BRAF inhibitors in colorectal cancer: Toward a differentiation therapy?
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ABSTRACT:
SUBMITTER: Herr R
PROVIDER: S-EPMC4905336 | biostudies-other | 2015 Oct-Dec
REPOSITORIES: biostudies-other
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Project description:Mutations in the BRAF proto-oncogene, which encodes the B-Raf kinase, are associated with more aggressive, less-differentiated and therapy-resistant colorectal cancers (CRC). However, the molecular mechanisms responsible for these correlations remain unknown. Here, we report the characterization of human isogenic CRC cell line models (Caco-2, HT29, Colo-205) in which we modulate the expression of the B-RafV600E oncoprotein either by conditional cDNA or shRNA expression. Using these models in conventional and three dimensional tissue culture systems, we demonstrate that genetic depletion of endogenous B-RafV600E decreases cellular motility and invasion, while it induces hallmarks of differentiated epithelia such as the formation of functional adherens and tight junctions. Importantly, these effects are recapitulated by exposing these lines to B-Raf (PLX4720, vemurafenib, dabrafenib) or MEK inhibitors (trametinib). Furthermore, loss of endogenous B-RafV600E in HT29 xenografts does not only stall tumor growth, but also induces epithelial structures with marked expression of Cdx-2, a prognostic marker and master regulator of intestinal morphogenesis. By performing the first transcriptome profiles of B-Raf inhibitor treated 3D cultures of a primary adenocarcinoma and a metastasis derived CRC cell line, we establish functional links between B-RafV600E and proteins of known and potentially new prognostic relevance. We propose that B-Raf/MEK/ERK pathway inhibitors could be used to induce CRC differentiation and thereby to limit metastatic disease. To measure the time resolved gene responses, RNA was isolated from PLX4720 or DMSO treated Colo-205 and HT29 3D culture cell lysates at days 1,3 and 10 and days 1,3, and 8, respectively. The time points for HT29 cells were taken in biological duplicates.
2014-12-21 | E-GEOD-50791 | biostudies-arrayexpress
Project description:Mutations in the BRAF proto-oncogene, which encodes the B-Raf kinase, are associated with more aggressive, less-differentiated and therapy-resistant colorectal cancers (CRC). However, the molecular mechanisms responsible for these correlations remain unknown. Here, we report the characterization of human isogenic CRC cell line models (Caco-2, HT29, Colo-205) in which we modulate the expression of the B-RafV600E oncoprotein either by conditional cDNA or shRNA expression. Using these models in conventional and three dimensional tissue culture systems, we demonstrate that genetic depletion of endogenous B-RafV600E decreases cellular motility and invasion, while it induces hallmarks of differentiated epithelia such as the formation of functional adherens and tight junctions. Importantly, these effects are recapitulated by exposing these lines to B-Raf (PLX4720, vemurafenib, dabrafenib) or MEK inhibitors (trametinib). Furthermore, loss of endogenous B-RafV600E in HT29 xenografts does not only stall tumor growth, but also induces epithelial structures with marked expression of Cdx-2, a prognostic marker and master regulator of intestinal morphogenesis. By performing the first transcriptome profiles of B-Raf inhibitor treated 3D cultures of a primary adenocarcinoma and a metastasis derived CRC cell line, we establish functional links between B-RafV600E and proteins of known and potentially new prognostic relevance. We propose that B-Raf/MEK/ERK pathway inhibitors could be used to induce CRC differentiation and thereby to limit metastatic disease.
