Li-Fraumeni Syndrome Patient-derived LFS50 Progression Cell Series as an Experimental Model for Breast Cancer Prevention Research

poster abstractLi-Fraumeni syndrome (LFS) is a cancer predisposition syndrome associated with germline mutations in the tumor suppressor gene TP53. Breast cancer (BC) is the most common tumor amongst women with LFS, who have increased risk for premenopausal BC before age 40 and a lifetime risk of 49% by the age of 60. Non-malignant, human mammary epithelial cells (HMECs) were derived from the contralateral breast tissue of LFS patient (LFS50) undergoing BC surgery. The LFS50 HMEC progression series comprises of pre-immortal (HME50), spontaneously immortalized (HME50-5E), hTERT-immortalized (HME50hTERT or HME50hT), and tumorigenic (HMET) which can be modeled to represent breast cancer progression. Gene expressions of the LFS50 series were profiled using HG-U133_Plus_2 Affymetrix chip. By hierarchical clustering, the LFS50 cells were observed to have significant differential expression of genes and ANOVA results revealed that EMT-related genes (e.g., epithelial membrane protein 3, p= 6.84911E-19; E-cadherin, p= 8.66098E-19; and Keratin 5, p= 9.73095E-19) to be the most differentially expressed amongst the LFS50 cells. Ingenuity Pathway Analysis (IPA) confirmed that Ecadherin and Keratin 5 were the top most differentially expressed genes as well as G2/M DNA Damage Checkpoint Regulation (p= 2.67E-05), Estrogen-mediated S-phase Entry (p=3.32E-04) Mitotic Roles of Polo-Like Kinase (p=5.5E-04) as few of the top canonical pathways. Furthermore, to identify the type of breast cancer that LFS50 series could model, the triple negative breast cancer (TNBC) subtyping database tool predicted that each of the LFS50 strains could be classified as a different subtype. Finally, as a proof of principle for drug targeting, treatment of the LFS50 series with PRIMA-1, a p53 rescue drug, using 3D cultures resulted in a reduction in acini size of the pre-invasive LFS50 cells (p<0.05). Therefore, this progression series can serve as a resource for drug target discovery and breast cancer prevention research

The telomerase inhibitor imetelstat alone, and in combination with trastuzumab, decreases the cancer stem cell population and self-renewal of HER2+ breast cancer cells

Cancer stem cells (CSCs) are thought to be responsible for tumor progression, metastasis, and recurrence. HER2 overexpression is associated with increased CSCs, which may explain the aggressive phenotype and increased likelihood of recurrence for HER2+ breast cancers. Telomerase is reactivated in tumor cells, including CSCs, but has limited activity in normal tissues, providing potential for telomerase inhibition in anti-cancer therapy. The purpose of this study was to investigate the effects of a telomerase antagonistic oligonucleotide, imetelstat (GRN163L), on CSC and non-CSC populations of HER2+ breast cancer cell lines. The effects of imetelstat on CSC populations of HER2+ breast cancer cells were measured by ALDH activity and CD44/24 expression by flow cytometry as well as mammosphere assays for functionality. Combination studies in vitro and in vivo were utilized to test for synergism between imetelstat and trastuzumab. Imetelstat inhibited telomerase activity in both subpopulations. Moreover, imetelstat alone and in combination with trastuzumab reduced the CSC fraction and inhibited CSC functional ability, as shown by decreased mammosphere counts and invasive potential. Tumor growth rate was slower in combination-treated mice compared to either drug alone. Additionally, there was a trend toward decreased CSC marker expression in imetelstat-treated xenograft cells compared to vehicle control. Furthermore, the observed decrease in CSC marker expression occurred prior to and after telomere shortening, suggesting that imetelstat acts on the CSC subpopulation in telomere length-dependent and -independent mechanisms. Our study suggests addition of imetelstat to trastuzumab may enhance the effects of HER2 inhibition therapy, especially in the CSC population

EFFECTS OF RESVERATROL ON PACLITAXEL-SENSITIVE AND –RESISTANT TRIPLE NEGATIVE BREAST CANCER CELLS

poster abstractTreatment of drug-resistant cancer cells remains a difficult problem in cancer therapy because most resistant cells can pump out drugs or upregulate other survival pathways to bypass a targeted therapy. The poly-phenol natural compound, resveratrol, has been shown to inhibit cell growth of multiple cancer types, but it is not cytotoxic to normal cells. However, the effects of resveratrol in triple negative breast cancer cells as well as cancers that are resistant to the common cancer drug, paclitaxel, are not well under-stood. In this study, the effects of resveratrol were investigated in the triple negative breast cancer cell line MDA-MB-231 as well as a novel paclitaxel-resistant MDA-MB-231 derived line generated in our laboratory. Both cell lines exhibited a reduction in cell proliferation after resveratrol treatment, with the paclitaxel-resistant cells to a greater extent. In addition, resveratrol decreased the ability of both cell lines to form colonies when plated at low density indicating reduced cell survival capacity. Resveratrol treatment also increased the amount of DNA fragmentation associated with cell death in both cell lines, again with the paclitaxel resistant cells being more sensitive. By protein expression analyses, we observed that in both the parental and resistant cell lines, resveratrol may be acting by through NAD-dependent deacetylase sirtuin (SIRT1) activity by decreasing the expression of the in-hibitor-of-apoptosis protein, survivin, as well as increasing the activator-of-cell death, caspase 7. These data suggest that resveratrol can inhibit prolif-eration and induce cell death in triple negative breast cancer cells, including paclitaxel-resistant cells. In addition, these results provide rationale for the use of resveratrol as an important starting point for the development of a novel anti-cancer agent for drug resistant, aggressive cancers as well as in combination with other anti-cancer drugs without significant toxicity to nor-mal cells

