung development; however, it is also a critical factor in lung cancer. TTF-1 is amplified in lung cancers, suggesting that it is a gain-of-function lung oncogene. Conversely, TTF-1 counters epithelial to mesenchymal transition in cell-based studies and inhibits progression of primary lung adenocarcinomas to metastases in an animal model of lung adenocarcinomas. The unifying theory regarding TTF-1 is that it exhibits both pro-oncogenic and anti-metastatic function depending on the cellular context. Occludin is the first discovered constituent of the epithelial tight junction; in recent years, a functional role of occludin as a tumor suppressor has begun to emerge. Here, we demonstrate that TTF-1 transactivated the expression of the epithelial tight junction molecules occludin (OCLN) and claudin-1 (CLDN1). We show that transcriptional activation occurred through a direct interaction of TTF-1 with the OCLN and CLDN1 promoters. Furthermore, in cells that lack TTF-1, exogenous TTF-1 expression dampened the inhibitory effect of TGF-ß on occludin and claudin-1 content. Using cells derived from a genetically engineered mouse model of lung adenocarcinomas, we observed that silenced TTF-1 expression down-regulated occludin, which we supported with additional siRNA experiments. Finally, TTF-1 knockdown conferred human lung cancer cells resistance to anoikis, and expression of occludin restored cellular sensitivity to anoikis. Overexpression of occludin impeded migration and induced anoikis in lung carcinoma cells. Collectively, these data suggest that TTF-1 transcriptionally regulates occludin, which represents another avenue of TTF-1-mediated metastasis suppression.

MicroRNAs and lung cancers: from pathogenesis to clinical implications.

Lung cancer is the leading cause of cancer-related deaths in the US and worldwide. Better understanding of the disease is warranted for improvement in clinical management. Here we summarize the functions of small-RNA-based, posttranscriptional gene regulators, i.e. microRNAs, in the pathogenesis of lung cancers. We discuss the microRNAs that play oncogenic as well as tumor suppressive roles. We also touch on the value of microRNAs as markers for diagnosis, prognosis and the promising field of microRNA-based novel therapies for lung cancers.

Cooperation between Rb and Arf in suppressing mouse retinoblastoma.

Retinoblastoma is a pediatric cancer that has served as a paradigm for tumor suppressor gene function. Retinoblastoma is initiated by RB gene mutations, but the subsequent cooperating mutational events leading to tumorigenesis are poorly characterized. We investigated what these additional genomic alterations might be using human retinoblastoma samples and mouse models. Array-based comparative genomic hybridization studies revealed deletions in the CDKN2A locus that include ARF and P16INK4A, both of which encode tumor suppressor proteins, in both human and mouse retinoblastoma. Through mouse genetic analyses, we found that Arf was the critical tumor suppressor gene in the deleted region. In mice, inactivation of one allele of Arf cooperated with Rb and p107 loss to rapidly accelerate retinoblastoma, with frequent loss of heterozygosity (LOH) at the Arf locus. Arf has been reported to exhibit p53-independent tumor suppressor roles in other systems; however, our results showed no additive effect of p53 and Arf coinactivation in promoting retinoblastoma. Moreover, p53 inactivation completely eliminated any selection for Arf LOH. Thus, our data reveal important insights into the p53 pathway in retinoblastoma and show that Arf is a key collaborator with Rb in retinoblastoma suppression.

Molecular biology of adenoid cystic carcinoma.

BACKGROUND: Adenoid cystic carcinoma (ACC) is an unusual salivary gland malignancy that remains poorly understood. Standard treatment, including surgery with postoperative radiation therapy, has attained reasonable local control rates, but the propensity for distant metastases has limited any improvement in survival over time. Our understanding of the molecular mechanisms driving ACC is quite rudimentary, due to the infrequent nature of its occurrence. METHODS: An extensive literature review was performed on salivary gland ACCs and basic science research findings. RESULTS: This review highlights many findings that are emerging about the carcinogenesis of ACC including cytogenetics, tumor suppressor genes, oncogenes, epigenetic alterations, mitochondrial alterations, and biomarker studies. CONCLUSION: Although there have been many discoveries, much still remains unknown about this rare malignancy. © 2011 Wiley Periodicals, Inc. Head Neck, 2011.

MiR-365 regulates lung cancer and developmental gene thyroid transcription factor 1.

Thyroid transcription factor 1 (TTF-1 or NKX2-1) is an essential fetal lung developmental factor, which can be recurrently activated by gene amplification in adult lung cancer. We have discovered the first microRNA (i.e., miR-365) that directly regulates TTF-1 by interacting with its 3'-untranslated region. By gene expression profiling, we identified other putative targets of miR-365 and miR-365*. In line with the microRNA/target relationship, the expression patterns of miR-365 and TTF-1 were in an inverse relationship in human lung cancer. Exploration of human lung cancer genomics data uncovered that TTF-1 gene amplification was significantly associated with DNA copy number loss at one of the two genomic loci encoding the precursor RNA of mature miR-365 (i.e., mir-365-1). This implies the existence of genetic selection pressure to lose the repressive miR-365 that would otherwise suppress amplified TTF-1. We detected a signaling loop between transforming growth factor beta (TGFb) and miR-365 and this loop reinforced suppression of TTF-1 via miR-365. Mir-365 also targeted an epithelial mesenchymal transition (EMT)-promoting gene HMGA2. In summary, these data connect the lung transcriptional program to the microRNA network.

miR-17~92 cooperates with RB pathway mutations to promote retinoblastoma.

The miR-17~92 cluster is a potent microRNA-encoding oncogene. Here, we show that miR-17~92 synergizes with loss of Rb family members to promote retinoblastoma. We observed miR-17~92 genomic amplifications in murine retinoblastoma and high expression of miR-17~92 in human retinoblastoma. While miR-17~92 was dispensable for mouse retinal development, miR-17~92 overexpression, together with deletion of Rb and p107, led to rapid emergence of retinoblastoma with frequent metastasis to the brain. miR-17~92 oncogenic function in retinoblastoma was not mediated by a miR-19/PTEN axis toward apoptosis suppression, as found in lymphoma/leukemia models. Instead, miR-17~92 increased the proliferative capacity of Rb/p107-deficient retinal cells. We found that deletion of Rb family members led to compensatory up-regulation of the cyclin-dependent kinase inhibitor p21Cip1. miR-17~92 overexpression counteracted p21Cip1 up-regulation, promoted proliferation, and drove retinoblastoma formation. These results demonstrate that the oncogenic determinants of miR-17~92 are context-specific and provide new insights into miR-17~92 function as an RB-collaborating gene in cancer.