2014-12-21 | GSE50791 | GEO
Project description:Colorectal cancer (CRC) is the second-leading cause of cancer-related deaths globally. BRAF mutation is present in about 10% of CRC patients and is associated with a poor response to chemotherapy. These patients have a relatively poor prognosis. This review aims to assess the efficacy and safety of BRAF inhibitors in BRAF-mutated CRC patients. A literature search was performed on PubMed and Embase, and clinical trials relevant to BRAF inhibitors in CRC were included. Data were extracted for efficacy and safety variables. Two randomized clinical trials (n = 765) and eight non-randomized trials (n = 281) were included based on inclusion criteria. In RCTs, an overall response was reported in 23% of the patients treated with BRAF inhibitor-based regimens compared to 2.5% with control regimens. The hazard ratio of overall survival was also significantly better with triplet encorafenib therapy at 0.52 (95% CI = 0.39-0.70). In single-arm trials, ORR was 17% and 34% in two-drug and three-drug regimens, respectively. BRAF inhibitor-based regimens were safe and effective in the treatment of BRAF-mutated CRC. Large-scale randomized trials are needed to find a suitable population for each regimen. PROSPERO registration No. CRD42023471627.
| S-EPMC10779564 | biostudies-literature
Project description:Oncogenic mutations in BRAF are believed to initiate serrated colorectal cancers; however, the mechanisms of BRAF-driven colon cancer are unclear. We find that oncogenic BRAF paradoxically suppresses stem cell renewal and instead promotes differentiation. Correspondingly, tumor formation is inefficient in BRAF-driven mouse models of colon cancer. By reducing levels of differentiation via genetic manipulation of either of two distinct differentiation-promoting factors (Smad4 or Cdx2), stem cell activity is restored in BRAFV600E intestines, and the oncogenic capacity of BRAFV600E is amplified. In human patients, we observe that reduced levels of differentiation in normal tissue is associated with increased susceptibility to serrated colon tumors. Together, these findings help resolve the conditions necessary for BRAF-driven colon cancer initiation. Additionally, our results predict that genetic and/or environmental factors that reduce tissue differentiation will increase susceptibility to serrated colon cancer. These findings offer an opportunity to identify susceptible individuals by assessing their tissue-differentiation status.
| S-EPMC5747303 | biostudies-literature
Project description:Autophagy is the basic catabolic mechanism that involves cell degradation of unnecessary or dysfunctional cellular components. Autophagy has a controversial role in cancer--both in protecting against tumor progression by isolation of damaged organelles, or by potentially contributing to cancer growth. The impact of autophagy in RAS induced transformation still remains to be further analyzed based on the differential effect of RAS isoforms and tumor cell context. In the present study, the effect of KRAS/BRAF/PIK3CA oncogenic pathways on the autophagic cell properties and on main components of the autophagic machinery like p62 (SQSTM1), Beclin-1 (BECN1) and MAP1LC3 (LC3) in colon cancer cells was investigated. This study provides evidence that BRAF oncogene induces the expression of key autophagic markers, like LC3 and BECN1 in colorectal tumor cells. Herein, PI3K/AKT/MTOR inhibitors induce autophagic tumor properties, whereas RAF/MEK/ERK signalling inhibitors reduce expression of autophagic markers. Based on the ineffectiveness of BRAFV600E inhibitors in BRAFV600E bearing colorectal tumors, the BRAF related autophagic properties in colorectal cancer cells are further exploited, by novel combinatorial anti-cancer protocols. Strong evidence is provided here that pre-treatment of autophagy inhibitor 3-MA followed by its combination with BRAFV600E targeting drug PLX4720 can synergistically sensitize resistant colorectal tumors. Notably, colorectal cancer cells are very sensitive to mono-treatments of another autophagy inhibitor, Bafilomycin A1. The findings of this study are expected to provide novel efficient protocols for treatment of otherwise resistant colorectal tumors bearing BRAFV600E, by exploiting the autophagic properties induced by BRAF oncogene.
