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Item 4168 Understanding ECM-Based Drug Resistivity in Breast Cancer(Cambridge University Press, 2020-07-29) Libring, Sarah; Shinde, Aparna; Boodaghidizaji, Miad; Plummer, Alexandra; Ardekani, Arezoo; Wendt, Michael; Solorio, Luis; Medicine, School of MedicineOBJECTIVES/GOALS: Cell-cell (CC) and cell-matrix interactions (CM) are known to affect drug sensitivity of cancer cells, but are not effectively recapitulated using 2D platforms. This research aims to determine how cell and matrix interactions confer drug resistivity in 3 distinct culturing models: 2D (no CM/limited CC), 3D spheroids (CC) and 3D fibronectin (both). METHODS/STUDY POPULATION: We examined four breast cancer cell types. The cells were derived from a nonmetastatic primary tumor (HMLE-E2) or overt bone-metastasis (BM). Transglutaminase 2 (TGM2), a matrix crosslinking protein, is overexpressed in metastatic bone tumors and may play a key role in matrix-conferred drug resistivity. In a gain-of-function model, TGM2 was upregulated in HMLE-E2 cells and compared to shTGM2 knockdown BM cells. Growth rates were analyzed using metabolic activity over 8 days, and drug sensitivity to Neratinib (0-1000 nM) was analyzed via cell titer. To account for the different transport properties of the 3 distinct culture environments, we developed a mathematical model for each condition, allowing us to normalize the drug sensitivity results across models to effectively compare true biological resistivity. RESULTS/ANTICIPATED RESULTS: We observed that increased cellular levels of TGM2 significantly increase the growth rate and drug resistivity of cells on fibronectin matrices. Interestingly, in 2D cultures, TGM2 expression was correlated with higher Neratinib resistivity but did not affect growth rates. In spheroid models without a significant matrix component, that rely solely on cell-cell junctions, high levels of TGM2 were correlated with lower survival rates. Lower levels of TGM2 are correlated with a more epithelial phenotype, and using our mathematical model we have identified significant transport differences between high and low TGM2 spheroids. We theorize that the low TGM2 spheroids have denser packing, which lowers the rate of diffusion and, thus reduces the effective concentration of the drug to the majority of the cells. DISCUSSION/SIGNIFICANCE OF IMPACT: Our studies indicate that the cellular response to drugs can be altered by changes in both transport properties of the tissue and the CM interactions. By systematically investigating the effects of CC interactions and CM interactions, we can use mathematical models to delineate physical means of drug resistivity from a biologically driven resistance.Item Abrogating cholesterol esterification suppresses growth and metastasis of pancreatic cancer(SpringerNature, 2016-12-15) Li, J.; Gu, D.; Lee, SS-Y.; Song, B.; Bandyopadhyay, S.; Chen, S.; Konieczny, SF.; Ratliff, TL.; Liu, X.; Xie, J.; Cheng, J-X.; Department of Pediatrics, IU School of MedicineCancer cells are known to execute reprogramed metabolism of glucose, amino acids and lipids. Here, we report a significant role of cholesterol metabolism in cancer metastasis. By using label-free Raman spectromicroscopy, we found an aberrant accumulation of cholesteryl ester in human pancreatic cancer specimens and cell lines, mediated by acyl-CoA cholesterol acyltransferase-1 (ACAT-1) enzyme. Expression of ACAT-1 showed a correlation with poor patient survival. Abrogation of cholesterol esterification, either by an ACAT-1 inhibitor or by shRNA knockdown, significantly suppressed tumor growth and metastasis in an orthotopic mouse model of pancreatic cancer. Mechanically, ACAT-1 inhibition increased intracellular free cholesterol level, which was associated with elevated endoplasmic reticulum stress and caused apoptosis. Collectively, our results demonstrate a new strategy for treating metastatic pancreatic cancer by inhibiting cholesterol esterification.Item Bidirectional regulation of YAP and ALDH1A1(2015-08-10) Martien, Matthew F.; Wells, Clark D.; Hurley, Thomas D.; Quilliam, Lawrence A.Breast cancer is the second leading cause of cancer death for women in the United States. Approximately, 1 in 5 women will recur with cancer within 10 years of completing treatment and recent publications have suggested that breast cancer stem cells confer resistance to therapy. These reports highlight aldehyde dehydrogenase 1A1 (ALDH1A1) and Yes-associated protein (YAP) as a biomarker and key mediator of the stem cell phenotype respectively. To further understand how YAP and ALDH1A1 facilitate chemoresistance, this study investigated how ALDH1A1 specific