BRAF Expression Research Articles

Significance of BRAF and Nrf2 in evaluation of serous ovarian tumors

Background Ovarian carcinoma is the second leading cause of cancer-related deaths among women. Many treatment strategies have been developed; however, the prognosis is still poor. This study aims to evaluate the immunohistochemical expression of the serine/threonine-protein kinase B-raf (BRAF) and nuclear factor erythroid 2-related factor 2 (Nrf2) in serous ovarian tumors and to correlate their expression with clinicopathological features of the cases. Materials and methods This uncontrolled retrospective study was performed on 50 cases: benign (n=10), borderline (n=12), and malignant serous ovarian neoplasm (n=28). The malignant serous tumors included nine low-grade cases and 19 high-grade cases. The immunohistochemical expression of Nrf2 and BRAF was correlated with clinicopathological features of prognostic importance. Results The mean of BRAF expression is increased in borderline but it is almost equal for low-grade and high-grade carcinoma cases, which was statistically significant (P Conclusion BRAF and Nrf2 could play a significant role in the step of carcinogenesis and then in progression of serous ovarian tumors.

Expression and clinical significance of PD-L1 and BRAF expression in nasopharyngeal carcinoma

BackgroundThe prognostic value of programmed death-ligand 1 (PD-L1) and BRAF expression in nasopharyngeal carcinoma (NPC) is not well-defined. In this study we investigated alterations in PD-L1, BRAF and EGFR by using immunohistochemistry analysis in a cohort of consecutively enrolled NPC patients.MethodsA retrospective review of 154 NPC patients form our previous study (BMC Cancer. 2013; 13:226) were conducted. Survival and prognostic impacts were analyzed based on PD-L1, BRAF and EGFR expression levels.ResultsOne hundred fifty four patients were included in this study. PD-L1 expression was detected in 87.7% of patients; 14.3% had 1–5% PD-L1 expression, 47.4% had 5–49% expression while 26% had ≥50% expression Higher PD-L1 expression was significantly associated with shorter PFS and OS. The median PFS was 25 months (95% CI 15.7–34.3 months) and OS was 35 months (95% CI 22.60–47.4 months) for patients with PD-L1 expression ≥50%; both median PFS and OS were not yet reached for patients with PD-L1 expression < 50%. PFS was significantly higher in BRAF mutation positive patients (5-year PFS: 55.1% vs. 30.8%, P = 0.044).ConclusionTumor PD-L1 expression and BRAF mutation are associated with poor outcomes in patients with NPC. This study was retrospectively registered in ClinicalTrials.gov (NCT03989297) on 2019-6-18.

Abstract 3170: BRAF expression is associated with poor survival in colorectal cancers independent of BRAF (V600E) mutation

Abstract The RAS pathway is known to be commonly activated and a key determinant of colorectal cancer (CRC) biology, with mutational activation of RAS/RAF genes observed in ~50% of CRC patients. The relationship between gene mutation and expression is, however, poorly understood. We analyzed how the expression of RAS/RAF genes vs. their respective mutations impacted outcomes. The relationship to other rarely mutated RAS/RAF genes (HRAS, ARAF, RAF1 (i.e. c-RAF)) was also examined in these tumors. While KRAS expression was positively associated with KRAS mutations (r=0.26, p&amp;lt;0.0001), BRAF expression was, unexpectedly, negativelycorrelated with BRAF (V600E) mutation (r= -0.18, p=0.0001). Thus, a substantial number of patients express relatively high levels of BRAF absent of BRAF mutations. Additional analysis indicates that lower BRAF expression was significantly correlated with MSI or the CMS1 subtype that is strongly associated with BRAF (V600E). Intriguingly, despite negative association with mutant BRAF (V600E) that is well-established to be associated with poor prognosis, higher BRAF expression was also associated with poor overall survival, as indicated by Kaplan-Meier survival analysis of BRAF expression by quartiles in either 458 CRCs (Logrank test for trend p=0.0262) in 406 WT BRAF CRCs (p=0.0102). Note that the expression of all other five RAS/RAF genes (HRAS, KRAS, NRAS, ARAF and RAF1) was not associated with survival. Further analysis with the gene expression signatures measuring RAS pathway activation reveals that unlike BRAF (V600E) (or KRAS mutations or KRASexpression), BRAF expression appeared to be negatively correlated with RAS pathway activation signature scores. Taken together, these data suggest that higher BRAF expression in CRC patients may activate an “alternative” pathway to the canonical pathway directed by mutant BRAF (V600E) as yet another means to deliver tumor progression. Citation Format: Mingli Yang, Michael J. Schell, Timothy J. Yeatman. BRAF expression is associated with poor survival in colorectal cancers independent of BRAF (V600E) mutation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3170.

