Search

MT1-MMP overexpression

Purpose:BRAF and MEK inhibitors (BRAFi and MEKi) are actively used for the treatment of metastatic melanoma in patients with BRAFV600E mutation in their tumors. However, the development of resistance to BRAFi and MEKi remains a difficult clinical challenge with limited therapeutic options available to these patients. In this study, we investigated the mechanism and potential therapeutic utility of combination BRAFi and adoptive T-cell therapy (ACT) in melanoma resistant to BRAFi.Experimental Design:Investigations were performed in vitro and in vivo with various human melanoma cell lines sensitive and resistant to BRAFi as well as patient-derived xenografts (PDX) derived from patients. In addition, samples were evaluated from patients on a clinical trial of BRAFi in combination with ACT.Results:Herein we report that in human melanoma cell lines, senstitive and resistant to BRAFi and in PDX from patients who progressed on BRAFi and MEKi therapy, BRAFi caused transient upregulation of mannose-6-phosphate receptor (M6PR). This sensitized tumor cells to CTLs via uptake of granzyme B, a main component of the cytotoxic activity of CTLs. Treatment of mice bearing resistant tumors with BRAFi enhanced the antitumor effect of patients' TILs. A pilot clinical trial of 16 patients with metastatic melanoma who were treated with the BRAFi vemurafenib followed by therapy with TILs demonstrated a significant increase of M6PR expression on tumors during vemurafenib treatment.Conclusions:BRAF-targeted therapy sensitized resistant melanoma cells to CTLs, which opens new therapeutic opportunities for the treatment of patients with BRAF-resistant disease.See related commentary by Goff and Rosenberg, p. 2682

Time-lapse imaging of A375(Fucci2)-BR cells treated with the control.

Table S1 Mean density values of crystal violet staining of 16 melanoma cell lines treated with 6-thio-dG and PLX4720. Table S2 RNA-seq data related to A375 cells treated with 6-thio-dG. Table S3 RNA-seq data related to A375 cells treated with BIBR 1532. Table S4 Apoptotic index and ssGSEA scores of 11 gene sets in 18 CCLE melanoma cell lines. Table S5 RPPA data related to A375 cells treated with 6-thio-dG. Table S6 Mean density values of crystal violet staining of 6 therapy-resistant melanoma cell lines treated with 6-thio-dG. Table S7 RPPA data related to the comparison between LOX-IMVI parental and BR cells. Table S8 RPPA data related to LOX-IMVI parental cells treated with 6-thio-dG. Table S9 RPPA data related to LOX-IMVI BR cells treated with 6-thio-dG. Table S10 RPPA data related to LOX-IMVI BR xenografts treated with 6-thio-dG. Table S11 The clinical information from patients with metastatic melanoma treated with immunotherapies.

Supplemental Figure 1. Temporal regulation and the effect of combination of B-raf inhibitor on M6PR expression. Supplemental Figure 2. Effect of combination of B-raf and MEK inhibitors on M6PR expression. Supplemental Figure 3. WM35 cells resistant to PLX4720. Supplemental Figure 4. Effect of M6PR overexpression and deletion on melanoma cell sensitivity to PLX4720 in MTT test. Supplemental Figure 5. Expression of surface molecules on melanoma cells with manipulated expression of M6PR. Supplemental Table 1 Clinical characteristics of patients enrolled to the study Supplemental Table 2. Grade 3+ Adverse Events in treated patients

Purpose: Telomerase promoter mutations are highly prevalent in human tumors including melanoma. A subset of patients with metastatic melanoma often fail multiple therapies, and there is an unmet and urgent need to prolong disease control for those patients.Experimental Design: Numerous preclinical therapy-resistant models of human and mouse melanoma were used to test the efficacy of a telomerase-directed nucleoside, 6-thio-2′-deoxyguanosine (6-thio-dG). Integrated transcriptomics and proteomics approaches were used to identify genes and proteins that were significantly downregulated by 6-thio-dG.Results: We demonstrated the superior efficacy of 6-thio-dG both in vitro and in vivo that results in telomere dysfunction, leading to apoptosis and cell death in various preclinical models of therapy-resistant melanoma cells. 6-thio-dG concomitantly induces telomere dysfunction and inhibits the expression level of AXL.Conclusions: In summary, this study shows that indirectly targeting aberrant telomerase in melanoma cells with 6-thio-dG is a viable therapeutic approach in prolonging disease control and overcoming therapy resistance. Clin Cancer Res; 24(19); 4771–84. ©2018 AACR.See related commentary by Teh and Aplin, p. 4629

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 integrinβ1 (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 TGFβ dependent, as inhibition of TGFβ 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.

