Abstract Purpose: The overexpression and loss of polarity of Mucin1 (MUC1) has been documented in aggressive breast cancer. We have identified guaifenesin as a novel treatment to reduce or eliminate MUC1 expression on human breast cancer cells. Methods: MCF-7 and ZR-75-1 human breast cancer cells were grown for 48 hours in varying concentrations of guaifenesin to determine if a dose dependent effect on MUC1 protein expression resulted with treatment. In addition, cells were also harvested, RNA isolated, reverse transcribed and PCR was performed using ABI quantitative real time PCR machine. In another set of experiments, MCF-7 cells were plated and grown for 72 hours in the absence or presence of 12.5 mM guaifenesin and the cells were harvested and stained with propidium iodine for analysis by flow cytometry. The Cell-Titer Glo assay was performed on MCF-7 and ZR-75-1 cells incubated for 48 hours in the presence of guaifenesin at varying concentrations and the LD50 was determined. In a final set of experiments, MCF-7 cells, incubated in the presence or absence of guaifenesin, were analyzed for their ability to migrate /proliferate in vitro by using a standard scratch assay. For in vivo studies ovarectomized female nude mice were injected with MCF-7-luciferase tagged cells into the mammary fat pad 24 hours after an estrogen pellet was implanted into the right flank. Treatment (vehicle or 400 mg/kg/daily) was initiated after 10 days and tumor growth was monitored through Kodak imaging system. Six weeks after the treatment tumors were isolated, measured and processed for immunohistochemistry to analyze the expression of MUC1, TUNEL and CD31. Results: Initially, MUC1 gene expression was confirmed in MCF-7 and ZR-75-1 human breast cancer cell lines. Following confirmation, the LD50 was determined to be 12.5 mM. MUC1 gene expression was successfully decreased in a dose-dependent manner in both MCF-7 and ZR-75-1 cell lines when treated with guaifenesin. When MCF-7 cells are treated with 12.5mM guaifenesin, G1 arrest occurs within 24 hours and by 72 hours, 94.2% of cells are in G1 compared to 76.2% in control (drug free) conditions. In addition, there was a significant decrease in the ability of guaifenesin treated MCF-7 cells to migrate into the scratch compared to the control (untreated) cells. In vivo studies revealed that guaifenesin significantly decreased the breast tumor weight and volume which was associated with a low expression of MUC1 in tumor sections as evaluated by immunohistochemistry. Conclusion: These findings suggest that guaifenesin may provide a novel approach to inhibit breast cancer growth. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1561.
Background: Malignant clear cell renal carcinoma (ccRCC) is an aggressive tumor that is highly resistant to chemotherapy and radiation. Current therapeutic approaches to management of ccRCC have not significantly improved patient survival, therefore novel therapies are needed. The von Hippel-Lindau tumor suppressor gene is frequently mutated in ccRCC resulting in unregulated transcriptional activity of hypoxia-inducible factors (HIF) 1α and 2α. HIF-mediated transcription leads to increased growth factor expression and growth factor receptor (GFR)-mediated signaling. NFκB and STAT3 are phosphorylated in response to GFR activation and modulate gene expression, which promotes cell growth and invasion. Activated NFκB and STAT3 expression is associated with ccRCC pathogenesis.
Supplementary Data from Blockade of MGMT Expression by O<sup>6</sup> Benzyl Guanine Leads to Inhibition of Pancreatic Cancer Growth and Induction of Apoptosis
Triple-Negative Breast Cancer (TNBC) is a type of breast cancer with absence of expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2). It represents 10-20% of all breast cancer cases, and is associated with frequent relapse and poor prognosis due to the lack of targeted therapy. Amongst all TNBC, metastatic TNBC is associated with the worse prognosis and has the fewest therapeutic options. Therefore, identifying molecular drivers for TNBC metastasis and developing potential targeted therapies will be highly beneficial for patients with metastatic TNBC.We classified 8 TNBC cell lines into two groups, more invasive (n = 3) vs. less invasive (n = 5), by assessing their abilities to both invade through 3D Matrigel and migrate distantly, characterized through in vitro cell invasion and migration assays, respectively. We compared the two groups using transcriptome profiles from Cancer Cell Line Encyclopedia (CCLE) and found that transglutaminase 2 (TGM2) was significantly upregulated in more invasive cell lines (fold change = 4.6, p < 0.001). Overexpression of TGM2 at both RNA and protein levels was confirmed experimentally in more invasive TNBC cells. These findings indicated that TGM2 might play a role in TNBC metastasis, but its functionality in invasiveness has not been evaluated by previous studies. In our study, suppressing TGM2 expression either by TGM2 inhibitor, cystamine dihydrochloride (CD), or by silencing TGM2 (siRNA) was found to significantly reduce the amount of invaded cells and inhibit cell migration, suggesting that TGM2 contributes to both cell invasion and cell migration in TNBC.Our analysis also showed a significant correlation (r = 0.94, p = 0.001) between expression levels of TGM2 and actin filament-associated protein 1 (AFAP-1). AFAP1, which regulates actin cytoskeleton integrity and was found to contribute to tumorigenic growth by regulating focal contacts in other cancer types, was suppressed when TGM2 expression was downregulated, suggesting that TGM2 may potentiate cell metastasis through upregulation of AFAP-1 expression in TNBC. We also observed co-localization of AFAP-1 and actin filaments and that downregulation of TGM2 dramatically reduced actin filament assembly.These observations identify a novel role of TGM2 in promoting TNBC cell metastasis and a new machinery of TGM2 in regulating microfilaments through AFAP-1. To conclude, TGM2 could act as a powerful biomarker and a molecular target circumventing TNBC metastasis. (Judy A. Tjoe and Jun Yin are considered to be co-corresponding authors for this study)Citation Format: Mitchell Piacsek, Andrea Sand, Richard A. Rovin, Dmitry Bosenko, Santhi D. Konduri, Judy A. Tjoe, Jun Yin. Transglutaminase 2 (TGM2) overexpression contributes to triple-negative breast cancer metastasis through AFAP1-dependent pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 168.
