Romana Moench

TLR7 and TLR8 expression increases tumor cell proliferation and promotes chemoresistance in human pancreatic cancer

Chronic inflammation as an important epigenetic and environmental factor for putative tumorigenesis and tumor progression may be associated with specific activation of Toll-like receptors (TLR). Recently, carcinogenesis has been suggested to be dependent on TLR7 signaling. In the present study, we determined the role of both TLR7 and TLR8 expression and signaling in tumor cell proliferation and chemoresistance in pancreatic cancer. Expression of TLR7/TLR8 in UICC stage I–IV pancreatic cancer, chronic pancreatitis, normal pancreatic tissue and human pancreatic (PANC1) cancer cell line was examined. For in vitro/in vivo studies TLR7/TLR8 overexpressing PANC1 cell lines were generated and analyzed for effects of (un-)stimulated TLR expression on tumor cell proliferation and chemoresistance. TLR expression was increased in pancreatic cancer, with stage-dependent upregulation in advanced tumors, compared to earlier stages and chronic pancreatitis. Stimulation of TLR7/TLR8 overexpressing PANC1 cells resulted in elevated NF-κB and COX-2 expression, increased cancer cell proliferation and reduced chemosensitivity. More importantly, TLR7/TLR8 expression increased tumor growth in vivo. Our data demonstrate a stage-dependent upregulation of both TLR7 and TLR8 expression in pancreatic cancer. Functional analysis in human pancreatic cancer cells point to a significant role of both TLRs in chronic inflammation-mediated TLR7/TLR8 signaling leading to tumor cell proliferation and chemoresistance.

Exclusive inhibition of PI3K/Akt/mTOR signaling is not sufficient to prevent PDGF-mediated effects on glycolysis and proliferation in colorectal cancer.

Platelet-derived growth factor (PDGF) and signaling via its receptors plays a crucial role in tumor cell proliferation and thus may represent an attractive target besides VEGF/EGFR-based antibody therapies. In this study we analyzed the influence of PDGF in colorectal cancer. PDGF was expressed intensively in early and even more intensively in late stage primary CRCs. Like VEGF, PDGF enhanced human colon cancer proliferation, and increased oxidative glycolytic activity, and activated HIF1α and c-Myc in vitro. PDGF activated the PI3K/Akt/mTOR pathway while leaving MAPK signaling untouched. Further dissection showed that inhibition of Akt strongly impeded cancer cell growth while inhibition of PI3K did not. MAPK analysis suggested an inhibitory crosstalk between both pathways, thus explaining the different effects of the Akt and PI3K inhibitors on cancer cell proliferation. PDGF stimulates colon cancer cell proliferation, and prevents inhibitor induced apoptosis, resulting in tumor growth. Therefore inhibition of PDGF signaling seems to be a promising target in colorectal cancer therapy. However, due to the multifaceted nature of the intracellular PDGF signaling, careful intervention strategies are needed when looking into specific signaling pathways like PI3K/Akt/mTOR and MAPK.

Toll Like Receptor 2, 4, and 9 Signaling Promotes Autoregulative Tumor Cell Growth and VEGF/PDGF Expression in Human Pancreatic Cancer

Toll like receptor (TLR) signaling has been suggested to play an important role in the inflammatory microenvironment of solid tumors and through this inflammation-mediated tumor growth. Here, we studied the role of tumor cells in their process of self-maintaining TLR expression independent of inflammatory cells and cytokine milieu for autoregulative tumor growth signaling in pancreatic cancer. We analyzed the expression of TLR2, -4, and -9 in primary human cancers and their impact on tumor growth via induced activation in several established pancreatic cancers. TLR-stimulated pancreatic cancer cells were specifically investigated for activated signaling pathways of VEGF/PDGF and anti-apoptotic Bcl-xL expression as well as tumor cell growth. The primary pancreatic cancers and cell lines expressed TLR2, -4, and -9. TLR-specific stimulation resulted in activated MAP-kinase signaling, most likely via autoregulative stimulation of demonstrated TLR-induced VEGF and PDGF expression. Moreover, TLR activation prompted the expression of Bcl-xL and has been demonstrated for the first time to induce tumor cell proliferation in pancreatic cancer. These findings strongly suggest that pancreatic cancer cells use specific Toll like receptor signaling to promote tumor cell proliferation and emphasize the particular role of TLR2, -4, and -9 in this autoregulative process of tumor cell activation and proliferation in pancreatic cancer.