Supplementary materials and methods

Supplementary Fig. 1: Related to Fig. 2 (A) The percentage of apoptotic and dead cells indicated as PSVue 643+ cells in each of 12 BRAF-mutant melanoma cell lines treated with PLX4720 or 6-thio-dG at indicated doses for 120 hours. Cells were then harvested and co-stained with PSVue643 and Propidium iodide (PI). The average of 2 biological replicates was plotted. (B) SA-β-gal staining of 4 BRAF-mutant melanoma cell lines treated with 6-thio-dG at 5μM for 9 days. A representative image of 3 biological replicates was shown for each experimental sample. (C and D) Mouse weights of 1205Lu (C) and A375 (D) xenografts in each treatment group were shown. upplementary Fig. 2: Related to Fig. 4 (A) Long-term cell growth assay of 4 melanoma cell lines that acquired resistance to MAPKi treated with 6-thio-dG at indicated doses for 12 days. Cells were then fixed and stained with 3 crystal violet. A representative image of 2 biological replicates was shown for each experimental condition. (B-D) Tumor volumes of WM9 BR (B), UACC-903 BR (C) and A2058 CR (D) xenografts that were treated with the vehicle control and 6-thio-dG at indicated doses. (E) The ssGSEA plot of 11 MSigDB gene sets related to telomere and telomerase that were significantly altered in LOX-IMVI BR cells treated with BIBR 1532 (BIBR) or 6-thio-dG (6dG). (F-I) The heatmaps of RPPA data depicting 30 proteins that were most significantly down-regulated in four representative BR cell lines treated with 6-thio-dG, including A375 BR (F), WM3936-1 (G), 1205Lu BR (H) and WM1552C BR (I). (J) Western blotting of proteins that were downregulated in LOX-IMVI BR cells treated with 6-thio-dG (6dG). (K) The immnohistochemical analysis of AXL was performed in A375 tumors that were treated with the control, 6-thio-dG and Dabrafenib. Supplementary Fig. S3: Related to Fig. 5 (A-C) The heatmaps of two telomere transcriptional gene signatures, two melanoma-specific gene sets and two MAPK pathway-related gene sets in three datasets in which transcriptomes of paired pre- and post-treatment tumor biopsies derived from patients who progressed on MAPKi were profiled. 5 Supplementary Fig. S4: Related to Fig. 5 6 The heatmaps of two telomere transcriptional gene signatures, two melanoma-specific gene sets and two MAPK pathway-related gene sets in paired pre-, on- and post-treatment tumor biopsies derived from 16 patients who were treated with immunotherapies. Supplementary Fig. S5: Related to Fig. 6 (A-E) The heatmaps of RPPA data depicting 30 proteins that were most significantly downregulated in 5 short-term cultures or cell lines derived from immunotherapy-resistant tumors that were treated with 6-thio-dG, including 13-456-3-3 (A), 15-1761-1-2 (B), WM4265-1 (C), WM4265-2 (D) and G43 (E).

Targeting MAPK pathway using a combination of BRAF and MEK inhibitors is an efficient strategy to treat melanoma harboring BRAF-mutation. The development of acquired resistance is inevitable due to the signaling pathway rewiring. Combining western blotting, immunohistochemistry, and reverse phase protein array (RPPA), we aim to understanding the role of the mTORC1 signaling pathway, a center node of intracellular signaling network, in mediating drug resistance of BRAF-mutant melanoma to the combination of BRAF inhibitor (BRAFi) and MEK inhibitor (MEKi) therapy. The mTORC1 signaling pathway is initially suppressed by BRAFi and MEKi combination in melanoma but rebounds overtime after tumors acquire resistance to the combination therapy (CR) as assayed in cultured cells and PDX models. In vitro experiments showed that a subset of CR melanoma cells was sensitive to mTORC1 inhibition. The mTOR inhibitors, rapamycin and NVP-BEZ235, induced cell cycle arrest and apoptosis in CR cell lines. As a proof-of-principle, we demonstrated that rapamycin and NVP-BEZ235 treatment reduced tumor growth in CR xenograft models. Mechanistically, AKT or ERK contributes to the activation of mTORC1 in CR cells, depending on PTEN status of these cells. Our study reveals that mTOR activation is essential for drug resistance of melanoma to MAPK inhibitors, and provides insight into the rewiring of the signaling networks in CR melanoma.

