Molecularly targeted therapeutics have revolutionized the treatment of BRAFV600E-driven malignant melanoma, but the rapid development of resistance to BRAF kinase inhibitors (BRAFi) presents a significant obstacle. Here, we describe the identification of experimental melanoma therapeutics overcoming NRAS-based BRAFi-resistance employing a stringent genetic model of BRAFi-resistance, i.e. isogenic melanoma cell lines that differ only by NRAS mutational status (BRAFi-sensitive A375-BRAFV600E/NRASQ61 versus BRAFi-resistant A375 BRAFV600E/NRASQ61K). As a result of unbiased candidate screening, the clinical antimalarial mefloquine (MQ) was the only apoptogenic agent causing BRAFi-resistant A375 melanoma cell death at low micromolar concentrations. Comparative gene expression-array analysis (A375-BRAFV600E/NRASQ61 versus A375-BRAFV600E/NRASQ61K) revealed that MQ is a dual inducer of endoplasmic reticulum (ER) and redox stress responses that precede MQ-induced loss of viability. ER-trackerTM DPX fluorescence imaging and electron microscopy indicated ER swelling, accompanied by rapid induction of ER stress signaling (phospho-eIF2α, XBP-1s, ATF4). Fluo-4 AM-fluorescence indicated the occurrence of cytosolic calcium overload observable within seconds of MQ exposure. In a bioluminescent murine model employing intracranial injection of A375-Luc2 (BRAFV600E/NRASQ61K), an oral MQ regimen efficiently antagonized growth of brain metastases. Taken together, these data suggest feasibility of identifying valid candidates for drug repurposing aiming at chemotherapeutic elimination of malignant melanoma cells targeting BRAFV600E/NRASQ61K cells even if metastasized to the brain.

BRAF inhibitor (BRAFi) resistance compromises long term survivorship of malignant melanoma patients. Mutant NRASQ61K with constitutive activation of PI3K/AKT/mTOR signaling is a major mediator of BRAFi resistance, and BRAFi therapy can accelerate pre-existing RAS-mutant malignancies including NRAS-mutant leukemias in melanoma patients. Here, employing NanoString transcriptomic analysis of isogenic (A375-BRAFV600E versus A375-BRAFV600E/NRASQ61K) malignant melanoma cells we demonstrate that BRAFi treatment selectively targets BRAFV600E/NRASQ61K cells with induction of Epithelial to Mesenchymal Transition (EMT) gene expression, paradoxically promoting invasiveness and metastasis in vivo. Cancer progression nCounterTM pathway analysis identified 'EMT' and 'Proliferative Control' gene expression networks specific to BRAFV600E/NRASQ61K status. Strikingly, in contrast to BRAFi (vemurafenib, VEM)-induced antiproliferative and antiinvasive effects in BRAFV600E cells, VEM enhanced proliferation and invasiveness of BRAFV600E/NRASQ61K cells. RT2Profiler PCR array analysis confirmed VEM-upregulation of genes promoting EMT and proliferation [AKT1, MMP3, PDGFRB, RAC1, SPARC, ZEB1, ZEB2 (≤ 350-fold; p<0.05)] detectable only in BRAFV600E/NRASQ61K cells, while causing the expected downregulation of EMT-driver genes [CDH2, FN1, FOXC2, IGFBP4, MMP9, VIM, WNT5A (≤ 40-fold; p<0.05)] only in the BRAFV600E isogenic variant. Phenotypic transwell migration assays confirmed the seemingly opposing effects of VEM treatment on melanoma cell invasiveness [achieving blockade (BRAFV600E) or enhancement (BRAFV600E/NRASQ61K)]. In a bioluminescent SCID mouse metastasis model using A375-luc isogenic variants, VEM treatment (50 mg/kg; p.o., q.d.) enhanced lung tumor burden imposed by BRAFV600E/NRASQ61K cells, while blocking metastasis of BRAFV600E cells. Our data provide preclinical evidence that identifies a BRAFi-driven upregulation of EMT-related gene expression potentially enhancing invasiveness and metastasis in human BRAFV600E/NRASQ61K melanoma.