Upregulated Heat Shock Proteins After Hyperthermic Chemotherapy Point to Induced Cell Survival Mechanisms in Affected Tumor Cells From Peritoneal Carcinomatosis

In patients with peritoneal carcinomatosis cytoreductive surgery combined with hyperthermic intraperitoneal chemotherapy (HIPEC) represents a promising treatment strategy. Here, we studied the role of hyperthermic chemotherapy on heat shock protein (HSP) expression and induction of tumor cell death and survival. HSP27, HSP70, and HSP90 combined with effects on tumor cell proliferation and chemosensitivity were analyzed in human colon cancer. Hyperthermic chemotherapy resulted in significant HSP27/HSP70 and HSP90 gene/protein overexpression in analyzed HT-29/SW480/SW620 colon cancer cells and peritoneal metastases from patients displaying amplified expression of proliferation markers, proliferating cell nuclear antigen and antiapoptotic protein Bcl-xL. Moreover, functionally increased chemoresistance against 5-fluorouracil/mitomycin C and oxaliplatin after hyperthermic chemotherapy points to induced survival mechanisms in cancer cells. In conclusion, the results indicate that intracellular HSP-associated antiapoptotic and proliferative effects after hyperthermic chemotherapy negatively influence beneficial effects of hyperthermic chemotherapy-induced cell death. Therefore, blocking HSPs could be a promising strategy to further improve the rate of tumor cell death and outcome of patients undergoing HIPEC therapy.

Clinical Significance of Disseminated Pluripotent Tumor Cell SignatureExpression in the Bone Marrow from Patients with Colorectal Cancer

Purpose: Disseminated tumor cells (DTCs) are critically involved in tumor relapse and survival in several invasive tumors. We previously showed that the ATP-binding cassette (ABC) transporter, ABCB5, is a chemoresistance mediator expressed on specific cell subsets in colorectal cancer (CRC) and other malignancies. This study evaluated the molecular signature expression and its clinical relevance of DTCs in bone marrow from patients with colon cancer. Methods: This study included 49 consecutive patients (UICC stage I-IV) that underwent curatively intended or palliative surgery for CRC. We analyzed cells from bone marrow aspirates obtained before surgery and derived from patients that had completed minimally a 5-year follow-up. The gene expression of ABCB5 in comparison to CD133 (molecule for identifying cancer initiating cells), Lgr5 (an intestinal stem cell marker) as well as Cytokeratin (CK) 20 (terminally differentiated tumor cells of epithelial origin) in these cells was evaluated. Results: Bone marrow analysis showed differential expression between the analyzed genes. ABCB5 and Lgr5 and to lesser extent CD133 and CK20 genes were significantly expressed in the analyzed cells from bone marrow aspirates while only ABCB5 and Lgr5 were significantly negative associated with tumor progress and overall survival. Conclusion: Overexpression of ABCB5 and Lgr5 in bone marrow negatively influenced patient survival pointing to a specific chemo resistant and pluripotent cell subgroup of DTCs in the bone marrow. ABCB5 like Lgr5 positive cells seem to be involved in limited tumor related patient survival, suggesting that ABCB5- and Lgr5-positive cells may be relevant for specific clinical intervention strategies.