Clinical and biological significance of adamantinomatous craniopharyngioma with CTNNB1 mutation.

The Wnt/β-catenin signaling pathway is strongly implicated in the pathogenesis of adamantinomatous craniopharyngioma (adaCP). However, there is no evidence that the CTNNB1 mutation activates the target gene of Wnt/β-catenin signaling, and it is unknown whether it affects the tumorigenesis of adaCP. To assess the effect of the CTNNB1 mutation of adaCP, the authors analyzed the correlation between the mutation and clinical, radiological, pathological, and biological findings. Between 2003 and 2015, 42 patients (24 male and 18 female, median age 42 years) with either papillary craniopharyngioma (papCP) or adaCP underwent tumor resection at the authors' institution. BRAF V600E and CTNNB1 in papCP and adaCP samples were sequenced by next-generation sequencing and the Sanger method, and mRNA expression levels of Axin2 and BMP4 were evaluated by RT-PCR. Axin2, BMP4, β-catenin, and BRAF expression were evaluated by immunohistochemistry. Other data were collected from clinical reports. The BRAF V600E mutation was detected in all 10 cases of papCP (100%). CTNNB1 exon 3 mutations were detected in 21 of 31 (68%) cases of adaCP, excluding 1 case for which there were no available sequence data. The mRNA expression level of Axin2 was significantly higher in adaCPs with a CTNNB1 mutation than in those without (p < 0.05). The immunohistochemical findings of Axin2 and BMP4 did not correlate with CTNNB1 mutation positivity. When patients who received adjuvant radiation therapy were excluded, progression-free survival was shorter in the mutation-positive group than in the mutation-negative group (log-rank test, p = 0.031). Examination of clinical characteristics and immunohistochemical findings of adaCPs showed that there was no significant correlation between CTNNB1 mutation positivity and age, sex, tumor volume, gross-total resection, optic tract edema, calcification, or T1 signal intensity of cyst fluid on MRI, β-catenin, and MIB-1 index. These results raise the possibility that the CTNNB1 mutation in adaCP may be associated with disease recurrence, and genes related to the Wnt/β-catenin signaling pathway might represent a therapeutic target.

Loss of nuclear localization of thyroid transcription factor 1 and adverse outcomes in papillary thyroid cancer

Function of the thyroid follicular cell depends on nuclear expression of thyroid transcription factor 1 (TTF1). Regulation of this key protein regulating iodide transport is not well known, but its loss is linked to the most lethal of thyroid malignancies. We examined TTF1 nuclear expression in the context of adverse pathological features, disease recurrence, and BRAF status in papillary thyroid carcinomas with (n = 182) and without (n = 303) nodal metastases. Overall nuclear expression level of TTF1 was strong and diffuse in approximately 73%, whereas 27% exhibited lower levels or a paucity of nuclear staining. In the same cohort, approximately 59% exhibited the BRAF mutation. On univariate analysis, low levels of TTF1 nuclear expression was linked to vascular invasion, extrathyroidal extension, and nodal metastases. Multivariate analysis indicated that low levels of TTF1 were most strongly linked to nodal metastases and vascular invasion. Interestingly, TTF1 levels were not linked to the BRAF mutation. TTF1 staining alone predicted disease recurrence, but when combined with BRAF status, the 2 markers exhibited a more marked influence. Patients lacking the BRAF mutation and exhibiting normal levels of TTF1 exhibited very low levels of disease recurrence (11% at 10 years). Conversely, patient tumors with low levels of TTF1 and the BRAF mutation recurred in 31% of cases in the same time frame. The mixed expression of BRAF under varying levels of differentiation may explain, in part, the contradictory studies regarding the impact of BRAF mutations on patient prognosis and also indicates a complex genomic signature for dedifferentiated thyroid cancer.

