Endometrial cancer is the most commonly diagnosed gynaecological malignancy. Unfortunately, 15–20% of women demonstrate persistent or recurrent tumours that are refractory to current chemotherapies. ...We previously identified activating mutations in fibroblast growth factor receptor 2 (FGFR2) in 12% (stage I/II) to 17% (stage III/IV) endometrioid ECs and found that these mutations are associated with shorter progression‐free and cancer‐specific survival. Although FGFR inhibitors are undergoing clinical trials for treatment of several cancer types, little is known about the mechanism by which they induce cell death. We show that treatment with BGJ398, AZD4547 and PD173074 causes mitochondrial depolarization, cytochrome c release and impaired mitochondrial respiration in two FGFR2‐mutant EC cell lines (AN3CA and JHUEM2). Despite this mitochondrial dysfunction, we were unable to detect caspase activation following FGFR inhibition; in addition, the pan‐caspase inhibitor Z‐VAD‐FMK was unable to prevent cell death, suggesting that the cell death is caspase‐independent. Furthermore, while FGFR inhibition led to an increase in LC3 puncta, treatment with bafilomycin did not further increase lipidated LC3, suggesting that FGFR inhibition led to a block in autophagosome degradation. We confirmed that cell death is mitochondrial‐dependent as it can be blocked by overexpression of Bcl‐2 and/or Bcl‐XL. Importantly, we show that combining FGFR inhibitors with the BH3 mimetics ABT737/ABT263 markedly increased cell death in vitro and is more effective than BGJ398 alone in vivo, where it leads to marked tumour regression. This work may have implications for the design of clinical trials to treat a wide range of patients with FGFR‐dependent malignancies.
Bcl‐2 family members bound to Bax and Bak in FGFR‐mutant cancer cells maintain mitochondrial function and promote survival. FGFR inhibitor BGJ398 induces Bim expression which sequesters Bcl‐2 family members from Bax and Bak, leading to MOMP and cytochrome c release. Combining BGJ398 with the Bcl‐2/XL inhibitor ABT263 lowers the apoptotic threshold and enhances mitochondrial‐dependent cell death.
Patients with late stage resected cutaneous melanoma have poor overall survival (OS) and experience irreversible adverse events from systemic therapy. There is a clinical need to identify biomarkers ...to predict outcome. Performing germline/tumour whole-exome sequencing of 44 stage III/IV melanoma patients we identified pathogenic germline mutations in CDKN2A, CDK4, ATM, POLH, MRE11A, RECQL4 and XPC, affecting 7/44 patients. These mutations were associated with poor OS (p = 0.0082). We confirmed our findings in The Cancer Genome Atlas (TCGA) human skin cutaneous melanoma cohort where we identified pathogenic variants in 40/455 patients (p = 0.0203). Combining these cohorts (n = 499) further strengthened these findings showing germline carriers had worse OS (p = 0.0009). Additionally, we determined whether tumour mutation burden (TMB) or BRAF status were prognostic markers of survival. Low TMB rate (< 20 Mut/Mb; p = 0.0034) and BRAF p.V600 mutation (p = 0.0355) were associated with worse progression-free survival. Combining these biomarkers indicated that V600 mutant patients had significantly lower TMB (p = 0.0155). This was confirmed in the TCGA (n = 443, p = 0.0007). Integrative analysis showed germline mutation status conferred the highest risk (HR 5.2, 95% CI 1.72-15.7). Stage IV (HR 2.5, 0.74-8.6) and low TMB (HR 2.3, 0.57-9.4) were similar, whereas BRAF V600 status was the weakest prognostic biomarker (HR 1.5, 95% CI 0.44-5.2).
To identify 'melanoma-specific' microRNAs (miRNAs) we used an unbiased microRNA profiling approach to comprehensively study cutaneous melanoma in relation to other solid malignancies, which revealed ...233 differentially expressed (≥ 2 fold, p < 0.05) miRNAs. Among the top 20 most significantly different miRNAs was hsa-miR-514a-3p. miR-514a is a member of a cluster of miRNAs (miR-506-514) involved in initiating melanocyte transformation and promotion of melanoma growth. We found miR-514a was expressed in 38/55 (69%) melanoma cell lines but in only 1/34 (3%) other solid cancers. To identify miR-514a regulated targets we conducted a miR-514a-mRNA 'pull-down' experiment, which revealed hundreds of genes, including: CTNNB1, CDK2, MC1R, and NF1, previously associated with melanoma. NF1 was selected for functional validation because of its recent implication inacquired resistance to BRAFV600E-targeted therapy. Luciferase-reporter assays confirmed NF1 as a direct target of miR-514a and over-expression of miR-514a in melanoma cell lines inhibited NF1 expression, which correlated with increased survival of BRAFV600E cells treated with PLX4032. These data provide another mechanism for the dysregulation of the MAPK pathway which may contribute to the profound resistance associated with current RAF-targeted therapies.