Abstract 1237: PDGF induces cell growth and changes in glucose metabolism in colon cancer in the absence of PDGF receptors

Background: The Platelet derived growth factor (PDGF) and its receptors play a major role in inducing proliferation, migration, and angiogenesis by activating intracellular PI3K/Akt/mTOR signaling events in different solid tumors and therefore represent attractive targets in tumor therapy. Recently we showed a PDGF-induced activation of metabolism and proliferation in HT29 colon cancer cells in the absence of specific PDGF receptors. The aim of this study was to analyze PDGFR and VEGFR expression and possible alternative PDGF binding partners in colorectal cancer (CRC). Methods: Human HT29 colon cancer cells were cultured and stimulated with PDGF in a time-dependent manner. Additionally, VEGFR2 and EGFR inhibition was performed to analyze alternative PDGF signaling events. Whole cell or RNA extracts were analyzed by Western Blot and RT-q-PCR for receptors and PI3K/Akt/mTOR signaling. To investigate the effects of specific receptor inhibition on proliferation, MTS proliferation assays were performed. Moreover, mRNA levels of PDGFR and VEGFR in tumors from patients with CRC were analyzed by RT-qPCR. Results: Human UICC stage I-IV tumors exhibited a significantly increased PDGFRβ and VEGFR1,2 gene expression. As observed previously, HT29 colon cancer cells showed only positivity for VEGFR1,2, but no PDGFR protein expression. Despite that, stimulation with PDGF resulted in increased proliferation and metabolic changes. In contrast, Caco-2 and SW480 colon cancer cells showed PDGFR and VEGFR expression on protein level. Interestingly, PDGF stimulation increased VEGFR1 and 2 gene expression in HT29 colon cancer cells and secondly inhibition of VEGFR2 and EGFR showed alterations in proliferation and Akt signaling in PDGF stimulated cancer cells. Conclusion: Despite the absence of PDGF receptors in HT29 colon cancer cells, PDGF induced proliferating and metabolic effects in the tumor cells, suggesting an alternative binding partner on the tumor cell surface. Further investigation of the alternative PDGF binding partners could be of clinical relevance in CRC. Note: This abstract was not presented at the meeting. Citation Format: Romana Moench, Tanja Grimmig, Christoph-Thomas Germer, Ana Maria Waaga-Gasser, Martin Gasser. PDGF induces cell growth and changes in glucose metabolism in colon cancer in the absence of PDGF receptors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1237. doi:10.1158/1538-7445.AM2017-1237

Abstract 4879: Cancer cell-mediated signaling of TLR 2, 4, and 9 causes activation of PI3K/Akt/mTOR pathway and induces tumor cell proliferation in pancreatic cancer

Background: Toll like receptor (TLR) ligands are in clinical use for the immunotherapy of different cancers. They are supposed to induce an inflammatory immune response against the tumor. Interestingly, several studies showed that TLRs were expressed by cancer cells in different tumor entities and that their activation can contribute to an inflammatory microenvironment. Yet, for pancreatic cancer, TLR expression and its impact in tumor cells is only poorly understood. Therefore this study analyzed the influence of TLR2, 4, and 9 expression and activation on tumor cell signaling and proliferation in pancreatic cancer. Methods: The expression of TLR 2, 4, and 9 was analyzed in vitro in several established as well as primary human pancreatic cancer cell lines by qRT-PCR and Western Blot. TLR stimulation was then performed in BxPC-3, MIA Paca2, and PacaDD135 cells using the TLR ligands oligodeoxyribonucleotide2006 (ODN2006), lipoteichonic acid of Staphylococcus aureus (LTA-SA) and High-Mobility Group Box 1 (HMGB1). Expression of MyD88 and pAkt was then analyzed by Western Blot. Functional analysis on proliferation (ATP assay) and cytokine expression (Luminex) was additionally performed. Results: Expression of TLR2, 4 and/or 9 was demonstrated in all investigated human pancreatic cancer cell lines. Receptor activation by single or combined use of TLR ligands resulted in increased MyD88 and pAkt (Ser473) expression. Additionally, up-regulated anti-apoptotic protein Bcl-2 expression was found in stimulated cells, but not in the unstimulated cells. Furthermore, TLR activation resulted in the production of the interleukin(IL)-6, IL-8 and tumor necrosis factor a (TNF-a). Interestingly, tumor cell proliferation was increased within 24 and 48 hours of stimulation. Conclusion: Our results demonstrate TLR2, 4 and/or 9 expression in all human pancreatic cancer cell lines. Additionally, our findings on TLR activation suggest chronic inflammation-mediated TLR signaling to negatively influence tumor cell apoptosis and to shift the cytokine release in pancreatic cancer towards an inflammatory microenvironment. These findings emphasize the particular role of TLR2, 4 and 9 and their activation in pancreatic cancer, outlining their relevance as potential targets for cancer therapy. Citation Format: Jennifer Kreckel, Tanja Grimmig, Romana Moench, Christoph T. Germer, Martin Gasser, Ana Maria Waaga-Gasser. Cancer cell-mediated signaling of TLR 2, 4, and 9 causes activation of PI3K/Akt/mTOR pathway and induces tumor cell proliferation in pancreatic cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4879.