Melanoma Progression Inhibits Pluripotency and Differentiation of Melanoma-Derived iPSCs Produces Cells with Neural-like Mixed Dysplastic Phenotype

SummaryMelanomas are known to exhibit phenotypic plasticity. However, the role cellular plasticity plays in melanoma tumor progression and drug resistance is not fully understood. Here, we used reprogramming of melanocytes and melanoma cells to induced pluripotent stem cell (iPSCs) to investigate the relationship between cellular plasticity and melanoma progression and mitogen-activated protein kinase (MAPK) inhibitor resistance. We found that melanocyte reprogramming is prevented by the expression of oncogenic BRAF, and in melanoma cells harboring oncogenic BRAF and sensitive to MAPK inhibitors, reprogramming can be restored by inhibition of the activated oncogenic pathway. Our data also suggest that melanoma tumor progression acts as a barrier to reprogramming. Under conditions that promote melanocytic differentiation of fibroblast- and melanocyte-derived iPSCs, melanoma-derived iPSCs exhibited neural cell-like dysplasia and increased MAPK inhibitor resistance. These data suggest that iPSC-like reprogramming and drug resistance of differentiated cells can serve as a model to understand melanoma cell plasticity-dependent mechanisms in recurrence of aggressive drug-resistant melanoma.

MiR-9-5p Increases Sensitivity to Cisplatin in Thyroid Cancer Cells by Down-Regulating BRAF Expression

Objective: To explore the expression of miR-9-5p and BRAF in cisplatin resistant strain thyroid cancer cells and reversal effect of drug resistance as well as the possible mechanism. Methods: The cisplatin-resistant thyroid cancer cells (FTC-133/DDP and TPC-1/DDP) were respectively divided into 3 groups as NC, DDP and DDP + miRNA groups. Measuring cell proliferation by MTT assay and cell apoptosis by flow cytometry; Evaluating invasion cell number and wound healing rates by transwell and wound healing assay. The relative proteins (BRAF, Mek and Erk1/2) were measured by WB assay. The correlation between miR-9-5p and BRAF by dual-luciferase reporter assay in FTC-133/DDP and TPC-1/DDP cells. Results: In FTC-133/DDP and TPC-1/DDP cells experiment, compared with DDP group, with miR-9-5p supplement, the cell proliferation rats were significantly depressed with cell apoptosis increasing (P &lt; 0.001, respectively); invasion cell number and wound healing rats were significantly down-regulation (P &lt; 0.001, respectively) in DDP + miRNA groups. Meanwhile, the BRAF, Mek and Erk1/2 proteins expressions were significantly depressed in DDP + miRNA groups were significantly suppressed compared with those in DDP groups (P &lt; 0.001, respectively). By dual-luciferase reporter assay, BRAF was the target gene of miR-9-5p in FTC133/DDP and TPC-1/DDP cells. Conclusion: miR-9-5p increases sensitivity to cisplatin in thyroid cancer cells by down-regulating BRAF expression.

Protein expression of KIT, BRAF, GNA11, GNAQ and RASSF1 in feline diffuse iris melanomas

Feline iris melanoma, the most common feline intraocular tumour, has a reported metastatic rate of 19–63%. However, there is a lack of knowledge about its molecular biology. Previous studies have reported that feline iris melanomas do not harbour mutations comparable to common mutations found in their human counterpart. Nevertheless, there are differences in the gene expression patterns. The aim of this study was to investigate the protein expression of B-RAF oncogene serine/threonine kinase (BRAF), G protein subunit alpha q (GNAQ) and 11 (GNA11), KIT proto-oncogene receptor tyrosine kinase (KIT), and Ras association family member 1 (RASSF1) in feline iris melanomas.Fifty-seven formalin-fixed paraffin embedded (FFPE) iris melanomas and 25 FFPE eyes without ocular abnormalities were stained with antibodies against the respective proteins using immunofluorescence. Averaged pixel intensities/μm2 and percentage of stained area from total tissue area were measured and the results were compared. Compared to the control group, iris melanomas showed overexpression of BRAF, GNAQ, GNA11 and KIT. The higher expression of BRAF, GNAQ, GNA11 and KIT in feline iris melanomas suggest that these proteins may play a key role in the development of feline iris melanomas and KIT may present a possible target for future therapies in cats with feline iris melanomas.

