RESUMO
BACKGROUND & AIMS: About 15% of intrahepatic cholangiocarcinomas (iCCAs) express fibroblast growth factor receptor 2 (FGFR2) fusion proteins (FFs), usually alongside mutational inactivation of TP53, CDKN2A or BAP1. In FFs, FGFR2 residues 1-768 fuse to sequences encoded by a diverse array of partner genes (>60) causing oncogenic FF activation. While FGFR-specific tyrosine kinase inhibitors (F-TKI) provide clinical benefit in FF+ iCCA, responses are partial and/or limited by resistance mechanisms, such as the V565F substitution in the FGFR2 gatekeeper residue. Improving on FF targeting in iCCA therefore remains a critical unmet need. Herein, we aimed to generate a murine model of FF-driven iCCA and use this to uncover actionable FF-associated dependencies. METHODS: Four iCCA FFs carrying different fusion sequences were expressed in Tp53-/- mouse liver organoids. Tumorigenic properties of genetically modified liver organoids were assessed by transplantation into immuno-deficient mice. Cellular models derived from neoplastic lesions were exploited for pre-clinical studies. RESULTS: Transplantation of FF-expressing liver organoids yielded tumors diagnosed as CCA based on histological, phenotypic and transcriptomic analyses. The penetrance of this tumorigenic phenotype was influenced by FF identity. Tumor organoids and 2D cell lines derived from CCA lesions were addicted to FF signaling via Ras-Erk, regardless of FF identity or V565F mutation. Dual blockade of FF and the Ras-Erk pathway by concomitant pharmacological inhibition of FFs and Mek1/2 provided greater therapeutic efficacy than single agent F-TKI in vitro and in vivo. CONCLUSIONS: FF-driven iCCA pathogenesis was successfully modeled on a Tp53-/- murine background, revealing biological heterogeneity among structurally different FFs. Double blockade of FF-ERK signaling deserves consideration for precision-based approaches against human FF+ iCCA. LAY SUMMARY: Intrahepatic cholangiocarcinoma (iCCA) is a rare cancer that is difficult to treat. A subtype of iCCA is caused by genomic alterations that generate oncogenic drivers known as FGFR2 fusions. Patients with FGFR2 fusions respond to FGFR inhibitors, but clinical responses are often of modest duration. We used animal and cellular models to show that FGFR2 fusions require the activity of a downstream effector named Mek1/2. We found that dual blockade of FGFR2 fusions and Mek1/2 was more effective than isolated inhibition of FGFR2 fusions, pointing to the potential clinical utility of dual FGFR2-MEK1/2 blockade in patients with iCCA.
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Colangiocarcinoma/etiologia , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/antagonistas & inibidores , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/genética , Proteína Supressora de Tumor p53/efeitos dos fármacos , Análise de Variância , Animais , Linhagem Celular/metabolismo , Colangiocarcinoma/genética , Modelos Animais de Doenças , Camundongos , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
The telomeric protein TRF2 is overexpressed in several human malignancies and contributes to tumorigenesis even though the molecular mechanism is not completely understood. By using a high-throughput approach based on the multiplexed Luminex X-MAP technology, we demonstrated that TRF2 dramatically affects VEGF-A level in the secretome of cancer cells, promoting endothelial cell-differentiation and angiogenesis. The pro-angiogenic effect of TRF2 is independent from its role in telomere capping. Instead, TRF2 binding to a distal regulatory element promotes the expression of SULF2, an endoglucosamine-6-sulfatase that impairs the VEGF-A association to the plasma membrane by inducing post-synthetic modification of heparan sulfate proteoglycans (HSPGs). Finally, we addressed the clinical relevance of our findings showing that TRF2/SULF2 expression is a worse prognostic biomarker in colorectal cancer (CRC) patients.
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Neoplasias do Colo/metabolismo , Sulfotransferases/genética , Proteína 2 de Ligação a Repetições Teloméricas/metabolismo , Microambiente Tumoral , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Linhagem Celular Tumoral , Neoplasias do Colo/irrigação sanguínea , Neoplasias do Colo/patologia , Proteoglicanas de Heparan Sulfato/química , Proteoglicanas de Heparan Sulfato/metabolismo , Heparina/metabolismo , Humanos , Masculino , Camundongos , Camundongos Nus , Metástase Neoplásica , Neovascularização Patológica , Sulfatases , Sulfotransferases/biossíntese , Proteína 2 de Ligação a Repetições Teloméricas/deficiência , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Telomere repeat binding factor 2 (TRF2) has been increasingly recognized to be involved in telomere maintenance and DNA damage response. Here, we show that TRF2 directly binds SIRT6 in a DNA independent manner and that this interaction is increased upon replication stress. Knockdown of SIRT6 up-regulates TRF2 protein levels and counteracts its down-regulation during DNA damage response, leading to cell survival. Moreover, we report that SIRT6 deactetylates in vivo the TRFH domain of TRF2, which in turn, is ubiquitylated in vivo activating the ubiquitin-dependent proteolysis. Notably, overexpression of the TRF2cT mutant failed to be stabilized by SIRT6 depletion, demonstrating that the TRFH domain is required for its post-transcriptional modification. Finally, we report an inverse correlation between SIRT6 and TRF2 protein expression levels in a cohort of colon rectal cancer patients. Taken together our findings describe TRF2 as a novel SIRT6 substrate and demonstrate that acetylation of TRF2 plays a crucial role in the regulation of TRF2 protein stability, thus providing a new route for modulating its expression level during oncogenesis and damage response.