Abstract 2945: Cell stress during HIPEC causes heat shock protein induction and reduced chemosensitivity in human colon cancer

Background: Hyperthermic intraperitoneal chemotherapy (HIPEC) is a promising procedure for the treatment of peritoneal carcinomatosis (PC). Heat shock proteins (HSPs) and other proteins involved in cellular repair mechanisms seem to induce cytoprotective processes during HIPEC therapy. Therefore, the aim of this study was to analyze the effects of HIPEC-related conditions on tumor cell proliferation and the expression of HSPs in human colon cancer. Methods: Human colon cancer cell lines HT29, SW480 and SW620 were exposed to different temperatures (37°C, 41°C, and 43°C) as well as defined cytostatic agents (Oxaliplatin, Mitomycin C, and 5-Fluorouracil). After cellular regeneration (30 min, 24 h, 48 h and 72 h) RNA isolation and whole cell extraction was performed. Gene and protein expression analysis of HSP27, 70, 72 and 90 as well as PCNA, Ki-67, BCl-2 and BCl-Xl were carried out using RT-qPCR and Western blot. Additionally, MTS cell proliferation assays were performed 24 h, 48 h, 72 h and 96 h post treatment. Moreover, AnnexinV apoptosis assays were conducted. Results: All colon cancer cells exposed to hyperthermic conditions showed initially up-regulated HSP gene expression. Highest expression was found after exposure to 43°C. Combined cytostatic and hyperthermic treatment demonstrated additional increase in HSP27 expression and in other HSPs to a lesser degree. Tumor cells exposed to cytostatic agents showed overall higher HSP gene expression compared to cells without chemotoxic treatment. Similar effects were detected for the expression of the proliferation marker PCNA and anti-apoptotic protein BCl-Xl. Apoptosis assay demonstrated decreased numbers of apoptotic cells at 43°C compared to normothermia. Additionally, proliferation assays revealed reduced chemosensitivity in cells treated with hyperthermia. Conclusion: Desired effects of hyperthermia used in HIPEC therapy to achieve anti-proliferative and apoptosis inducing effects seem to be negatively influenced by cell stress mediated repair mechanisms in colon cancer. Our in vitro findings suggest analyzed HSPs to be significantly involved in this hyperthermia and chemotoxicity mediated cellular repair mechanisms. While initial increase in HSP expression can counteract cytotoxic effects during HIPEC therapy their prolonged expression may promote lasting resistance to cellular stress. Citation Format: Tanja Grimmig, Kerstin Kloos, Rebecca Thumm, Romana Moench, Christoph T. Germer, Ana Maria Waaga-Gasser, Martin Gasser. Cell stress during HIPEC causes heat shock protein induction and reduced chemosensitivity in human colon cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2945.