Immunohistochemical assessment of growth factor signaling molecules: MAPK, Akt, and STAT3 pathways in oral epithelial precursor lesions and squamous cell carcinoma.

Several growth factors and their receptors, such as epidermal growth factor receptor, have been studied as prognostic biomarkers for many epithelial malignancies. The signal transduction cascade of those receptors includes RAS/RAF/ERK, PI3K/Akt/mTOR, and STAT3 pathways. The aim of this study was to investigate the expression levels of several key proteins of those pathways in patients with oral squamous cell carcinoma (OSCC) and oral epithelial precursor lesions (OEPLs), and to correlate the expressions of these proteins with clinicopathologic features and prognosis. Fifteen leukoplakia (LP), 15 low-grade epithelial dysplasia, 15 high-grade epithelial dysplasia (HD), and 132 OSCC specimens were immunohistochemically examined for KRAS, HRAS, NRAS, BRAF, pERK1/2, pAkt, pmTOR, and pSTAT3 expression. Immunoreactivity for these molecules predominantly occurred in regions OEPL basal to prickle layers and in most OSCC cells. KRAS and NRAS expression was significantly lower in OSCC than in OEPLs, while pAkt and pmTOR showed higher expression in OSCC than in OEPLs. pERK1/2 expression was significantly higher in HD than in LP. In OSCC, KRAS and NRAS immunoreactivity was significantly higher in advanced age and male gender. In addition, higher immunoreactivity was shown in pERK1/2 in female gender and advanced TNM stage, pAkt in advanced T classification and cases without postoperative metastasis, pmTOR in advanced mode of invasion, and pSTAT3 in invasion depth. Correlations between these markers and clinicopathological variables were also noted. MAPK, Akt, and STAT3 pathways might play diverse roles in oral carcinogenesis.

Role of P53, E-cadherin and BRAF as predictors of regional nodal recurrence for papillary thyroid cancer

BackgroundRegional nodal recurrence (RNR) in patients diagnosed with papillary thyroid carcinoma (PTC) has increased. Variable immunohistochemical (IHC) markers have been studied for predicting the likelihood of PTC for recurrence. We aimed to clarify the IHC expression of p53, Ecadherin and BRAF as potential markers of RNR in PTC. Method145 (73 study group and 72 control group) patients with PTC were analyzed retrospectively between January 2010 and June 2017. Further classification to a specific histological variant was done, and IHC expression of p53, Ecadherin and BRAF was analyzed both in the primary tumor and in nodal recurrence. ResultsRegarding the risk of RNR, we found certain clinicopathologic features as elder age ≥55 years, tumor size >1 cm, presence of microscopic extrathyroid extension, presence of lymphovascular emboli, and conventional papillary subtype. Furthermore, IHC results for negative E-cadherin, and positive P53 and BRAF are significant risk factors, while radioactive iodine (RAI) adjuvant therapy decrease recurrence risk. ConclusionWe found several risk factors for RNR in PTC diagnosed patients, all of which are easily achievable in clinical settings. In this regard, we suggested that patients with specific clinicopathologic and immunohistochemical features have strict follow up for early detection of RNR as it has a great impact on their survival.

Clinicopathologic Characterization of Bilateral Testicular Germ Cell Tumors With Immunohistochemical Evaluation of Mismatch Repair and BRAF (V600E) Genes Mutations.