Molecular Mechanisms of Paclitaxel Resistance and Resveratrol Sensitivity in MDA-MB-231 Breast Cancer Cells

poster abstractTreatment of drug-resistant cancer cells remains a difficult problem in cancer therapy because most resistant cells can pump out drugs or upregulate other survival pathways to bypass a targeted therapy. To study cancers that are resistant to the common cancer drug, paclitaxel, a novel paclitaxel-resistant cell line was generated from the breast cancer cell line MDA-MB-231. A “spiking” method of paclitaxel treatment was used to select for a population of cells that are resistant to the drug. This method mimics the development of resistance in recurrent tumors in patients. However, it is difficult to study such a heterogeneous population. To better study these cells, the paclitaxel-resistant cell line was cloned using a limiting dilution method to provide more homogeneous populations of resistant cells. The 29 clones obtained exhibited a paclitaxel IC50 range of 8 μM to 78 μM which was equivalent to a 200- to 2000-fold increase in resistance compared to the parent line. It has been suggested that the polyphenol natural compound, resveratrol, which has been shown to inhibit cell growth of multiple cancer types, may be useful as a combination anti-cancer treatment or novel therapeutic for drug-resistant cancer cells. The parent line, the heterogeneous resistant line, the least paclitaxel-resistant clone and the most paclitaxelresistant clone were similarly sensitive to resveratrol treatment. We observed that treatment with 10-100 μM concentrations of resveratrol in all cell lines showed a reduction in cell proliferation and increased apoptosis within 72 hours (p<0.05), with the paclitaxel-resistant cells to a greater extent. In addition, resveratrol decreased the ability of the parent, heterogeneous resistant and the highest resistant clone cells to form colonies (an indication of reduced cell survival capacity). This resistant cell line and its clones provide a powerful tool to study paclitaxel-resistance in and therapeutics for breast cancer

Engaging Medical Students in Research or Scholarly Activities as Part of the Curriculum at Indiana University

Presented as a poster at 2020 IUSM Education Day

Medical genetics and epigenetics of telomerase

Telomerase is a specialized reverse transcriptase that extends and maintains the terminal ends of chromosomes, or telomeres. Since its discovery in 1985 by Nobel Laureates Elizabeth Blackburn and Carol Greider, thousands of articles have emerged detailing its significance in telomere function and cell survival. This review provides a current assessment on the importance of telomerase regulation and relates it in terms of medical genetics. In this review, we discuss the recent findings on telomerase regulation, focusing on epigenetics and non-coding RNAs regulation of telomerase, such as microRNAs and the recently discovered telomeric-repeat containing RNA transcripts. Human genetic disorders that develop due to mutations in telomerase subunits, the role of single nucleotide polymorphisms in genes encoding telomerase components and diseases as a result of telomerase regulation going awry are also discussed. Continual investigation of the complex regulation of telomerase will further our insight into the use of controlling telomerase activity in medicine

Global gene expression of histologically normal primary skin cells from BCNS subjects reveals "single-hit" effects that are influenced by rapamycin

Studies of dominantly heritable cancers enabled insights about tumor progression. BCNS is a dominantly inherited disorder that is characterized by developmental abnormalities and postnatal neoplasms, principally BCCs. We performed an exploratory gene expression profiling of primary cell cultures derived from clinically unaffected skin biopsies of BCNS gene-carriers (PTCH1 +/-) and normal individuals. PCA and HC of untreated keratinocytes or fibroblasts failed to clearly distinguish BCNS samples from controls. These results are presumably due to the common suppression of canonical HH signaling in vitro. We then used a relaxed threshold (p-value <0.05, no FDR cut-off; FC 1.3) that identified a total of 585 and 857 genes differentially expressed in BCNS keratinocytes and fibroblasts samples, respectively. A GSEA identified pancreatic β cell hallmark and mTOR signaling genes in BCNS keratinocytes, whereas analyses of BCNS fibroblasts identified gene signatures regulating pluripotency of stem cells, including WNT pathway. Significantly, rapamycin treatment (FDR<0.05), affected a total of 1411 and 4959 genes in BCNS keratinocytes and BCNS fibroblasts, respectively. In contrast, rapamycin treatment affected a total of 3214 and 4797 genes in normal keratinocytes and normal fibroblasts, respectively. The differential response of BCNS cells to rapamycin involved 599 and 1463 unique probe sets in keratinocytes and fibroblasts, respectively. An IPA of these genes in the presence of rapamycin pointed to hepatic fibrosis/stellate cell activation, and HIPPO signaling in BCNS keratinocytes, whereas mitochondrial dysfunction and AGRN expression were uniquely enriched in BCNS fibroblasts. The gene expression changes seen here are likely involved in the etiology of BCCs and they may represent biomarkers/targets for early intervention