Assuntos
Dano ao DNA , Sirtuínas/metabolismo , Proteína 2 de Ligação a Repetições Teloméricas/metabolismo , Acetilação , Antineoplásicos Fitogênicos/farmacologia , Camptotecina/farmacologia , Linhagem Celular , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Humanos , Imuno-Histoquímica , Modelos Moleculares , Poli(ADP-Ribose) Polimerases/metabolismo , Ligação Proteica , Conformação Proteica , Estabilidade Proteica , Proteólise/efeitos dos fármacos , Proteínas Recombinantes de Fusão/metabolismo , Sirtuínas/química , Especificidade por Substrato , Proteína 2 de Ligação a Repetições Teloméricas/química , Proteína 2 de Ligação a Repetições Teloméricas/genética , UbiquitinaçãoRESUMO
OBJECTIVE: Cancer stem cells (CSCs) are responsible for tumour formation and spreading, and their targeting is required for tumour eradication. There are limited therapeutic options for advanced colorectal cancer (CRC), particularly for tumours carrying RAS-activating mutations. The aim of this study was to identify novel CSC-targeting strategies. DESIGN: To discover potential therapeutics to be clinically investigated as single agent, we performed a screening with a panel of FDA-approved or investigational drugs on primary CRC cells enriched for CSCs (CRC-SCs) isolated from 27 patients. Candidate predictive biomarkers of efficacy were identified by integrating genomic, reverse-phase protein microarray (RPPA) and cytogenetic analyses, and validated by immunostainings. DNA replication stress (RS) was increased by employing DNA replication-perturbing or polyploidising agents. RESULTS: The drug-library screening led to the identification of LY2606368 as a potent anti-CSC agent acting in vitro and in vivo in tumour cells from a considerable number of patients (â¼36%). By inhibiting checkpoint kinase (CHK)1, LY2606368 affected DNA replication in most CRC-SCs, including RAS-mutated ones, forcing them into premature, lethal mitoses. Parallel genomic, RPPA and cytogenetic analyses indicated that CRC-SCs sensitive to LY2606368 displayed signs of ongoing RS response, including the phosphorylation of RPA32 and ataxia telangiectasia mutated serine/threonine kinase (ATM). This was associated with mutation(s) in TP53 and hyperdiploidy, and made these CRC-SCs exquisitely dependent on CHK1 function. Accordingly, experimental increase of RS sensitised resistant CRC-SCs to LY2606368. CONCLUSIONS: LY2606368 selectively eliminates replication-stressed, p53-deficient and hyperdiploid CRC-SCs independently of RAS mutational status. These results provide a strong rationale for biomarker-driven clinical trials with LY2606368 in patients with CRC.