The incidence of bilateral testicular germ cell tumor (TGCT) is 1% to 5%. Despite the high rate of treatment success, resistance to chemotherapy has a detrimental effect. Some studies found MMR and BRAF gene mutations to be associated with chemotherapy resistance, which has not been found by others. However, the role of microsatellite instability (MSI) and BRAF mutations in bilateral disease has not been investigated. In this article, we studied the clinicopathologic characteristics and immunohistochemical expressions of MMR and BRAF in 13 patients with bilateral TGCT. Bilateral tumors were found in 4% of patients in our data. The mean ages at the first and subsequent diagnoses were 26.9 and 28.3 years, respectively. Eleven patients had metachronous disease; and the mean period between both tumors was 4.9 years. Six had mixed GCTs (MGCT) initially and later developed contralateral seminoma, 3 had bilateral MGCTs; 1 initially had pure embryonal carcinoma and subsequently MGCT and finally, 1 patient had initial seminoma and contralateral germ cell neoplasia in situ only. Of the patients with synchronous GCT, 1 had a MGCT and contralateral non-seminoma and 1 had seminoma and contralateral MGCT. In metachronous cases, 40% and 78% had an initial and subsequent stage of pT1, respectively. Hormonal and/or metastatic recurrence was observed in 30% of metachronous tumors. Six patients received chemotherapy, including patients with metastasis. No progression occurred after therapy. MLH1, PMS2, MSH2, and MSH6 staining was retained in all tumors. No BRAF staining was found. In conclusion, we found no association between bilateral TGCT and the MMR/MSI pathway and that subsequent metachronous tumors behaved much more indolently.

SAT-042 Pituitary Expression Of Isl1 Prevents Ectopic, Foxa1 And Foxj1 Mediated Differentiation Of Progenitors Into Rathke’S Cleft Cysts

Islet 1 (ISL1) is a LIM homeodomain transcription factor expressed in the developing pituitary gland. Its expression overlaps with the progenitor markers SOX2 and PROP1 and with the differentiation markers NR5A1 and CGA, or alpha-subunit. We conditionally knocked out Isl1 using several Cre recombinase drivers and found increased pituitary progenitor cell apoptosis and reduced thyrotroph and gonadotroph differentiation, which results in pituitary-thyroid axis dysfunction and reduced body size in adolescent mice. Elimination of Isl1 with Prop1-Cre results in the development of multiple Rathke’s cleft cysts in pituitaries of mutant mice postnatally, with 100% penetrance. Rathke’s cleft cysts are common intracranial lesions that are thought to originate from embryonic oral epithelium. They can be asymptomatic or cause pituitary dysfunction, headache, visual problems, and diabetes insipidus. The Prop1-cre induced mouse Rathke's cleft cysts mimic the histology of human cysts in that they are comprised of single-layered, ciliated epithelial cells that express the characteristic pathologic markers, cytokeratin 8 and acetylated tubulin. Moreover, they lack expression of B-raf, a marker of craniopharyngiomas, another important pituitary pathology of embryological origin. RNA-Seq analysis of newborn, mutant mouse pituitaries identified ectopic expression of Foxj1 and Foxa1. Normally, the pioneer transcription factor FOXA1 antagonizes epithelial to mesenchymal-like transitions by positively regulating E-cadherin expression and maintaining epithelial-like fate, and FOXJ1 drives a ciliogenic gene expression program. This suggests that ectopic FOXA1 and FOXJ1 may drive Rathke's cleft cyst formation. To assess whether our model of Rathke's cleft cyst formation accurately predicts the mechanisms underlying this pathology in humans, we obtained tissues from patients that underwent surgery for problematic cysts, and we analyzed expression of FOXA1, FOXJ1 and progenitor markers SOX2 and SOX9. We found significant, consistent, ectopic expression of FOXA1 and FOXJ1 in human cysts and variable expression of progenitor markers, mirroring the findings in mice. Together, these studies support a model whereby expression of ISL1 in pituitary progenitors is necessary to drive differentiation into thyrotropes and gonadotropes, and without this essential driver, progenitors can activate FOXA1 and FOXJ1, permitting them to differentiate along an oral epithelial cell fate, producing mucinous cysts. Thus, Isl1 expression in pituitary progenitor cells is necessary to suppress ectopic cell fates and promote appropriate thyrotroph and gonadotroph differentiation. This pituitary-specific Isl1 mouse knockout sheds light on the etiology of Rathke's cleft cysts and the role. of ISL1 in normal pituitary development.

LncRNA BANCR suppresses cell viability and invasion and promotes apoptosis in non-small-cell lung cancer cells in vitro and in vivo.