Abstract 3917: PDGF induces cell growth and glycolysis in colon cancer

The platelet derived growth factor (PDGF) plays an important role in several solid tumors. Involved in cell migration and proliferation primarily of stromal cells in cancers PDGF represents a key target in cancer therapy. The aim of this study was to analyze the specific role of PDGF on tumor cell proliferation and metabolism in colorectal cancer (CRC). The human colon cancer cell line HT-29 was cultured and stimulated time-dependently with PDGF. Additionally, inhibition of the PI3k/Akt/mTOR-pathway was performed simultaneously to PDGF stimulation. Whole cell and RNA extracts were analyzed by Western Blot and RT-q-PCR for the PI3k/Akt/mTOR-pathway and components of cellular metabolism. To investigate the effects of PDGF on proliferation MTS assays were performed. Additionally, mRNA levels of PDGF and metabolic factors in tumors from patients with CRC were analyzed by RT-qPCR. PDGF stimulation resulted in increased HT29 cell proliferation compared to untreated controls. Blocking of Akt resulted in inhibition of pS6, activation of Retinoblastoma (Rb) and reduced tumor cell growth. Additionally, under stimulation a higher glycolytic rate was observed while oxygen consumption remained unaltered. Investigated tumors from patients with CRC showed a stage-dependent increase in PDGF expression and a boosted glycolytic rate. The cytokine PDGF induced proliferation accompanied with an altered glycolysis in HT-29 colon cancer. An increased glycolysis and tumor cell proliferation support accelerated cell proliferation and tumor growth. The growth reducing effect of Akt inhibition indicates the PI3K/Akt/mTOR-pathway to play a crucial role in CRC progression and therefore could be an important target in cancer therapy. Note: This abstract was not presented at the meeting. Citation Format: Romana Moench, Vinicius Kannen, Tanja Grimmig, Christoph T. Germer, Martin Gasser, Ana Maria Waaga-Gasser. PDGF induces cell growth and glycolysis in colon cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3917. doi:10.1158/1538-7445.AM2015-3917

Abstract 1244: Hyperthermia and chemotherapy mediated effects on tumor cell proliferation and heat shock protein expression in human colon cancer

In patients with peritoneal carcinomatosis (PC) of gastrointestinal cancer hyperthermic intraperitoneal chemotherapy (HIPEC) represents a promising treatment option integrated into multimodal concepts. Heat shock proteins (HSP) play a major role in cellular stress response conferring increased resistance in tumor cells. Therefore we analyzed HSP expression profiles in tumor cells exposed to hyperthermic and chemotherapeutic stress. To mimic HIPEC-like conditions HT29 colon cancer cells were exposed to varying hyperthermic conditions for 60 min with additional 5-fluorouracil (5-FU) treatment. HSP expression was analyzed 30 min, 24 h, 48 h and 72 h after treatment using Western Blot and RT-qPCR. Untreated cells cultivated at 37°C served as controls. Additionally, effects on tumor cell proliferation were determined 24 h, 48 h and 72 h after treatment by MTS-assay. Hyperthermia caused temperature dependent upregulation of HSP27, HSP70, HSP72, and HSP90 gene and protein expression that was further increased by additional cytotoxic 5-FU treatment. 5-FU initiated progressive rise in HSP gene expression up to 72 h after 1 h exposure at normothermia as well as under hyperthermia. After isolated hyperthermia tumor cell proliferation was recovered at 72 h. Antiproliferative effects of 5-FU could not be enhanced by further increased hyperthermia. Antiproliferative effects of hyperthermia induced during HIPEC therapy seem to be negatively influenced by highly conserved HSP mechanisms. Our findings suggest that HSP27, HSP70/72, and HSP90 are significantly involved in hyperthermia and chemotoxicity mediated cell stress repair mechanisms. While initial increase in HSP expression can counteract cytotoxic effects during HIPEC therapy long-term elevated expression levels may cause lasting resistance to cellular stress. Note: This abstract was not presented at the meeting. Citation Format: Tanja Grimmig, Rebeca Thumm, Romana Moench, Eva M. Moll, Christoph T. Germer, Ana Maria Waaga-Gasser, Martin Gasser. Hyperthermia and chemotherapy mediated effects on tumor cell proliferation and heat shock protein expression in human colon cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1244. doi:10.1158/1538-7445.AM2015-1244