Targeting Discoidin Domain Receptor 1 (DDR1) Signaling and Its Crosstalk with ß1-integrin Emerges as a Key Factor for Breast Cancer Chemosensitization upon Collagen Type 1 Binding.

Collagen type 1 (COL1) is a ubiquitously existing extracellular matrix protein whose high density in breast tissue favors metastasis and chemoresistance. COL1-binding of MDA-MB-231 and MCF-7 breast cancer cells is mainly dependent on ß1-integrins (ITGB1). Here, we elucidate the signaling of chemoresistance in both cell lines and their ITGB1-knockdown mutants and elucidated MAPK pathway to be strongly upregulated upon COL1 binding. Notably, Discoidin Domain Receptor 1 (DDR1) was identified as another important COL1-sensor, which is permanently active but takes over the role of COL1-receptor maintaining MAPK activation in ITGB1-knockdown cells. Consequently, inhibition of DDR1 and ERK1/2 act synergistically, and sensitize the cells for cytostatic treatments using mitoxantrone, or doxorubicin, which was associated with an impaired ABCG2 drug efflux transporter activity. These data favor DDR1 as a promising target for cancer cell sensitization, most likely in combination with MAPK pathway inhibitors to circumvent COL1 induced transporter resistance axis. Since ITGB1-knockdown also induces upregulation of pEGFR in MDA-MB-231 cells, inhibitory approaches including EGFR inhibitors, such as gefitinib appear promising for pharmacological interference. These findings provide evidence for the highly dynamic adaptation of breast cancer cells in maintaining matrix binding to circumvent cytotoxicity and highlight DDR1 signaling as a target for sensitization approaches.

ß1-Integrin binding to collagen type 1 transmits breast cancer cells into chemoresistance by activating ABC efflux transporters.

Molecular interactions of tumor cells with the microenvironment are regarded as onset of chemotherapy resistance, referred to as cell adhesion mediated drug resistance (CAM-DR). Here we elucidate a mechanism of CAM-DR in breast cancer cells in vitro. We show that human MCF-7 and MDA-MB-231 breast cancer cells decrease their sensitivity towards cisplatin, doxorubicin, and mitoxantrone cytotoxicity upon binding to collagen type 1 (COL1) or fibronectin (FN). The intracellular concentrations of doxorubicin and mitoxantrone were decreased upon cell cultivation on COL1, while cellular cisplatin levels remained unaffected. Since doxorubicin and mitoxantrone are transporter substrates, this refers to ATP binding cassette (ABC) efflux transporter activities. The activation of the transporters BCRP, P-gp and MRP1 was shown by fluorescence assays to distinguish the individual input of these transporters to resistance in presence of COL1 and related to their expression levels by western blot. An ABC transporter inhibitor was able to re-sensitize COL1-treated cells for doxorubicin and mitoxantrone toxicity. Antibody-blocking of ß1-integrin (ITGB1) induced sensitization towards the indicated cytostatic drugs by attenuating the increased ABC efflux activity. This refers to a key role of ITGB1 for matrix binding and subsequent transporter activation. A downregulation of α2ß1 integrin following COL1 binding appears as clear indication for the relationship between ITGB1 and ABC transporters in regulating resistance formation, while knockdown of ITGB1 leads to a significant upregulation of all three transporters. Our data provide evidence for a role of CAM-DR in breast cancer via an ITGB1 - transporter axis and offer promising therapeutic targets for cancer sensitization.

Inhibition of the PI3K but not the MEK/ERK pathway sensitizes human glioma cells to alkylating drugs.