Predictors of publication rate from 2018 and 2019 IMPRS abstracts: an exploratory analysis

INTRODUCTION/BACKGROUND: Since 2017 the IU School of Medicine has offered the opportunity to medical students to participate in the Indiana University Medical Student Program for Research and Scholarship (IMPRS). For many students, this is the first opportunity to conduct research in clinical medicine and have their research abstract available in the Proceedings of IMPRS journal, enhancing the visibility of their research and later publishing in a peer-reviewed journal. We determined and analyzed which abstracts from the IMPRS 2018 and 2019 program were then published as peer-reviewed articles. STUDY OBJECTIVE: Our purpose was to determine the proportion of abstracts presented at the IMPRS Summer Program Oral Presentation Symposium during 2018 and 2019 that were further published as full articles in peer-reviewed publications, the time lag to publication, and the factors associated with successful publication. We will identify potential trends or predictors of publication by comparing the years of presentation and publication, the journals where the abstract was published, the IMPRS research track, financial support received, IMPRS program award winning status, and research topics of the published abstracts. METHODS: A total of 241 abstracts were presented at the IMPRS oral Presentation Symposium during 2018 and 2019 (abstracts were identified from the records uploaded in the Proceedings of IMPRS website http://journals.iupui.edu/index.php/IMPRS). We used Pubmed, Google Scholar, Dimensions database, and Citation Finder app to search and find accurate citations of the final version of the published abstract. We combined the author names of the students and mentors with keywords from the abstracts. We used PubMed to gather the articles' medical subject heading (MeSH) terms to perform a co-occurrence analysis. We used Excel to aggregate, clean up and analyze the data and VOSviewer software to generate the topic analysis and visualization map. RESULTS AND CONCLUSION: Overall, 52 of the abstracts (publication rate of 21.6%) have been published in 49 peer-reviewed scholarly publications, by January 2022, with an average and median time between the oral presentation and the official date of publication of 21 months (IQR=9, 26-17). The articles identified were published in 44 journals and one book series; the journal Journal Impact Factor (JIF) ranged from 1.276 to 21.198 (mean 5.62). The journal with the most published articles (≥ 4) was the Journal of Surgical Research. The majority of papers presented at IMPRS 2018 and 2019 that end up with a publication belong to the “Laboratory and translational research” IMPRS research track (66.7%). This approach aims to contribute to a better understanding of the characteristics of the abstracts presented in the context of the IMPRS program that continues the process of publishing manuscripts in peer-reviewed scholarly publications. The findings provide relevant insight to the librarians concerning their involvement and support with the IMPRS program in advising students about initial steps toward scholarly publishing

SOX17 Regulates Conversion of Human Fibroblasts Into Endothelial Cells and Erythroblasts by Dedifferentiation Into CD34+ Progenitor Cells

BACKGROUND: The mechanisms underlying the dedifferentiation and lineage conversion of adult human fibroblasts into functional endothelial cells have not yet been fully defined. Furthermore, it is not known whether fibroblast dedifferentiation recapitulates the generation of multipotent progenitors during embryonic development, which give rise to endothelial and hematopoietic cell lineages. Here we established the role of the developmental transcription factor SOX17 in regulating the bilineage conversion of fibroblasts by the generation of intermediate progenitors. METHODS: CD34+ progenitors were generated after the dedifferentiation of human adult dermal fibroblasts by overexpression of pluripotency transcription factors. Sorted CD34+ cells were transdifferentiated into induced endothelial cells and induced erythroblasts using lineage-specific growth factors. The therapeutic potential of the generated cells was assessed in an experimental model of myocardial infarction. RESULTS: Induced endothelial cells expressed specific endothelial cell surface markers and also exhibited the capacity for cell proliferation and neovascularization. Induced erythroblasts expressed erythroid surface markers and formed erythroid colonies. Endothelial lineage conversion was dependent on the upregulation of the developmental transcription factor SOX17, whereas suppression of SOX17 instead directed the cells toward an erythroid fate. Implantation of these human bipotential CD34+ progenitors into nonobese diabetic/severe combined immunodeficiency (NOD-SCID) mice resulted in the formation of microvessels derived from human fibroblasts perfused with mouse and human erythrocytes. Endothelial cells generated from human fibroblasts also showed upregulation of telomerase. Cell implantation markedly improved vascularity and cardiac function after myocardial infarction without any evidence of teratoma formation. CONCLUSIONS: Dedifferentiation of fibroblasts to intermediate CD34+ progenitors gives rise to endothelial cells and erythroblasts in a SOX17-dependent manner. These findings identify the intermediate CD34+ progenitor state as a critical bifurcation point, which can be tuned to generate functional blood vessels or erythrocytes and salvage ischemic tissue