| S-EPMC4891035 | biostudies-literature
Project description:BackgroundCombinatorial inhibition of epidermal growth factor receptor (EGFR) and BRAF shows remarkable clinical benefits in patients with BRAF V600E-mutant metastatic colorectal cancer (mCRC). However, the tumor may inevitably develop resistance to the targeted therapy, thereby limiting the response rate and durability. This study aimed to determine the genetic alterations associated with intrinsic and acquired resistance to EGFR/BRAF inhibitors in BRAF V600E-mutant mCRC.MethodsTargeted sequencing of 520 cancer-related genes was performed in tumor tissues and in plasma samples collected from patients with BRAF V600E-mutant mCRC, who were treated with EGFR/BRAF ± MEK inhibitors, before and after the targeted treatment. Clinical benefit was defined as an objective response or a stable disease lasting longer than the median progression-free survival (PFS).ResultsIn all, 25 patients with BRAF V600E-mutant mCRC were included in this study. Those with RNF43 mutations (n = 8) were more likely to achieve clinical benefit from EGFR/BRAF inhibitors than those with wild-type RNF43 (87.5% versus 37.5%, p = 0.034). Genetic alterations in receptor tyrosine kinase genes (n = 6) were associated with worse PFS (p = 0.005). Among the 23 patients whose disease progressed after the EGFR/BRAF-targeted therapy, at least one acquired resistance-related mutation was detected in 12 patients. Acquired mutations were most frequently observed in the mitogen-activated protein kinase pathway-related genes (n = 9), including KRAS (G12D and Q61H/R), NRAS (Q61L/R/K and amplification), BRAF (amplification), and MEK1 (K57T). MET amplification and PIK3R1 Q579fs mutation emerged in three patients and one patient, respectively, after disease progression.ConclusionMultiple genetic alterations are associated with clinical benefits and resistance to EGFR/BRAF inhibitors in BRAF V600E-mutant mCRC. Our findings provide novel insights into strategies for overcoming resistance to EGFR/BRAF inhibitors in patients with BRAF V600E-mutant mCRC.
| S-EPMC9210093 | biostudies-literature
Project description:Patients with BRAF-mutated colorectal cancer (CRC) have a poor prognosis despite recent therapeutic advances such as combination therapy with BRAF, MEK, and epidermal growth factor receptor (EGFR) inhibitors. To identify microRNAs (miRNAs) that can improve the efficacy of BRAF inhibitor dabrafenib (DAB) and MEK inhibitor trametinib (TRA), we screened 240 miRNAs in BRAF-mutated CRC cells and identified five candidate miRNAs. Overexpression of miR-193a-3p, one of the five screened miRNAs, in CRC cells inhibited cell proliferation by inducing apoptosis. Reverse-phase protein array analysis revealed that proteins with altered phosphorylation induced by miR-193a-3p were involved in several oncogenic pathways including MAPK-related pathways. Furthermore, overexpression of miR-193a-3p in BRAF-mutated cells enhanced the efficacy of DAB and TRA through inhibiting reactivation of MAPK signaling and inducing inhibition of Mcl1. Inhibition of Mcl1 by siRNA or by Mcl1 inhibitor increased the antiproliferative effect of combination therapy with DAB, TRA, and anti-EGFR antibody cetuximab. Collectively, our study demonstrated the possibility that miR-193a-3p acts as a tumor suppressor through regulating multiple proteins involved in oncogenesis and affects cellular sensitivity to MAPK-related pathway inhibitors such as BRAF inhibitors, MEK inhibitors, and/or anti-EGFR antibodies. Addition of miR-193a-3p and/or modulation of proteins involved in the miR-193a-3p-mediated pathway, such as Mcl1, to EGFR/BRAF/MEK inhibition may be a potential therapeutic strategy against BRAF-mutated CRC.