BACKGROUND: Intrinsic chemoresistance of glioblastoma (GBM) is frequently owed to activation of the PI3K and MEK/ERK pathways. These signaling cascades are tightly interconnected however the quantitative contribution of both to intrinsic resistance is still not clear. Here, we aimed at determining the activation status of these pathways in human GBM biopsies and cells and investigating the quantitative impact of both pathways to chemoresistance. METHODS: Receptor tyrosine kinase (RTK) pathways in temozolomide (TMZ) treatment naive or TMZ resistant human GBM biopsies and GBM cells were investigated by proteome profiling and immunoblotting of a subset of proteins. Resistance to drugs and RTK pathway inhibitors was assessed by MTT assays. Apoptotic rates were determined by Annexin V staining and DNA damage with comet assays and immunoblotting. RESULTS: We analyzed activation of RTK pathways by proteome profiling of tumor samples of patients which were diagnosed a secondary GBM and underwent surgery and patients which underwent a second surgery after TMZ treatment due to recurrence of the tumor. We observed substantial activation of the PI3K and MEK/ERK pathways in both groups. However, AKT and CREB phosphorylation was reduced in biopsies of resistant tumors while ERK phosphorylation remained unchanged. Subsequent proteome profiling revealed that multiple RTKs and downstream targets are also activated in three GBM cell lines. We then systematically describe a mechanism of resistance of GBM cell lines and human primary GBM cells to the alkylating drugs TMZ and cisplatin. No specific inhibitor of the upstream RTKs sensitized cells to drug treatment. In contrast, we were able to restore sensitivity to TMZ and cisplatin by inhibiting PI3K in all cell lines and in human primary GBM cells. Interestingly, an opposite effect was observed when we inhibited the MEK/ERK signaling cascade with two different inhibitors. CONCLUSIONS: Temozolomide treatment naive and TMZ resistant GBM biopsies show a distinct activation pattern of the MEK/ERK and PI3K signaling cascades indicating a role of these pathways in resistance development. Both pathways are also activated in GBM cell lines, however, only the PI3K pathway seems to play a crucial role in resistance to alkylating agents and might serve as drug target for chemosensitization.

ETS-Transcription Factor ETV1 Regulates Stromal Expansion and Metastasis in Pancreatic Cancer.

BACKGROUND & AIMS: The ETS-transcription factor ETV1 is involved in epithelial-mesenchymal transition during pancreatic development and is induced in mouse pancreatic intraepithelial neoplasia (PanIN) and pancreatic ductal adenocarcinoma (PDAC). We investigated the function of ETV1 in stromal expansion of PDAC and metastasis, as well as its effects on a novel downstream target Sparc, which encodes a matricellular protein found in PDAC stroma that has been associated with invasiveness, metastasis and poor patient outcomes. METHODS: Pancreatic ductal cells were isolated from Pdx1Cre;Kras(G12D/+) mice (PanIN), Pdx1Cre;Kras(G12D/+);p53(fl/+) and Pdx1Cre;Kras(G12D/+);p53(fl/+);Rosa26(YFP) mice (PDAC), and Pdx1Cre;Kras(G12D/+);p53(fl/+);Sparc(-/-) mice. Cells were grown in 3-dimensional organoid culture to analyze morphology, proliferation, and invasion. Human PanIN and PDAC tissues were evaluated for ETV1 expression. Orthotopic pancreatic transplants of ETV1-overexpressing PDAC and respective control cells were performed. RESULTS: ETV1 expression was significantly increased in human PanINs and, even more so, in primary and metastatic PDAC. Analyses of mouse orthotopic xenografts revealed that ETV1 induced significantly larger primary tumors than controls, with significantly increased stromal expansion, ascites and metastases. In 3-dimensional organoids, ETV1 disrupted cyst architecture, induced EMT, and increased invasive capacity. Furthermore, we identified Sparc as a novel functional gene target of Etv1 by luciferase assays, and SPARC and ETV1 proteins co-localized in vivo. Disruption of Sparc abrogates the phenotype of stromal expansion and metastasis found with ETV1 overexpression in vivo. We identified hyaluronan synthase 2 (Has2) as another novel downstream factor of Etv1; that may mediate ETV1's significant expansion of hyaluronic acid in PDAC stroma. Conversely, disruption of Etv1 in PDAC mice (Pdx1Cre;Kras(G12D/+);p53(fl/+);Rosa26(YFP);Cre;Etv1(fl/fl)) reduced levels of SPARC and hyaluronic acid in the stroma. CONCLUSIONS: ETV1 is critical in the desmoplastic stromal expansion and metastatic progression of pancreatic cancer in mice, mediated functionally in part through Sparc and Has2.