inhibition affected YAP activity and growth of basal-like breast cancer cells, which are enriched in cancer stem cells. Intriguingly, attenuation of growth by ALDH1A1 inhibition was observed when cells were plated on a reconstituted basement membrane. Further, the inhibition of cell growth correlated with cytosolic retention of YAP and a reduction in YAP signaling. In a complementary analysis, the overexpression of YAP correlated with an increased level of ALDH1A1 transcript. Results from this study indicate a novel mechanism by which basal-like breast cancer cells utilize YAP to maintain the stem cell phenotype and also suggest ALDH1A1 as a potential therapeutic target for breast cancer therapy.Item Development of a New Monochrome Multiplex qPCR Method for Relative Telomere Length Measurement in Cancer(Elsevier, 2018-05) Dahlgren, Paige N.; Bishop, Kanokwan; Dey, Shatovisha; Herbert, Brittney-Shea; Tanaka, Hiromi; Medical and Molecular Genetics, School of MedicineExcess telomere shortening has been observed in most cancer cells. The telomere quantitative polymerase chain reaction (qPCR) assay has become an important tool for epidemiological studies examining the effects of aging, stress, and other factors on the length of telomeres. Current telomere qPCR methods analyze the relative length of telomeres by amplifying telomere sequence products and normalizing with single-copy gene products. However, the current telomere qPCR does not always reflect absolute telomere length in cancer DNA. Because of genomic instability in cancer cells, we hypothesized that the use of single-copy genes (scg) is less accurate for normalizing data in cancer DNA and that new primer sets are required to better represent relative telomere length in cancer DNA. We first confirmed that cancer cells had a different copy ratio among different scg, implying that DNA is aneuploid. By using the new primer sets that amplify multiple-copy sequences (mcs) throughout the genome, the telomere qPCR results showed that the mcs primers were interchangeable with the scg primers as reference primers in normal DNA. By comparing results from the traditional southern blotting method (as kilobases) and results from monochrome multiplex qPCR using the mcs primers (as T/M ratios), we verified that the T/M ratio is highly correlated with absolute telomere length from the southern blot analysis. Together, the mcs primers were able to represent the telomere lengths accurately in cancer DNA samples. These results would allow for analyses of telomeres within cancerous DNA and the development of new, less invasive diagnostic tools for cancer.Item Item An inhibitor of the mitotic kinase, MPS1, is selective towards pancreatic cancer cells(2014) Bansal, Ruchi; Grimes, Brenda R.; Herbert, Brittney-Shea; Dlouhy, Stephen RobertThe abysmal five year pancreatic cancer survival rate of less than 6% highlights the need for new treatments for this deadly malignancy. Cytotoxic drugs normally target rapidly dividing cancer cells but unfortunately often target stem cells resulting in toxicity. This warrants the development of compounds that selectively target tumor cells. An inhibitor of the mitotic kinase, MPS1, which has been shown to be more selective towards cancer cells than non-tumorigenic cells, shows promise but its effects on stem cells has not been investigated. MPS1 is an essential component of the Spindle Assembly Checkpoint and is proposed to be up-regulated in cancer cells to maintain chromosomal segregation errors within survivable limits. Inhibition of MPS1 kinase causes cancer cell death accompanied by massive aneuploidy. Our studies demonstrate that human adipose stem cells (ASCs) and can tolerate higher levels of a small molecule MPS1 inhibitor than pancreatic cancer cells. In contrast to PANC-1 cancer cells, ASCs and telomerase-immortalized pancreatic ductal epithelial cells did not exhibit elevated chromosome mis-segregation after treatment with the MPS1 inhibitor for 72hrs. In contrast, PANC-1 pancreatic cancer cells exhibited a large increase in chromosomal mis-segregation under similar conditions. Furthermore, growth of ASCs was minimally affected post treatment whereas PANC-1 cells were severely growth impaired suggesting a favorable therapeutic index. Our studies, demonstrate that MPS1 inhibition is selective towards pancreatic cancer cells and that stem cells are less affected in vitro. These data suggest MPS1 inhibition should be further investigated as a new treatment approach in pancreatic cancer.Item Targeting telomerase in HER2 positive breast cancer: role of cancer stem cells(2015-02) Koziel, Jillian Elizabeth; Herbert, Brittney-Shea; Chan, Rebecca; Schneider, Bryan P.; Tanaka, HiromiCancer stem cells (CSCs) are proposed to play a major role in tumor progression, metastasis, and recurrence. The Human Epidermal growth factor Receptor 2 (HER2) gene is amplified and/or its protein product overexpressed in approximately 20% of breast cancers. 