Targeting Extracellular Matrix Remodeling Restores BRAF Inhibitor Sensitivity in BRAFi-resistant Melanoma.

PURPOSE: The extracellular matrix (ECM) is an intriguing, yet understudied component of therapy resistance. Here, we investigated the role of ECM remodeling by the collagenase, MT1-MMP, in conferring resistance of v-Raf murine sarcoma viral oncogene homolog B1 (BRAF)-mutant melanoma to BRAF inhibitor (BRAFi) therapy. EXPERIMENTAL DESIGN: Publicly available RNA-sequencing data and reverse phase protein array were used to determine the relevance of MT1-MMP upregulation in BRAFi-resistant melanoma in patients, patient-derived xenografts, and cell line-derived tumors. Short hairpin RNA (shRNA)-mediated knockdown of MT1-MMP, inhibition via the selective MT1-MMP/MMP2 inhibitor, ND322, or overexpression of MT1-MMP was used to assess the role of MT1-MMP in mediating resistance to BRAFi. RESULTS: MT1-MMP was consistently upregulated in posttreatment tumor samples derived from patients upon disease progression and in melanoma xenografts and cell lines that acquired resistance to BRAFi. shRNA- or ND322-mediated inhibition of MT1-MMP synergized with BRAFi leading to resensitization of resistant cells and tumors to BRAFi. The resistant phenotype depends on the ability of cells to cleave the ECM. Resistant cells seeded in MT1-MMP uncleavable matrixes were resensitized to BRAFi similarly to MT1-MMP inhibition. This is due to the inability of cells to activate integrinß1 (ITGB1)/FAK signaling, as restoration of ITGB1 activity is sufficient to maintain resistance to BRAFi in the context of MT1-MMP inhibition. Finally, the increase in MT1-MMP in BRAFi-resistant cells is TGFß dependent, as inhibition of TGFß receptors I/II dampens MT1-MMP overexpression and restores sensitivity to BRAF inhibition. CONCLUSIONS: BRAF inhibition results in a selective pressure toward higher expression of MT1-MMP. MT1-MMP is pivotal to an ECM-based signaling pathway that confers resistance to BRAFi therapy.

Author Correction: Early Cellular Responses of Prostate Carcinoma Cells to Sepantronium Bromide (YM155) Involve Suppression of mTORC1 by AMPK.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

JAB1/COPS5 is a putative oncogene that controls critical oncoproteins deregulated in prostate cancer.

Recent evidence support that the c-Jun activation domain-binding protein 1 (JAB1)/COPS5 has an oncogenic function in various tissues. We show that JAB1 amplification in human prostate cancer (PCa) correlates with reduced overall survival and disease-free progression. Immunohistochemical staining shows enhanced expression of JAB1 in the cytoplasmic compartment of PCa cells compared to the normal prostate epithelium, indicating the activity/function of JAB1 is altered in PCa. To test the function of JAB1 in PCa, we efficiently silenced JAB1 expression using four unique shRNAs in three PCa cell lines (LNCaP, C4-2, and PC-3) and an immortalized prostate epithelial cell line, RWPE-1. Our data clearly show that silencing JAB1 robustly suppresses the growth of PCa cells, but not RWPE-1 cells, suggesting that PCa cells become addicted to JAB1. To study the potential mechanism by which JAB1 controls PCa growth, we profiled gene expression changes by whole transcriptome microarray analysis of C4-2 cells silenced for JAB1 using a pool of 3 shRNAs compared to scrambled shRNA control. We identified 1268 gene changes ≥1.5 fold by silencing JAB1 in C4-2. Western blot confirmation and bioinformatics pathway analyses support that PCa cells become addicted to JAB1 through controlling the following signaling pathways: cell cycle, p53 signaling, DNA replication, TGF-ß/BMP, MAPK, TNF, and steroid hormone biosynthesis. We propose that UGT2B28, UGT2B10, UGT2B11, Skp2, EZH2, MDM2, BIRC5 (Survivin), UBE2C, and Smads 1/5/8, which are all associated with the abovementioned key oncogenic pathways, may play critical roles in the putative oncogenic function of JAB1 in PCa.

Early Cellular Responses of Prostate Carcinoma Cells to Sepantronium Bromide (YM155) Involve Suppression of mTORC1 by AMPK.

The imidazolium compound YM155, first discovered as a potent inhibitor of Survivin, effectively kills many carcinomas in preclinical models. However, the upstream signaling mechanism triggered by YM155 remains unclear. Here we studied early signaling responses in vitro in prostate and renal cancer cell lines in a dose-dependent manner. We found that YM155 rapidly activates the retinoblastoma protein, correlating with the loss of expression of all three Cyclin Ds. Using Western blot, various selective chemical inhibitors and q-PCR, we show that YM155-mediated decrease in protein levels of Cyclin Ds, Survivin and Mcl-1 is independent of transcription or proteasomal control mechanisms. Moreover, we provide the first evidence that YM155 changes the phosphorylation status of known mTOR-target proteins involved in translational control, namely ribosomal protein S6 (rS6) and 4E-BP1. Our data support that YM155 achieves this by blocking mTORC1 via the phosphorylation of Raptor at S792 through activated AMPKα (T172). Furthermore, we also used a polysome profile, supporting that YM155 markedly suppresses cap-dependent translation of mRNAs which include Survivin, Cyclin D1 and Mcl-1. We provide the first evidence that YM155 functions as a potent activator of AMPKα, a robust suppressor of mTORC1 and an attenuator of global protein synthesis.

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.

Inhibiting Notch1 enhances immunotherapy efficacy in melanoma by preventing Notch1 dependent immune suppressive properties.

We have previously shown that Notch1 plays a critical role in modulating melanoma tumor cell growth and survival. Here we show that Notch1 also contributes to an immune-suppressive tumor microenvironment (TME). Notch1 inhibition reduces immune suppressive cells (i.e. MDSCs and Tregs) while allowing the recruitment of functional CD8(+) T cells, leading to a decrease in the Tregs/CD8(+) ratio, a key parameter in assessing positive responses to immune-checkpoint inhibitors. Inhibition of Notch1 improves the antitumor activity of nivolumab and ipilimumab, particularly when given in combination. Mechanistically, tumor-associated Notch1 regulates the expression of several chemokines involved in MDSCs and Tregs recruitment. Among them, CCL5, IL6 and IL8, or MIP2 in mouse, were consistently reduced by Notch1 depletion in several human and mouse melanoma cell lines. Notch1 controls the transcription of IL8 and IL6; and the secretion of CCL5 likely by inhibiting the expression of SNAP23, a member of the SNAREs family of proteins involved in cell exocytosis. Inhibition of SNAP23 decreases CCL5 secretion similarly to Notch1 inhibition. Hence, targeting Notch1 would affect both melanoma intrinsic growth/survival properties, and provide an immune-responsive TME, thus improving immune therapy efficacy.