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Gemcitabine induced TIMP-1 attenuates therapy response and promotes tumour growth and liver metastasis in pancreatic cancer

ABSTRACT: Gemcitabine constitutes one of the backbones for chemotherapy treatment in pancreatic ductal adenocarcinoma (PDAC) but patients often show poor or complete lack of response to this agent. Molecular markers downstream of Gemcitabine treatment in pre-clinical models may provide an insight into resistance mechanisms. We identified potential secretory biomarkers of Gemcitabine resistance (response) in the transgenic KRasG12D; Trp53R172H; Pdx-1 Cre (KPC) mouse model of PDAC using cytokine arrays. We validated the oncogenic role of the cytokine tissue inhibitor of matrix metalloproteinases 1 (TIMP-1) in primary pancreatic tumours and metastasis using both in vitro techniques and animal models. We identified potential pathways affected downstream of TIMP-1 using the Illumina Human H12 array. Our findings were validated in both primary and metastatic models of pancreatic cancer. Gemcitabine increases inflammatory cytokines including TIMP-1 in the KPC mouse model. TIMP-1 is upregulated in patients with pancreatic intraepithelial neoplasias grade 3 and PDAC lesions relative to matched normal pancreatic tissue. Additionally, we demonstrate that TIMP-1 plays a role in tumour proliferation and angiogenesis, while inhibition resensitises to gemcitabine and radiotherapy. Strikingly, serum TIMP-1 levels support the formation of liver metastasis through the recruitment of immunosuppressive cell populations, such as CD11b+Gr1+ myeloid cells and CD4+CD25+FOXP3+ Tregs to the hepatic microenvironment. Gemcitabine treatment results in upregulation of the pro-tumourigenic/pro-metastatic cytokine TIMP-1, which partially explains the therapeutic resistance and poor responses to this therapy in PDAC. Our study provides a rationale for the development and testing of TIMP-1 specific inhibitors in addition to chemo/radiotherapy.

ORGANISM(S): Homo sapiens

PROVIDER: GSE94891 | GEO | 2017/08/08

SECONDARY ACCESSION(S): PRJNA374729

REPOSITORIES: GEO

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Project description:Background & Aims: Pancreatic ductal adenocarcinomas (PDAC) are characterized by fibrosis and an abundance of cancer-associated fibroblasts (CAFs). We investigated strategies to disrupt interactions among CAFs, the immune system, and cancer cells, focusing on adhesion molecule cadherin 11 (CDH11), which has been associated with other fibrotic disorders and is expressed by activated fibroblasts. Methods: We compared levels of CDH11 mRNA in human pancreatitis and pancreatic cancer tissues and cells, compared with normal pancreas, and measured levels of CDH11 protein in human and mouse pancreatic lesions and normal tissues. We crossed p48-Cre;LSL-KrasG12D/+;LSL-Trp53R172H/+ (KPC) mice with CDH11-knockout mice and measured survival times of offspring. Pancreata were collected and analyzed by histology, immunohistochemistry, and (single-cell) RNA sequencing; RNA and proteins were identified by imaging mass cytometry. Some mice were given injections of PD1 antibody or gemcitabine and survival was monitored. Pancreatic cancer cells from KPC mice were subcutaneously injected into Cdh11+/+ and Cdh11–/– mice and tumor growth was monitored. Pancreatic cancer cells (mT3) from KPC mice (C57BL/6), were subcutaneously injected into Cdh11+/+ (C57BL/6J) mice and mice were given injections of antibody against CDH11, gemcitabine, or small molecule inhibitor of CDH11 (SD133) and tumor growth was monitored. Results: Levels of CDH11 mRNA and protein were significantly higher in CAFs than in pancreatic cancer epithelial cells, human or mouse pancreatic cancer cell lines, or immune cells. KPC/Cdh11+/– and KPC/Cdh11–/– mice survived significantly longer than KPC/Cdh11+/+ mice. Markers of stromal activation entirely surrounded pancreatic intraepithelial neoplasias in KPC/Cdh11+/+ mice and incompletely in KPC/Cdh11+/– and KPC/Cdh11–/– mice, whose lesions also contained fewer FOXP3+ cells in the tumor center. Compared with pancreatic tumors in KPC/Cdh11+/+ mice, tumors of KPC/Cdh1+/– mice had increased markers of antigen processing and presentation; more lymphocytes and cytokines associated with lymphocyte infiltration; decreased extracellular matrix components; and reductions in markers and cytokines associated with immunosuppression. Administration of the PD1 antibody did not prolong survival of KPC mice with 0, 1, or 2 alleles of Cdh11. Gemcitabine extended survival only of KPC/Cdh11+/– and KPC/Cdh11–/– mice or reduced subcutaneous tumor growth in mT3 engrafted Cdh11+/+ mice given in combination with the CDH11 antibody. A small molecule inhibitor of CDH11 reduced growth of pre-established mT3 subcutaneous tumors only if T and B cells were present in mice. Conclusions: Knockout or inhibition of CDH11, which is expressed by CAFs in the pancreatic tumor stroma, reduces growth of pancreatic tumors, increases their response to gemcitabine, and significantly extends survival of mice. CDH11 promotes immunosuppression and extracellular matrix deposition, and might be developed as a therapeutic target for pancreatic cancer.

Project description:Pancreatic ductal adenocarcinoma (PDAC) is a nearly uniformly lethal malignancy, with most patients facing an adverse clinical outcome. Given the pivotal role of aberrant Notch signaling in the initiation and progression of PDAC, we investigated the effect of MRK-003, a potent and selective γ-secretase inhibitor, in preclinical PDAC models. We used a panel of human PDAC cell lines, as well as patient-derived PDAC xenografts, to determine whether pharmacological targeting of the Notch pathway could inhibit pancreatic tumor growth and potentiate gemcitabine sensitivity. In vitro, MRK-003 treatment downregulated the canonical Notch target gene Hes-1, significantly inhibited anchorage independent growth, and reduced the subset of CD44+CD24+ and aldehyde dehydrogenase (ALDH)+ cells that have been attributed with tumor initiating capacity. Ex vivo pretreatment of PDAC cells with MRK-003 in culture significantly inhibited the subsequent engraftment in immunocompromised mice. In vivo, MRK-003 monotherapy significantly blocked tumor growth in 5 of 9 (56%) patient-derived PDAC xenografts. Moreover, a combination of MRK-003 and gemcitabine showed enhanced antitumor effects compared to gemcitabine alone in 4 of 9 (44%) PDAC xenografts. Baseline gene expression analysis of the treated xenografts indicated that upregulation of nuclear factor kappa B (NFκB) pathway components was associated with the sensitivity to single MRK-003, while upregulation in B-cell receptor (BCR) signaling and nuclear factor erythroid-derived 2-like 2 (NRF2) pathway correlated with response to the combination of MRK-003 with gemcitabine. The preclinical findings presented here provide further rationale for small molecule inhibition of Notch signaling as a therapeutic strategy in PDAC. Pancreatic ductal adenocarcinoma xenografts were grown in Athymic Nude-Foxn1nu mice. RNA was extracted and profiled in Affymetrix platform to identify genes correlating with sensitivity to MRK-003

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Gemcitabine induced TIMP-1 attenuates therapy response and promotes tumour growth and liver metastasis in pancreatic cancer

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