| S-EPMC8409311 | biostudies-literature
Project description:Metastatic BRAFV600E colorectal cancer (CRC) carries an extremely poor prognosis and is in urgent need of effective new treatments. While the BRAFV600E inhibitor encorafenib in combination with the EGFR inhibitor cetuximab (Enc+Cet) was recently approved for this indication, overall survival is only increased by 3.6 months and objective responses are observed in only 20% of patients. We have found that a limitation of Enc+Cet treatment is the failure to efficiently induce apoptosis in BRAFV600E CRCs, despite inducing expression of the pro-apoptotic protein BIM and repressing expression of the pro-survival protein MCL-1. Here, we show that BRAFV600E CRCs express high basal levels of the pro-survival proteins MCL-1 and BCL-XL, and that combining encorafenib with a BCL-XL inhibitor significantly enhances apoptosis in BRAFV600E CRC cell lines. This effect was partially dependent on the induction of BIM, as BIM deletion markedly attenuated BRAF plus BCL-XL inhibitor-induced apoptosis. As thrombocytopenia is an established on-target toxicity of BCL-XL inhibition, we also examined the effect of combining encorafenib with the BCL-XL -targeting PROTAC DT2216, and the novel BCL-2/BCL-XL inhibitor dendrimer conjugate AZD0466. Combining encorafenib with DT2216 significantly increased apoptosis induction in vitro, while combining encorafenib with AZD0466 was well tolerated in mice and further reduced growth of BRAFV600E CRC xenografts compared to either agent alone. Collectively, these findings demonstrate that combined BRAF and BCL-XL inhibition significantly enhances apoptosis in pre-clinical models of BRAFV600E CRC and is a combination regimen worthy of clinical investigation to improve outcomes for these patients.
| S-EPMC10907349 | biostudies-literature
Project description:Retinoic acid (RA) signaling is an important and conserved pathway that regulates cellular proliferation and differentiation. Furthermore, perturbed RA signaling is implicated in cancer initiation and progression. However, the mechanisms by which RA signaling contributes to homeostasis, malignant transformation, and disease progression in the intestine remain incompletely understood. Here, we report, in agreement with previous findings, that activation of the Retinoic Acid Receptor and the Retinoid X Receptor results in enhanced transcription of enterocyte-specific genes in mouse small intestinal organoids. Conversely, inhibition of this pathway results in reduced expression of genes associated with the absorptive lineage. Strikingly, this latter effect is conserved in a human organoid model for colorectal cancer (CRC) progression. We further show that RXR motif accessibility depends on progression state of CRC organoids. Finally, we show that reduced RXR target gene expression correlates with worse CRC prognosis, implying RA signaling as a putative therapeutic target in CRC.
| S-EPMC8633980 | biostudies-literature
Project description:Although recent clinical trials of BRAF inhibitor combinations have demonstrated improved efficacy in BRAF-mutant colorectal cancer, emergence of acquired resistance limits clinical benefit. Here, we undertook a comprehensive effort to define mechanisms underlying drug resistance with the goal of guiding development of therapeutic strategies to overcome this limitation. We generated a broad panel of BRAF-mutant resistant cell line models across seven different clinically relevant drug combinations. Combinatorial drug treatments were able to abrogate ERK1/2 phosphorylation in parental-sensitive cells, but not in their resistant counterparts, indicating that resistant cells escaped drug treatments through one or more mechanisms leading to biochemical reactivation of the MAPK signaling pathway. Genotyping of resistant cells identified gene amplification of EGFR, KRAS, and mutant BRAF, as well as acquired mutations in KRAS, EGFR, and MAP2K1 These mechanisms were clinically relevant, as we identified emergence of a KRAS G12C mutation and increase of mutant BRAF V600E allele frequency in the circulating tumor DNA of a patient at relapse from combined treatment with BRAF and MEK inhibitors. To identify therapeutic combinations capable of overcoming drug resistance, we performed a systematic assessment of candidate therapies across the panel of resistant cell lines. Independent of the molecular alteration acquired upon drug pressure, most resistant cells retained sensitivity to vertical MAPK pathway suppression when combinations of ERK, BRAF, and EGFR inhibitors were applied. These therapeutic combinations represent promising strategies for future clinical trials in BRAF-mutant colorectal cancer. Cancer Res; 76(15); 4504-15. ©2016 AACR.
| S-EPMC4970882 | biostudies-literature