Chemical tanning is widely regarded as a safe alternative to solar UV-induced skin tanning, but the cutaneous biology impacted by chemical tanning remains largely unexplored. Chemical tanning is based on the formation of melanin-mimetic cutaneous pigments ('melanoidins') from spontaneous glycation reactions between epidermal amino acid/protein components and reactive sugars including the glycolytic ketose dihydroxyacetone (DHA). Here, we have examined cutaneous effects of acute DHA exposure on cultured human keratinocytes, epidermal reconstructs, and mouse skin employing gene epression array analysis and immunodetection. In human HaCaT keratinocytes, DHA (1-20 mM; 6 h) did not impair viability while causing a stress response with activation of phospho-protein signal transduction [p-p38, p-Hsp27, p-eIF2α] and gene expression (HSPA6, HMOX1, CRYAB, CCL3), not observed in response to the tanning-inactive DHA-control glycerol. Formation of DHA-specific advanced glycation endproducts resulting from posttranslational protein adduction was confirmed by mass spectrometric detection of N-ε-(carboxyethyl)-L-lysine and N7-carboxyethyl-L-arginine. Skin cells with CRISPR-Cas9 elimination of the carbonyl stress response gene GLO1 (glyoxalase 1) displayed hypersensitivity to DHA cytotoxicity. A topical DHA regimen elicited a similar stress response in human epidermal reconstructs [EpidermTM; 1-10% DHA in carrier; 6-24 h] as revealed by expression array (HSPA1A, HSPA6, HSPD1, IL6, DDIT3, EGR1) and IHC analysis (CEL, HO-1, p-Hsp27). In SKH1 mouse skin, gene expression analysis confirmed a topical DHA-induced stress response substantiated by IHC-detection of CEL and p-Hsp27. Given the worldwide use of chemical tanners including DHA in consumer products these prototype data deserve further molecular exploration in living human skin.

Epithelial-mesenchymal transition (EMT) is a key molecular pathway and promising therapeutic target in human melanomagenesis. Employing a phenotypic screen that examines therapeutic suppression of EMT in A375 malignant melanoma cells, we have now identified a drug-like chemical entity, 4-[(7-chloro-2-methoxy-1,5-dihydrobenzo[b] [1,5]naphthyridin-10-yl)imino]-2,6-bis(pyrrolidin-ylmethyl)cyclohexa-2,5-dien 1-one (PYD), as a potent EMT inhibitor. In a SCID mouse metastasis model employing time-resolved bioluminescent detection of lung tumorigenesis after tail-vain injection of stable A375-luciferase transfectants (monitored over three weeks post-injection), lung tumor burden imposed by A375-luc2 cells was attenuated by PYD-based systemic intervention (100 mg/kg; p.o., q.d., 3 d regimen), a curative outcome achievable without causation of any organ toxicity or negative impact on body weight. Differential transcriptomic analysis futher substantiated PYD-induced inhibition of EMT-related gene expression [upregulated: ECAD, IL1RN NUDT13, SNAI3; downregulated: BMP1, CTNNB1, FN1, FZD7, MMP2, MMP9, MYC, NCAD, SNAI2, TFRC, TWIST1, VIM, WNT5A, ZEB1, ZEB2 (up to tenfold; p < 0.05)]. Phenotypic transwell migration and MatrigelTM 3D-invasion assays, confirmed by PCR- and ELISA-based detection of MMP9 downregulation, revealed PYD as a micromolar inhibitor of melanoma cell invasiveness. Moreover, prolonged PYD exposure (24-48 h) caused melanoma cell apoptosis with complete rescue by pan-caspase inhibiton (zVAD-fmk). Taken together, these data provide strong preclinical evidence in support of the heretofore unexplored therapeutic efficacy of PYD blocking melanoma cell invasiveness and metastasis in vitro and in vivo.