Purpose: BRAF and MEK inhibitors (BRAFi and MEKi) are actively used for the treatment of metastatic melanoma in patients with BRAFV600E mutation in their tumors. However, the development of resistance to BRAFi and MEKi remains a difficult clinical challenge with limited therapeutic options available to these patients. In this study, we investigated the mechanism and potential therapeutic utility of combination BRAFi and adoptive T-cell therapy (ACT) in melanoma resistant to BRAFi. Experimental Design: Investigations were performed in vitro and in vivo with various human melanoma cell lines sensitive and resistant to BRAFi as well as patient-derived xenografts (PDX) derived from patients. In addition, samples were evaluated from patients on a clinical trial of BRAFi in combination with ACT. Results: Herein we report that in human melanoma cell lines, senstitive and resistant to BRAFi and in PDX from patients who progressed on BRAFi and MEKi therapy, BRAFi caused transient upregulation of mannose-6-phosphate receptor (M6PR). This sensitized tumor cells to CTLs via uptake of granzyme B, a main component of the cytotoxic activity of CTLs. Treatment of mice bearing resistant tumors with BRAFi enhanced the antitumor effect of patients' TILs. A pilot clinical trial of 16 patients with metastatic melanoma who were treated with the BRAFi vemurafenib followed by therapy with TILs demonstrated a significant increase of M6PR expression on tumors during vemurafenib treatment. Conclusions: BRAF-targeted therapy sensitized resistant melanoma cells to CTLs, which opens new therapeutic opportunities for the treatment of patients with BRAF-resistant disease. See related commentary by Goff and Rosenberg, p. 2682

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 integrinβ1 (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 TGFβ dependent, as inhibition of TGFβ 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.

Non-muscle invasive bladder cancer (NMIBC) patients often have fewer treatment options, and suffer the progression of disease due to mechanisms that are not clear, as well as due to its diversity. This study was designed to explore the molecular mechanism of bladder cancer through an RNA-seq. In addition to conventional analyses, we also simplified the network through modularization using the WGCNA algorithm, with the help of the topological overlapping matrix and hierarchical cluster tree, which are based on the PPI network of STRING. Furthermore, the hub genes were confirmed through survival analyses in the independent cohorts (n = 431). Among them, 15 genes were significantly associated with poor prognosis. Finally, we validated the results at mRNA and protein level using qRT-PCR, IHC and western blotting. Taken together, our research is important for the prediction, as well as the prospective clinical development of drug targets and biomarkers.

Purpose: Telomerase promoter mutations are highly prevalent in human tumors including melanoma. A subset of patients with metastatic melanoma often fail multiple therapies, and there is an unmet and urgent need to prolong disease control for those patients. Experimental Design: Numerous preclinical therapy-resistant models of human and mouse melanoma were used to test the efficacy of a telomerase-directed nucleoside, 6-thio-2′-deoxyguanosine (6-thio-dG). Integrated transcriptomics and proteomics approaches were used to identify genes and proteins that were significantly downregulated by 6-thio-dG. Results: We demonstrated the superior efficacy of 6-thio-dG both in vitro and in vivo that results in telomere dysfunction, leading to apoptosis and cell death in various preclinical models of therapy-resistant melanoma cells. 6-thio-dG concomitantly induces telomere dysfunction and inhibits the expression level of AXL. Conclusions: In summary, this study shows that indirectly targeting aberrant telomerase in melanoma cells with 6-thio-dG is a viable therapeutic approach in prolonging disease control and overcoming therapy resistance. Clin Cancer Res; 24(19); 4771–84. ©2018 AACR. See related commentary by Teh and Aplin, p. 4629

The aim of the present study was to analyze potential therapy targets for prostate cancer using integrated analysis of two gene expression profiles. First, gene expression profiles GSE38241 and GSE3933 were downloaded from the Gene Expression Omnibus database. Differentially expressed genes (DEGs) between prostate cancer and normal control samples were identified using the Linear Models for Microarray Data package. Pathway enrichment analysis of DEGs was performed using Gene Ontology and the Kyoto Encyclopedia of Genes and Genomes. Furthermore, protein-protein interaction (PPI) networks of DEGs were constructed, on the basis of the Search Tool for the Retrieval of Interacting Genes/Proteins database. The Molecular Complex Detection was utilized to perform module analysis of the PPI networks. In addition, transcriptional regulatory networks were constructed on the basis of the associations between transcription factors (TFs) and target genes. A total of 529 DEGs were identified, including 129 upregulated genes that were primarily associated with to the cell cycle. Additionally, 400 downregulated genes were identified, which were principally enriched in the pathways associated with vascular smooth muscle contraction and focal adhesion. Cell Division Cycle Associated 8, Cell Division Cycle 45, Ubiquitin Conjugating Enzyme E2 C and Thymidine Kinase 1 were identified as hub genes in the upregulated sub-network. Furthermore, the upregulated TF E2F, and the downregulated TF Early Growth Response 1, were identified to be critical in the transcriptional regulatory networks. The identified DEGs and TFs may have critical roles in the progression of prostate cancer, and may be used as target molecules for treating prostate cancer.