Background: As a leading cause of deaths worldwide, lung cancer is a collection of diseases with diverse etiologies which includes non-small-cell lung cancer (NSCLC). Increasing evidence reported that aberrant expression of BRAF activated non-coding RNA (BANCR) was involved in the tumorigenesis and progression of various malignancies.Purpose and methods: However, its role in NSCLC has not been completely clarified. In the present study, we identified the role of BANCR in the regulation of NSCLC cell viability, invasion, and apoptosis. Down-regulation of BANCR expression was significantly observed in different NSCLC cell lines (A549, H1299, H1650, H1975, SPC-A1, and PC-9), tumor tissue from NSCLC mouse model and 30 human NSCLC tissues compared with adjacent normal tissues.Result: Overexpression of BANCR in these six NSCLC cell lines attenuated the cell viability and invasion. An increased apoptotic level caused by BANCR overexpression was also detected and displayed a conversed influence on Bcl-2 and Bax expression in mRNA and protein level. Furthermore, we identified the effect of BANCR overexpression on tumor growth in NSCLC mouse model. The restoration of BANCR expression inhibits NSCLC.Conclusion: Taken together, our findings shed an insight on the novel molecular mechanisms of lung NSCLC oncogenesis and provide the information for new therapeutic approaches on the disease.

The Combination of RET, BRAF and Demographic Data Identifies Subsets of Patients with Aggressive Papillary Thyroid Cancer.

The use of BRAFV600E and RET/PTC1 as biomarkers to guide the extent of surgery in patients with papillary thyroid cancer (PTC) remains controversial. We assessed the combined use of demographic data (sex and age) with mRNA expression levels and/or mutational status (BRAFV600E and RET/PTC1) to identify potential subsets of patients with aggressive histopathological features (lymph node metastases and extrathyroidal extension). In a cohort of 126 consecutive patients, BRAFV600E and RET/PTC1 mutations were found in 52 and 18%, respectively. By conditional bivariate analysis (CBVA), a 'high activity' profile of BRAF (BRAFV600E positive or high expression) and 'low activity' profile of RET (RET/PTC1 negative or low expression) was associated with extrathyroidal extension (ETE) (OR 4.48). Alternatively, a 'high activity' profile of RET (RET/PTC1 positive or high expression) and 'low activity' profile of BRAF (BRAFV600E negative or low expression) were associated with lymph node metastasis (LNM) (OR 12.80). Furthermore, in patients younger than 55years, a low expression of BRAF was associated with LNM (OR 17.65) and the presence of BRAFV600E mutation was associated with ETE (OR 2.76). Our results suggest that the analysis of demographic and molecular variables by CBVA could contribute to identify subsets of patients with aggressive histopathologic features, providing a potential guide to personalised surgical management of PTC.

Pomalidomide hybrids act as proteolysis targeting chimeras: Synthesis, anticancer activity and B-Raf degradation

As the first intracellular signaling molecule and the most frequently mutated oncogene, B-Raf represents an important target in cancer therapy. Here we report several pomalidomide hybrids acting as proteolysis targeting chimeras (PROTACs) for the degradation of B-Raf. Due to its high expression of B-Raf, MCF-7 cells are sensitive to these compounds. Among them, compound 2 can effectively kill cancer cells via inducing cells apoptosis. As a B-Raf degrader, compound 2 can accelerate the degradation of B-Raf by recruiting ubiquitin-proteasome system, and further affects the expression of Mcl-1, a downstream protein of B-Raf. The anticancer mechanism of compound 2 is quite different from its mother compound and cancer cells seem to be more sensitive to the degrader, hinting that degradation of B-Raf by PROTAC is a potential way for cancer treatment.

Relationships among KRAS mutation status, expression of RAS pathway signaling molecules, and clinicopathological features and prognosis of patients with colorectal cancer.