Oesophageal adenocarcinoma (EAC) incidence is increasing and has a poor survival rate. Barrett's oesophagus (BE) is a precursor condition that is associated with EAC and often occurs in conjunction ...with chronic gastro-oesophageal reflux, however many individuals diagnosed with BE never progress to cancer. An understanding of the genomic features of BE and EAC may help with the early identification of at-risk individuals.
In this study, we assessed the genomic features of 16 BE samples using whole-genome sequencing. These included non-dysplastic samples collected at two time-points from two BE patients who had not progressed to EAC over several years. Seven other non-dysplastic samples and five dysplastic BE samples with high-grade dysplasia were also examined. We compared the genome profiles of these 16 BE samples with 22 EAC samples.
We observed that samples from the two non-progressor individuals had low numbers of somatic single nucleotide variants, indels and structural variation events compared to dysplastic and the remaining non-dysplastic BE. EAC had the highest level of somatic genomic variations. Mutational signature 17, which is common in EAC, was also present in non-dysplastic and dysplastic BE, but was not present in the non-progressors. Many dysplastic samples had mutations in genes previously reported in EAC, whereas only mutations in CDKN2A or in the fragile site genes appeared common in non-dysplastic samples. Rearrangement signatures were used to identify a signature associated with localised complex events such as chromothripsis and breakage fusion-bridge that are characteristic of EACs. Two dysplastic BE samples had a high contribution of this signature and contained evidence of localised rearrangements. Two other dysplastic samples also had regions of localised structural rearrangements. There was no evidence for complex events in non-dysplastic samples.
The presence of complex localised rearrangements in dysplastic samples indicates a need for further investigations into the role such events play in the progression from BE to EAC.
Identifying small extracellular vesicle (sEV) subpopulations based on their different molecular signatures could potentially reveal the functional roles in physiology and pathology. However, it is a ...challenge to achieve this aim due to the nano-sized dimensions of sEVs, low quantities of biological cargo each sEV carries, and our incomplete knowledge of identifying features capable of separating heterogeneous sEV subpopulations. Here, a sensitive, multiplexed, and nano-mixing-enhanced sEV subpopulation characterization platform (ESCP) is proposed to precisely determine the sEV phenotypic heterogeneity and understand the role of sEV heterogeneity in cancer progression and metastasis. The ESCP utilizes spatially patterned anti-tetraspanin-functionalized micro-arrays for sEV subpopulation sorting and nanobarcode-based surface-enhanced Raman spectroscopy for multiplexed read-outs. An ESCP has been used for investigating sEV phenotypic heterogeneity in terms of canonical sEV tetraspanin molecules and cancer-associated protein biomarkers in both cancer cell line models and cancer patient samples. Our data explicitly demonstrate the selective enrichment of tetraspanins and cancer-associated protein biomarkers, in particular sEV subpopulations. Therefore, it is believed that the ESCP could enable the evaluation and broader application of sEV subpopulations as potential diagnostic disease biomarkers.
The 5-year survival rate of pancreatic ductal adenocarcinoma (PDAC) is approximately 11% and has only improved marginally over the last three decades. For operable PDAC, resection and adjuvant ...FOLFIRINOX chemotherapy is standard of care. There is growing interest in perioperative regimens to improve outcomes. The non-randomized Phase II study "Gemcitabine and Abraxane for resectable Pancreatic cancer" (GAP) demonstrated the feasibility of perioperative gemcitabine/abraxane. Long-term survival in PDAC requires an effective immune response; hence, we undertook this translational study of the GAP trial cohort to identify immune-oncology biomarkers for clinical use.
We combined Nanostring nCounter technology with immunohistochemistry to investigate the correlation between gene expression and overall patient survival. Findings were investigated in samples from the International Cancer Genome Consortium (ICGC, n = 88) and the Australian Pancreatic Genome Initiative (APGI, n = 227).
We confirmed that human equilibrative nucleoside transporter 1 (hENT1) expression was not a prognostic marker in PDAC but patients with high levels of hENT1 were more likely to live longer than 24 months post-surgery. Additionally, CD274 (PD-L1) and two novel biomarkers of survival, cathepsin W (CTSW) and C-reactive protein (CRP), were identified in the GAP cohort (n = 19). CRP expression was confirmed in data from the ICGC. Although PD-L1 and CTSW proteins were not significant across all three cohorts, results show that low CRP mRNA and protein expression are associated with longer overall survival in all three patient groups.