This study aimed to explore the underlying microRNA (miRNA) targets in clear cell renal cell carcinoma (ccRCC). The expression profile with accession number GSE24952 was downloaded from the Gene Expression Omnibus database. Based on the dataset, the differentially expressed genes (DEGs) and miRNAs in ccRCC tissues and matched normal adjacent tissues were analyzed. The target genes of the differentially expressed miRNAs were then predicted. Expression levels of several key miRNAs and genes were detected by quantitative reverse transcription polymerase chain reaction (qRT-PCR). A total of 168 up- and 288 downregulated DEGs, and 26 up- and 54 downregulated differentially expressed miRNAs were identified. The target genes of miRNA-429 (TGFB1, CCND1, EGFR, and LAMC1) and miRNA-206 (CCND1 and EGFR) were upregulated. Based on the tumor suppressor (TS) gene and tumor-associated gene (TAG) databases, miRNA-142-5p was selected from the upregulated miRNAs. miRNA-429, miRNA-422a, miRNA-206, miRNA-132-3p, and miRNA-184 were selected from the downregulated miRNAs. Moreover, the miRNA regulation network revealed that CCND1 was the common target gene of miRNA-429, miRNA-206, and miRNA-184, and ATP1B1 was the common target gene of miRNA-140-3p and miRNA-142-5p. qRT-PCR revealed that the expression levels of miR-140-3p and CCND1 significantly increased, while that of ATP1B1 significantly decreased in 786-O cells compared with those in human renal tubular epithelial cells, which was in accordance with the predicted results of bioinformatic analysis. In conclusion, miRNA-429, miRNA-206, and miRNA-184 and their target gene CCND1, as well as miRNA-140-3p and miRNA-142-5p and their target gene ATP1B1, might play key roles in ccRCC progression and could be useful biomarkers during ccRCC development.

The aim was to expound the pathogenesis of prostate cancer and to identify the potentially biomarkers for prostate cancer (PC). DNA methylation microarray data GSE38240 containing 8 prostate cancer metastases and 4 normal prostate samples as well as gene expression profile data GSE26910 containing 6 prostate primary tumors and 6 normal samples were used. Differentially expressed genes (DEGs) and differently methylated sites of PC were screened and the regulatory network was constructed with DEGs-related transcription factors (TFs). The obtained hub genes were subjected to protein-protein interaction network analysis. Enrichment analysis of down-regulated DEGs were performed. Total 351 DEGs including 190 down-regulated and 161 up-regulated genes and 3234 differently methylated sites were identified. In total 69 DEGs-related TFs were found. Regulatory network contained 1301 nodes and 2527 connection pairs and that FOXA1 (forkhead box A1), BZRAP1-AS1 (benzodiazapine receptor associated protein 1 antisense RNA 1) and KRT8 (keratin 8) were the top three nodes of it. The enriched GO terms were mainly biological activity of the blood and cells-related. Total 29 DEGs (such as AGTR1, angiotensin II receptor, type 1) and 57 none-DEGs involved in the PPI network. Biological functions in blood circulation and the involved AGTR1 may play important roles in PC by gene-methylation. Besides, BZRAP1-AS1 may be novel biomarker related with PC.

Prostate cancer is the most common malignancy in males and easily develops to be aggressive which is closely related to the chronic inflammatory tumor microenvironment in situ. This study aimed to assess the immunoglobulin G (IgG) subclass of B cells and explore their interactions with T follicular helper (Tfh) subsets in prostate cancer patients. The percentages of peripheral blood naive B cells, memory B cells and mature B cells, as well as Tfh1, Tfh2 and Tfh17 cells were analyzed or sorted by FACSAria. The ratios of the different IgG subclasses (IgG1, IgG2, IgG3 and IgG4) were detected by ELISA, and the expression levels of CXCR3 and CCR6 were measured using RT-PCR and western blot analysis. Meanwhile a co-culture system of B and Tfh cells was to assess the effect of each Tfh subset on the antibody subclass switching of B cells in vitro. We observed higher percentages of 3 Tfh subsets and IgG4+ B cells in the patients with prostate cancer than that in the health controls and proved a positive correlation between Tfh2 and IgG4+ B cells. Then we verified that IL-4, IL-6, IL-10 and prostaglandin E2 (PGE2) effectively promoted antibody class switching of B cells, which may be mediated by inducing Tfh2 cells, yet the study was not completely dependent on Tfh cells. The results provide evidence of the B cell response to an immune suppressive environment by evaluating IgG4 antibodies, and established a relationship between IgG4+ B cells and Tfh2 cells. Clarification of lymphocyte functions in the inflammatory microenvironment of tumors will be of potential therapeutic value.