Hypochlorous acid (HOCl) is the active oxidizing principle released by standard swimming pool disinfectants used on a global scale, but the cutaneous consequences of human exposure to HOCl remain largely unknown, posing a major public health concern relevant to populations around the world. Here, for the first time, we have profiled the HOCl-induced cutaneous stress response in reconstructed human epidermis and SKH1 hairless mouse skin. In addition, we have investigated the molecular consequences of co-exposure to solar simulated ultraviolet (UV) radiation and HOCl, a procedure mimicking environmental exposure experienced by recreational swimmers. Performing gene expression array analysis (Oxidative Stress Plus RT2 ProfilerTM PCR Array) in organotypic human reconstructed epidermis (EpidermTM) exposed to topical HOCl (10-100 μM; 30 min-6 h), we have identified TXNRD2, a gene encoding the mitochondrial antioxidant enzyme thioredoxin reductase 2, as a novel key response factor and mechanistic determinant of chlorination stress sensitivity. Importantly, in SKH1 hairless mouse skin, UV/HOCl co-exposure studies revealed that the HOCl-induced cutaneous antioxidant response blocks inflammatory gene expression (COX2, NOS2) elicited by acute UV exposure, a finding consistent with emerging clinical evidence in support of a therapeutic role of topical hypochlorite preparations for the suppression of inflammatory skin conditions (atopic dermatitis, psoriasis). Taken together, these data indicate that HOCl co-exposure may block solar UV-induced skin inflammatory signaling. Given the public health relevance of human exposure to chlorination stress in the context of drinking water disinfection and recreational swimming pool use, an urgent need exists for further detailed molecular investigations.

Background Colon cancer (CC) is the third most commonly diagnosed cancer in the USA. While the overall incidence is declining, it is rising alarmingly in young patients (EOCC). CC in young patients tends to be more aggressive and often diagnosed at more advanced stages and portend poorer prognosis. Our recently published data showed that EOCC is a distinct disease with unique molecular features compared to late-onset CC (LOCC). The Cartilage Oligomeric Matrix Protein (COMP) was shown to be significantly upregulated in EOCC and correlated with poor survival. However, the role of COMP in CC tumorigenesis, especially in young patients, is not well understood. Thus, the aim of this study was to elucidate the role of COMP in CC tumorigenesis by modulating COMP levels in vitro and test how it affects proliferation. Then, patient samples were evaluated by testing the levels of proliferation marker Ki67. In addition, this study investigates whether higher transcriptional mRNA levels of COMP seen in more aggressive early-onset CC correlate with protein levels compared to late-onset CC. Methods COMP mRNA levels in fresh frozen colon tumors (young: n = 5; old: n = 5) were assessed by quantitative PCR (qPCR). Additionally, CC cell lines were profiled for COMP expression to choose an in vitro model to study the role of COMP in CC tumorigenesis. HT-29 (low COMP expression) and CaCo-2 (high COMP expression) cells were used for in vitro proliferation studies. Immunohistochemical (IHC) analysis was conducted to assess COMP and Ki67 protein levels in formalin-fixed paraffin-embedded (FFPE) colon tumors. Results Significantly higher COMP expression levels were observed in fresh frozen EOCC compared to LOCC tumors. This observation confirmed our previously reported results from NanoString gene expression assay using FFPE samples. Cell proliferation was significantly increased in HT-29 and CaCo-2 cells upon treatment with human recombinant COMP protein after 48 and 72 h (P < 0.05). This increase was more profound in HT-29 cells. Staining for COMP and Ki67 revealed high COMP protein levels in EOCC compared to LOCC patients. Conclusion COMP mRNA and protein levels are significantly higher in EOCC patients. Higher COMP levels correlate with increased proliferation suggesting a role in CC tumorigenesis.

