MT1-MMP-dependent ECM processing regulates laminB1 stability and mediates replication fork restart.

Radiotherapy remains a mainstay of treatment for a majority of cancer patients. We have previously shown that the membrane bound matrix metalloproteinase MT1-MMP confers radio- and chemotherapy resistance to breast cancer via processing of the ECM and activation of integrinß1/FAK signaling. Here, we further discovered that the nuclear envelope protein laminB1 is a potential target of integrinß1/FAK. FAK interacts with laminB1 contributing to its stability. Stable laminB1 is found at replication forks (RFs) where it is likely to allow the proper positioning of RF protection factors, thus preventing RF degradation. Indeed, restoration of laminB1 expression rescues replication fork stalling and collapse that occurs upon MT1-MMP inhibition, and reduces DNA damage in breast cancer cells. Together, these data highlight a novel mechanism of laminB1 stability and replication fork restart via MT1-MMP dependent extracelluar matrix remodeling.

Identification of RUNX1T1 as a potential epigenetic modifier in small-cell lung cancer.

Small-cell lung cancer (SCLC) can be subgrouped into common 'pure' and rare 'combined' SCLC (c-SCLC). c-SCLC features a mixed tumor histology of both SCLC and non-small-cell lung cancer (NSCLC). We performed targeted exome sequencing on 90 patients with SCLC, including two with c-SCLC, and discovered RUNX1T1 amplification specific to small cell tumors of both patients with c-SCLC, but in only 2 of 88 'pure' SCLC patients. RUNX1T1 was first identified in the fusion transcript AML1/ETO, which occurs in 12%-15% of acute myelogenous leukemia (AML). We further show higher expression of RUNX1T1 in the SCLC component of another c-SCLC tumor by in situ hybridization. RUNX1T1 expression was enriched in SCLC compared with all other cancers, including NSCLC, in both cell lines and tumor specimens, as shown by mRNA level and western blotting. Transcriptomic analysis of hallmark genes decreased by stable RUNX1T1 overexpression revealed a significant change in E2F targets. Validation experiments in multiple lung cancer cell lines showed that RUNX1T1 overexpression consistently decreased CDKN1A (p21) expression and increased E2F transcriptional activity, which is commonly altered in SCLC. Chromatin immunoprecipitation (ChIP) in these overexpressing cells demonstrated that RUNX1T1 interacts with the CDKN1A (p21) promoter region, which displayed parallel reductions in histone 3 acetylation. Furthermore, reduced p21 expression could be dramatically restored by HDAC inhibition using Trichostatin A. Reanalysis of ChIP-seq data in Kasumi-1 AML cells showed that knockdown of the RUNX1T1 fusion protein was associated with increased global acetylation, including the CDKN1A (p21) promoter. Thus, our study identifies RUNX1T1 as a biomarker and potential epigenetic regulator of SCLC.

The membrane tethered matrix metalloproteinase MT1-MMP triggers an outside-in DNA damage response that impacts chemo- and radiotherapy responses of breast cancer.

Breast cancer is the second leading cause of death among women in the US. Targeted therapies exist, however resistance is common and patients resort to chemotherapy. Chemotherapy is also a main treatment for triple negative breast cancer (TNBC) patients; while radiation is delivered to patients with advanced disease to counteract metastasis. Yet, resistance to both chemo- and radiotherapy is still frequent, highlighting a need to provide novel sensitizers. We discovered that MT1-MMP modulates DNA damage responses (DDR) in breast cancer. MT1-MMP expression inversely correlates to chemotherapy response of breast cancer patients. Inhibition of MT1-MMP sensitizes TNBC cells to IR and doxorubicin in vitro, and in vivo in an orthotopic breast cancer model. Specifically, depletion of MT1-MMP causes stalling of replication forks and Double Strand Breaks (DBSs), leading to increased sensitivity to additional genotoxic stresses. These effects are mediated by integrinß1, as a constitutive active integrinß1 reverts replication defects and protects cells depleted of MT1-MMP from IR and chemotherapy. These data highlight a novel DNA damage response triggered by MT1-MMP-integrinß1 and provide a new point of therapeutic targeting that may improve breast cancer patient outcomes.

Altered glutamine metabolism in platinum resistant ovarian cancer.

Ovarian cancer is characterized by an increase in cellular energy metabolism, which is predominantly satisfied by glucose and glutamine. Targeting metabolic pathways is an attractive approach to enhance the therapeutic effectiveness and to potentially overcome drug resistance in ovarian cancer. In platinum-sensitive ovarian cancer cell lines the metabolism of both, glucose and glutamine was initially up-regulated in response to platinum treatment. In contrast, platinum-resistant cells revealed a significant dependency on the presence of glutamine, with an upregulated expression of glutamine transporter ASCT2 and glutaminase. This resulted in a higher oxygen consumption rate compared to platinum-sensitive cell lines reflecting the increased dependency of glutamine utilization through the tricarboxylic acid cycle. The important role of glutamine metabolism was confirmed by stable overexpression of glutaminase, which conferred platinum resistance. Conversely, shRNA knockdown of glutaminase in platinum resistant cells resulted in re-sensitization to platinum treatment. Importantly, combining the glutaminase inhibitor BPTES with platinum synergistically inhibited platinum sensitive and resistant ovarian cancers in vitro. Apoptotic induction was significantly increased using platinum together with BPTES compared to either treatment alone. Our findings suggest that targeting glutamine metabolism together with platinum based chemotherapy offers a potential treatment strategy particularly in drug resistant ovarian cancer.