PDAC patients with long survival have higher hENT1 expression levels. Furthermore, CRP expression is a biomarker of poor prognosis following perioperative chemotherapy and resection in PDAC patients and thus may be useful for identifying patients who could benefit from more aggressive adjuvant strategies.
We previously identified miR-4731-5p (miR-4731) as a melanoma-enriched microRNA following comparison of melanoma with other cell lines from solid malignancies. Additionally, miR-4731 has been found ...in serum from melanoma patients and expressed less abundantly in metastatic melanoma tissues from stage IV patients relative to stage III patients. As miR-4731 has no known function, we used biotin-labelled miRNA duplex pull-down to identify binding targets of miR-4731 in three melanoma cell lines (HT144, MM96L and MM253). Using the miRanda miRNA binding algorithm, all pulled-down transcripts common to the three cell lines (n=1092) had potential to be targets of miR-4731 and gene-set enrichment analysis of these (via STRING v9.1) highlighted significantly associated genes related to the 'cell cycle' pathway and the 'melanosome'. Following miR-4731 overexpression, a selection (n=81) of pull-down transcripts underwent validation using a custom qRT-PCR array. These data revealed that miR-4731 regulates multiple genes associated with the cell cycle (e.g. CCNA2, ORC5L, and PCNA) and the melanosome (e.g. RAB7A, CTSD, and GNA13). Furthermore, members of the synovial sarcoma X breakpoint family (SSX) (melanoma growth promoters) were also down-regulated (e.g. SSX2, SSX4, and SSX4B) as a result of miR-4731 overexpression. Moreover, this down-regulation of mRNA expression resulted in ablation or reduction of SSX4 protein, which, in keeping with previous studies, resulted in loss of 2D colony formation. We therefore speculate that loss of miR-4731 expression in stage IV patient tumours supports melanoma growth by, in part; reducing its regulatory control of SSX expression levels.
Summary
Melanocytic behavior, survival, and proliferation are regulated through a complex system of cell–cell adhesion molecules. Pathologic changes leading to development of malignant melanoma, ...upset the delicate homeostatic balance between melanocytes and keratinocytes and can lead to altered expression of cell–cell adhesion and cell–cell communication molecules. Malignant transformation of melanocytes frequently coincides with loss of E‐cadherin expression. We now show loss of another member of the superfamily of classical cadherins, H‐cadherin (CDH13), which may be involved in the development of malignant melanoma. The provided data show that H‐cadherin expression is lost in nearly 80% of the analyzed melanoma cell lines. Knockdown of H‐cadherin using siRNA increases invasive capacity in melanocytes. Functional assays show that the re‐expression of H‐cadherin decreases migration and invasion capacity, as well as anchorage‐independent growth in comparison to control melanoma cells. Furthermore, melanoma cells, which re‐express H‐cadherin via stable transfection show a reduction in rate of tumor growth in a nu/nu mouse tumor model in comparison to the parental control transfected cell lines. Our study presents for the first time the down‐regulation of H‐cadherin in malignant melanomas and its possible functional relevance in maintenance healthy skin architecture.
Circulating Tumor DNA Aoude, Lauren G.; Brosda, Sandra; Ng, Jessica ...
The Journal of molecular diagnostics : JMD,
October 2023, 2023-10-00, Letnik:
25, Številka:
10
Journal Article
Recenzirano
Odprti dostop
For patients with BRAF wild-type stage III and IV melanoma, there is an urgent clinical need to identify prognostic biomarkers and biomarkers predictive of treatment response. Circulating tumor DNA ...(ctDNA) is emerging as a blood-based biomarker and has shown promising results for many cancers, including melanoma. The purpose of this study was to identify targetable, tumor-derived mutations in patient blood that may lead to treatment alternatives and improved outcomes for patients with BRAF-negative melanoma. Using a CAncer Personalized Profiling by deep Sequencing (CAPP-seq) pan-cancer gene panel, ctDNA from 150 plasma samples (n = 106 patients) was assessed, including serial blood collections for a subset of patients (n = 16). ctDNA variants were detected in 85% of patients, all in targetable pathways, such as vascular endothelial growth factor receptor, epidermal growth factor receptor, phosphatidylinositol 3-kinase/AKT, Bcl2/mammalian target of rapamycin (mTOR), ALK/MET, and cyclin-dependent kinase 4/6. Patients with stage IV melanoma with low ctDNA concentrations, <10 ng/mL, had significantly better disease-specific survival and progression-free survival. Patients with both a high concentration of ctDNA and any detectable ctDNA variants had the worst prognosis. In addition, these results indicated that longitudinal changes in ctDNA correlated with treatment response and disease progression determined by radiology. This study confirms that ctDNA may be used as a noninvasive liquid biopsy to identify recurrent disease and detect targetable variants in patients with late-stage melanoma.
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