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 support for the use of telomerase inhibition in anti-cancer therapy. Telomerase inhibition via an antagonistic oligonucleotide, imetelstat (GRN163L), has been shown to be effective in limiting cell growth in vitro and limiting tumor growth. Moreover, we have previously shown imetelstat can decrease metastases to the lungs, leading us to question if this is due to imetelstat targeting the CSC population. In this thesis, we investigated the effects of imetelstat on CSC and non-CSC populations of HER2+ breast cancer cell lines, as well as a triple negative breast cancer cell line, which lacks HER2 overexpression. Imetelstat inhibited telomerase activity in both CSC and non-CSC subpopulations. Moreover, imetelstat treatment alone and in combination with trastuzumab significantly reduced the CSC fraction and inhibited CSC functional ability, as shown by a significant decrease in 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. The decrease in CSC marker expression we observed occurred prior to and after telomere shortening, suggesting 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.Item Tumor collection/processing under physioxia uncovers highly relevant signaling networks and drug sensitivity(American Association for the Advancement of Science, 2022) Kumar, Brijesh; Adebayo, Adedeji K.; Prasad, Mayuri; Capitano, Maegan L.; Wang, Ruizhong; Bhat-Nakshatri, Poornima; Anjanappa, Manjushre; Simpson, Edward; Chen, Duojiao; Liu, Yunlong; Schilder, Jeanne M.; Colter, Austyn B.; Maguire, Callista; Temm, Constance J.; Sandusky, George; Doud, Emma H.; Wijeratne, Aruna B.; Mosley, Amber L.; Broxmeyer, Hal E.; Nakshatri, Harikrishna; Microbiology and Immunology, School of MedicinePreclinical studies of primary cancer cells are typically done after tumors are removed from patients or animals at ambient atmospheric oxygen (O2, ~21%). However, O2 concentrations in organs are in the ~3 to 10% range, with most tumors in a hypoxic or 1 to 2% O2 environment in vivo. Although effects of O2 tension on tumor cell characteristics in vitro have been studied, these studies are done only after tumors are first collected and processed in ambient air. Similarly, sensitivity of primary cancer cells to anticancer agents is routinely examined at ambient O2. Here, we demonstrate that tumors collected, processed, and propagated at physiologic O2 compared to ambient air display distinct differences in key signaling networks including LGR5/WNT, YAP, and NRF2/KEAP1, nuclear reactive oxygen species, alternative splicing, and sensitivity to targeted therapies. Therefore, evaluating cancer cells under physioxia could more closely recapitulate their physiopathologic status in the in vivo microenvironment.Item Type B Lactic Acidosis in a Patient with Mantle Cell Lymphoma(Hindawi, 2023-08-16) Nzenwa, Ikemsinachi C.; Berquist, Margaret; Brenner, Toby J.; Ansari, Aida; Al-Fadhl, Hamid D.; Aboukhaled, Michael; Patel, Shivani S.; Peck, Ethan E.; Al-Fadhl, Mahmoud D.; Thomas, Anthony V.; Zackariya, Nuha; Walsh, Mark M.; Bufill, Jose A.; Medicine, School of MedicineType B lactic acidosis is an uncommon medical emergency in which acid production overwhelms hepatic clearance. This specific etiology of lactic acidosis occurs without organ hypoperfusion and has been most commonly described in patients with hematologic malignancies but also in patients with solid tumors. The mechanism by which cancer cells switch their glucose metabolism toward increasingly anaerobic glycolytic phenotypes has been described as the "Warburg effect." Without treating the underlying malignancy, the prognosis for patients diagnosed with malignancy-related type B lactic acidosis is extremely poor. Here, we present a case of a 66-year-old male who was diagnosed with type B lactic acidosis secondary to mantle cell lymphoma. Bicarbonate drip was started to correct the lactic acidosis. The patient was also immediately treated with rituximab chemotherapy combined with rasburicase to avoid the hyperuricemia associated with tumor lysis syndrome. He responded to the early treatment and was discharged with normal renal function. Type B lactic acidosis secondary to hematologic malignancy is important to recognize. In order to successfully treat this syndrome, early diagnosis and simultaneous treatment of the imbalance of lactic acid levels and the underlying malignancy are necessary.