BACKGROUND Overall declines in incidence of rectal cancer (RC) in patients older than 50 years have been mostly attributed to improvement in treatment modalities and introduction of age-based screening. Recent studies, however, have shown a rise in the incidence of RC in patients younger than 50 years. The etiology of early-onset (EO) RC is not well understood. The aim of this study is to elucidate the molecular features of (EO) RC and show its uniqueness compared to late-onset (LO) disease. METHODS Two cohorts of patients with sporadic RC were identified. Tumors and matching non-involved tissues from six (EO) RC patients (< 50 years) and six (LO) RC patients (>65 years) were obtained from Pathology archives. Deparaffinized tissues were macro-dissected from FFPE sections, RNA isolated and used for expression profiling of 770 cancer related genes representing 13 canonical pathways. Statistical analysis was performed using the Gene Expression R-script module within the nCounter software v2.6. A gene was considered to be above background if the average count for the target gene was greater than the average counts for the eight negative control genes and if the P value of the t-test was less than 0.05. RESULTS When we compared rectal tumors to non-involved rectal tissues, changes in expression levels of 171 genes were statistically significant in early-onset group and 151 genes in late-onset group. Further comparative gene expression analysis between early- and late-onset rectal tumors normalized to their matching non-involved tissues revealed that changes in expression of 65 genes were unique to early-onset rectal tumors with 16 genes being up- and 49 genes down-regulated using the cutoff criteria of expression levels difference >2 fold and p-value <0.01. At the pathway level, MAPK signaling was the most deregulated pathway in early-onset rectal tumors compared to PI3K-AKT signaling pathway being the most deregulated in late-onset rectal tumors. CONCLUSIONS Results of this study suggest that sporadic early-onset rectal cancer is characterized by distinct molecular events compared to late-onset disease.

Introduction: Native Americans (NA) form a unique cohort of colon cancer (CC) patients among whom the variability in demographics and cancer characteristics remains unclear.Methods: We abstracted the national estimates for NA with CC using the Surveillance, Epidemiology, and End Result (SEER) database. Trend analysis of incidence, variation in location and patient demographic analysis were performed.Results: A total number of 26,674 NA with CC were reported during the 12-year study period. While the overall incidence of CC decreased by 12% during the study period, incidence increased by 38% in NA. Incidence of CC was more prevalent and higher increase (42%) seen in NA females than males (p = 0.02; 34%). Stage III tumors represented 29% of all CC, sigmoid colon the most common site location (38%) with 72% of all tumors being moderately differentiated. 55% tumors were localized in left, 36% in right and 9% in transverse colon. 92% of the NA were insured.Conclusion: Incidence of CC continues to rise in NA with majority of CC presented at higher stage and moderate differentiation. Published by Elsevier Inc.

UNLABELLED Lung cancer is one of the leading cause of cancer-related death around the world with the majority of diagnoses being non-small cell lung cancer (NSCLC). Given the poor survival rate and efficacy of current therapy for NSCLC, there is a need to identify and develop new therapeutic targets for treatment. We have observed significantly up-regulated levels of Fn14 in clinical samples of lung cancer relative to normal adjacent tissue. However, the functional role of Fn14 in these tumors is not understood yet. We used RT-PCR to establish the Fn14 expression profile in various NSCLC cell lines. Using isogenic variants of H460 NSCLC cell line with low, intermediate and high Fn14 expression as a cellular model, we determined that increased levels of integrin α6 in cells over-expressing Fn14 is suggestive of an important role of α6β1-fn14 interactions in motility of lung carcinoma and formation of metastases. Enhanced levels of Fn14 correlated with higher tumor cell migration and invasion in an MMP-1 dependent manner. Cells over-expressing Fn14 showed increased in vivo tumor formation with metastatic capacity to lymph nodes, lungs and liver. Thus, this research may be a step toward developing improved treatment strategies for NSCLC by improved detection and inhibition of metastases. KEYWORDS Fn14, TNFRSF12A, non-small cell lung cancer, H460 cells, motility, tumor formation and metastasis, integrin α6.