BACKGROUNDThe RAS/RAF/MEK/ERK and PI3K/AKT/mTOR signaling pathways all belong to mitogen-activated protein kinase (MAPK) signaling pathways, Mutations in any one of the upstream genes (such as the RAS gene or the BRAF gene) may be transmitted to the protein through transcription or translation, resulting in abnormal activation of the signaling pathway. This study investigated the relationship between the KRAS gene mutation and the clinicopathological features and prognosis of colorectal cancer (CRC), and the effect of KRAS mutations on its associated proteins in CRC, with an aim to clarify the cause of tumor progression and drug resistance caused by mutation of the KRAS gene.AIMTo investigate the KRAS gene and RAS pathway signaling molecules in CRC and to analyze their relationship with clinicopathological features and prognosisMETHODSColorectal cancer tissue specimens from 196 patients were analyzed for KRAS mutations using quantitative polymerase chain reaction and for KRAS, BRAF, MEK, and ERK protein expression levels using immunohistochemistry of tumor microarrays. To analyze differences of RAS pathway signaling molecule expression levels in different KRAS gene status, the relationships between these parameters and clinicopathological features, 4-year progression-free survival, and overall survival were analyzed by independent sample t test, Kaplan-Meier plots, and the log-rank test. Predictors of overall and disease-free survival were assessed using a Cox proportional hazards model.RESULTSOf the 196 patients, 62 (32%) carried mutations in codon 12 (53/62) or codon 13 (9/62) in exon 2 of the KRAS gene. KRAS, BRAF, ERK, and MEK protein expression was detected in 71.4%, 78.8%, 64.3%, and 50.8% of CRC tissues, respectively. There were no significant differences between KRAS mutation status and KRAS, BRAF, MEK, or ERK protein levels. Positive expression of KRAS and ERK was associated with poor tumor differentiation, and KRAS expression was also associated with age < 56 years. MEK expression was significantly associated with distant metastasis (P < 0.05). The 4-year progression-free survival rate, but not overall survival rate, was significantly higher in patients with KRAS-negative tumors than in those with KRAS-positive tumors (P < 0.05), whereas BRAF, MEK, and ERK expression was unrelated to survival. Multivariate analysis showed that only the expression of KRAS protein was a risk factor for tumor recurrence (P < 0.05). No other clinicopathological factors correlated with KRAS, BRAF, MEK, or ERK expression.CONCLUSIONKRAS gene mutations do not affect downstream protein expression in CRC. KRAS protein is associated with poor tumor differentiation, older age, and a risk of tumor recurrence.

Genetic Analysis Using a Gene Panel in 87 Caucasian Patients With Colorectal Cancer: Own Results and Review of Literature

Colorectal cancer (CRC) is the third most common cancer worldwide. The prognosis between left- and right-sided CRC differs, partly due to baseline differences as vascular supply. The purpose of the present study was to investigate whether there are genetic differences between left- and right-sided CRC. Eighty-seven patients with CRC (mean age: 61 years) were retrospectively included in the study. Blood samples were used for genetic analysis, by applying the sequencing research panel Ion AmpliSeq Colon and Lung Cancer Research Panel V2. Statistical analyses included Chi-square tests, Kaplan-Meier survival curves, and univariate/multivariate Cox-regression analyses. By testing the sequence of 22 genes included in the panel, a significant difference was detected between left- and right-sided CRC regarding the expression of BRAF and DDR2 genes, with mutations occurring more often in the right-sided CRC. In the multivariate setting, left-sided CRC only turned out as a significant positive prognostic parameter regarding progression-free survival, irrespective of the type of chemotherapy or BRAF and NRAS mutations. Tumour location was the only parameter proven to be an independent prognostic factor for CRC in the present study.

B-Raf deficiency impairs tumor initiation and progression in a murine breast cancer model.