Supplementary Data from Tolfenamic acid enhances pancreatic cancer cell and tumor response to radiation therapy by inhibiting survivin protein expression
Abstract O6 methylguanine DNA methyltransferease (MGMT) is overexpressed in majority of cancers including pancreatic cancer. MGMT has been the focus of significant research for its role in the repair of DNA damage caused by alkylating agents. In the present study, we tested the administration of the MGMT inhibitor O6-benzylguanine (BG) at non-toxic doses in combination with an alkylator TMZ (temozolomide) on pancreatic cancer cells (L3.6 pl and PANC1) in cell culture and xenograft model. Here, we report that TMZ decreases pancreatic cancer cell growth in a dose dependent manner. BG further sensitizes pancreatic cancer cells to TMZ and significantly inhibits pancreatic cancer growth. We also report that BG, either alone or in combination with TMZ, inhibits the expression of MGMT, Ki-67 and BIRC5 (survivin), which are involved in pancreatic tumorigenesis and cell division, and induces the expression of p21cip1 cell cycle inhibitor. Microarray analysis revealed that BG significantly decreased the transcription of a group of proto-oncogenes, growth factors, cell cycle and overall transcription regulators involved in tumorigenesis like ARHGDIB, IGFL1, KIF20A, CA9, MMP10 and E2F2 and increased the transcription of a variety of genes involved in tissue differentiation, cell cycle regulation and invasion inhibitors like GDF15, CDKN1A and TIMP1. In our pancreatic cancer xenograft model, BG +/- TMZ significantly inhibited pancreatic tumor growth in vivo compared to single agent TMZ or controls. Further, BG promotes p53 function leading to growth inhibition, in part substantiating the underlying mechanism of BIRC5 (survivin) inhibition and p21cip1 induction. In conclusion, our findings suggest that MGMT inhibition may potentially provide a novel therapeutic approach in pancreatic cancer by opening the option of using (oral) alkylators like TMZ. Citation Format: George C. Bobustuc, Jonathan Ticku, Anand Patel, Kalkunte S. Srivenugopal, Santhi D. Konduri. O6 methylguanine DNA methyltransferase inhibition sensitizes pancreatic cancer cells to temozolomide. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 810. doi:10.1158/1538-7445.AM2014-810
Malignant clear cell renal carcinoma (ccRCC) is an aggressive tumor highly resistant to chemotherapy and radiation. Current therapeutic approaches to management of ccRCC have not significantly improved patient survival, therefore novel therapies are needed. Activated NFκB and STAT3 expression is associated with ccRCC pathogenesis. The dietary polyphenol curcumin is a well-documented antitumor agent and a known inhibitor of NFκB and STAT3 activation. Given the lack of effective therapies that block ccRCC progression, our objective was to examine whether curcumin could suppress the growth and migration of ccRCC cells, and whether this suppression was mediated via inhibition of NFκB and STAT3 activity.
Tissue factor pathway inhibitor-2 (TFPI-2), a serine protease inhibitor abundant in the extracellular matrix, is expressed in high amounts in low-grade, non-invasive glioma cells but in low amounts in high-grade, highly invasive glioma cells. Overexpression of TFPI-2 by highly invasive glioma cells reduces their invasiveness and thus may be useful in cancer therapy. The mechanisms underlying the transcriptional regulation of TFPI-2 are not well elucidated. We previously reported that the -312 to +1 region of TFPI-2 was critical for the minimal, inducible regulation of TFPI-2 in gliomas. This region harbors sites for several transcription factors, including SP1 (-192 to -183 and -135 to -128), AP-1 (-310 to -300, -213 to -204, and -163 to -154), NF-kappaB (-229 to -221), an NF-kappaB-like site (-291 to -281), and Lyf-1 (-260 to -252). Here we transiently transfected low-grade Hs683 glioma cells with mutant constructs to clarify the role of these transcription factors in TFPI-2 regulation. Addition of phorbol 12-myristate 13-acetate, 1,2-diacyl-sn-glycerol, IFN-gamma, or IFN-alpha induced the expression of TFPI-2 wild-type promoter construct as well as TFPI-2 protein and mRNA in Hs683 cells. Mutations at either of two AP-1 sites (-310 to -300 and -163 to -154) or either of two SP1 sites (-192 to -183 and -135 to -128) resulted in reduced TFPI-2 activity, regardless of the presence of stimulator compounds, and reduction in DNA-protein binding (by electrophoretic mobility shift assay).