Copy number gains, point mutations and epigenetic silencing events are increasingly observed in genes encoding elements of the Ras/Raf/MEK/ERK signaling axis in human breast cancer. The three Raf kinases A-Raf, B-Raf, and Raf-1 have an important role as gatekeepers in ERK pathway activation and are often dysregulated by somatic alterations of their genes or by the aberrant activity of receptor tyrosine kinases (RTKs) and Ras-GTPases. B-Raf represents the most potent Raf isoform and a critical effector downstream of RTKs and RAS proteins. Aberrant RTK signaling is mimicked by the polyoma middle T antigen (PyMT), which activates various oncogenic signaling pathways, incl. the RAS/ERK axis, in a similar manner as RTKs in human breast cancer. Mammary epithelial cell directed expression of PyMT in mice by the MMTV-PyMT transgene induces mammary hyperplasia progressing over adenoma to metastatic breast cancer with an almost complete penetrance. To understand the functional role of B-Raf in this model for luminal type B breast cancer, we crossed MMTV-PyMT mice with animals that either lack B-Raf expression in the mammary gland or express the signaling impaired B-RafAVKA mutant. The AVKA mutation prevents phosphorylation of T599 and S602 in the B-Raf activation loop and thereby activation of the kinase by upstream signals. We demonstrate for the first time that B-Raf expression and activation is important for tumor initiation in vivo as well as for lung metastasis. Isogenic tumor cell lines generated from conditional Braf knock-out or knock-in mice displayed a reduction in EGF-induced ERK pathway activity as well as in proliferation and invasive growth in three-dimensional matrigel cultures. Our results suggest that B-Raf, which has been hardly studied in the context of breast cancer, represents a critical effector of the PyMT oncoprotein and invite for an assessment of its functional role in human breast cancer.

Growth suppression by dual BRAF(V600E) and NRAS(Q61) oncogene expression is mediated by SPRY4 in melanoma

The underlying forces that shape mutational patterns within any type of cancer have been poorly characterized. One of the best preserved exclusionary relationships is that between BRAF(V600E) and NRAS(Q61) in melanomas. To explore possible mechanisms which could explain this phenomenon, we overexpressed NRAS(Q61) in a set of BRAF(V600E) melanoma lines and vice versa. Controlled expression of a second activating oncogene led to growth arrest (“synthetic suppression”) in a subset of cells, which was accompanied by cell cycle arrest and senescence in several melanoma cell lines along with apoptosis. Through differential gene expression analysis, we identified SPRY4 as the potential mediator of this synthetic response to dual oncogene suppression. Ectopic introduction of SPRY4 recapitulated the growth arrest phenotype of dual BRAF(V600E)/NRAS(Q61) expression while SPRY4 depletion led to a partial rescue from oncogenic antagonism. This study thus defined SPRY4 as a potential mediator of synthetic suppression, which is likely to contribute to the observed exclusivity between BRAF(V600E) and NRAS(Q61R) mutations in melanoma. Further leverage of the SPRY4 pathway may also hold therapeutic promise for NRAS(Q61) melanomas.

Heme oxygenase 1 facilitates cell proliferation via the B-Raf-ERK signaling pathway in melanoma

BackgroundDespite therapeutic advancements (e.g. B-RAF inhibitors) targeting cutaneous melanoma, many cellular processes, including inducible heme oxygenase 1 (HO-1), counteract treatments for malignancies. So there is an urgent need to find biological treatment targets, develop new therapeutic approaches and achieve longer responses. This study aimed to explore the relationship of HO-1 and B-Raf via mediating ERK1/2 signaling on cell cycle in melanoma.MethodsImmunohistochemistry was applied to evaluate the levels of HO-1 and B-Raf expression in melanoma tissues and adjacent healthy tissues. Co-immunoprecipitation (Co-IP) assessed the interaction of HO-1 with B-Raf. Further study overexpression and knock-down of HO-1 in A375 cell lines, especially knockout HO-1 using CRISPR-Cas9, verified HO-1 regulate cell proliferation in vivo and in vitro. Finally, Western blot analysis and qRT-PCR were performed to investigate the mechanisms by which HO-1 mediates cell cycle by B-RAF-ERK1/2 signaling.ResultsFirst, histology and Co-IP show that HO-1 interacts with B-Raf directly in melanoma tissue. Further study illustrated that HO-1 overexpression promotes melanoma cell proliferation while HO-1 reduction represses melanoma cell proliferation because of HO-1 affects cell cycle. Mechanistic studies revealed that HO-1 was associated with a marked activation of B-RAF-ERK1/2 signaling and led to CDK2/cyclin E activation, thereby promoting melanoma proliferation.ConclusionsOur result reveals a previously unknown mechanism that the HO-1-B-RAF-ERK axis plays an important role in melanoma cell proliferation. Therapeutic target on HO-1 could be a novel method for treating melanoma.