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Antineoplásicos/farmacologia , Quinase 1 do Ponto de Checagem/efeitos dos fármacos , Neoplasias Colorretais/tratamento farmacológico , Células-Tronco Neoplásicas/efeitos dos fármacos , Pirazinas/farmacologia , Pirazóis/farmacologia , Linhagem Celular Tumoral , Quinase 1 do Ponto de Checagem/genética , Neoplasias Colorretais/genética , Replicação do DNA/efeitos dos fármacos , Humanos , Imuno-Histoquímica , Mutação , Células-Tronco Neoplásicas/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Proteína Supressora de Tumor p53/genéticaRESUMO
We have previously reported that nuclear expression of the Hippo transducer TAZ in association with Wnt pathway mutations negatively impacts survival outcomes in advanced gastric cancer (GC) patients. Here, we extended these previous findings by investigating another oncogenic cooperation, namely, the interplay between YAP, the TAZ paralogue, and p53. The molecular output of the YAP-p53 cooperation is dependent on TP53 mutational status. In the absence of mutations, the YAP-p53 crosstalk elicits a pro-apoptotic response, whereas in the presence of TP53 mutations it activates a pro-proliferative transcriptional program. In order to study this phenomenon, we re-analyzed data from 83 advanced GC patients treated with chemotherapy whose tissue samples had been characterized for YAP expression (immunohistochemistry, IHC) and TP53 mutations (deep sequencing). In doing so, we generated a molecular model combining nuclear YAP expression in association with TP53 missense variants (YAP+/TP53mut(mv)). Surprisingly, this signature was associated with a decreased risk of disease progression (multivariate Cox for progression-free survival: HR 0.53, 95% CI 0.30-0.91, p = 0.022). The YAP+/TP53mut(mv) model was also associated with better OS in the subgroup of patients who received chemotherapy beyond the first-line setting (multivariate Cox: HR 0.36, 95% CI 0.16-0.81, p = 0.013). Collectively, our findings suggest that the oncogenic cooperation between YAP and mutant p53 may translate into better survival outcomes. This apparent paradox can be explained by the pro-proliferative program triggered by YAP and mutant p53, that supposedly renders cancer cells more vulnerable to cytotoxic therapies.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Mutação de Sentido Incorreto , Fosfoproteínas/genética , Neoplasias Gástricas/genética , Proteína Supressora de Tumor p53/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Adulto , Idoso , Antineoplásicos/uso terapêutico , Proliferação de Células , Progressão da Doença , Intervalo Livre de Doença , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , Fosfoproteínas/metabolismo , Modelos de Riscos Proporcionais , Análise de Regressão , Neoplasias Gástricas/mortalidade , Fatores de Transcrição , Resultado do Tratamento , Proteína Supressora de Tumor p53/metabolismo , Proteínas de Sinalização YAPRESUMO
BACKGROUND: An extensive crosstalk co-regulates the Hippo and Wnt pathway. Preclinical studies revealed that the Hippo transducers YAP/TAZ mediate a number of oncogenic functions in gastric cancer (GC). Moreover, comprehensive characterization of GC demonstrated that the Wnt pathway is targeted by oncogenic mutations. On this ground, we hypothesized that YAP/TAZ- and Wnt-related biomarkers may predict clinical outcomes in GC patients treated with chemotherapy. METHODS: In the present study, we included 86 patients with advanced GC treated with first-line chemotherapy in prospective phase II trials or in routine clinical practice. Tissue samples were immunostained to evaluate the expression of YAP/TAZ. Mutational status of key Wnt pathway genes (CTNNB1, APC and FBXW7) was assessed by targeted DNA next-generation sequencing (NGS). Survival curves were estimated and compared by the Kaplan-Meier product-limit method and the log-rank test, respectively. Variables potentially affecting progression-free survival (PFS) were verified in univariate Cox proportional hazard models. The final multivariate Cox models were obtained with variables testing significant at the univariate analysis, and by adjusting for all plausible predictors of the outcome of interest (PFS). RESULTS: We observed a significant association between TAZ expression and Wnt mutations (Chi-squared p = 0.008). Combined TAZ expression and Wnt mutations (TAZpos/WNTmut) was more frequently observed in patients with the shortest progression-free survival (negative outliers) (Fisher p = 0.021). Uni-and multivariate Cox regression analyses revealed that patients whose tumors harbored the TAZpos/WNTmut signature had an increased risk of disease progression (univariate Cox: HR 2.27, 95% CI 1.27-4.05, p = 0.006; multivariate Cox: HR 2.73, 95% CI 1.41-5.29, p = 0.003). Finally, the TAZpos/WNTmut signature negatively impacted overall survival. CONCLUSIONS: Collectively, our findings indicate that the oncogenic YAP/TAZ-Wnt crosstalk may be active in GC, conferring chemoresistant traits that translate into adverse survival outcomes.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Mutação/genética , Proteínas Serina-Treonina Quinases/metabolismo , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologia , Via de Sinalização Wnt/genética , Idoso , Biomarcadores Tumorais/metabolismo , Feminino , Via de Sinalização Hippo , Humanos , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Estadiamento de Neoplasias , Modelos de Riscos Proporcionais , Análise de Sobrevida , Transativadores , Fatores de Transcrição , Proteínas com Motivo de Ligação a PDZ com Coativador Transcricional , Resultado do TratamentoRESUMO
The DNA damage response (DDR) network is exploited by cancer cells to withstand chemotherapy. Gastric cancer (GC) carries deregulation of the DDR and harbors genetic defects that fuel its activation. The ATM-Chk2 and ATR-Chk1-Wee1 axes are deputed to initiate DNA repair. Overactivation of these pathways in cancer cells may represent an adaptive response for compensating genetic defects deregulating G1 -S transition (e.g., TP53) and ATM/ATR-initiated DNA repair (e.g., ARID1A). We hypothesized that DDR-linked biomarkers may predict clinical outcomes in GC patients treated with chemotherapy. Immunohistochemical assessment of DDR kinases (pATM, pChk2, pChk1 and pWee1) and DNA damage markers (γ-H2AX and pRPA32) was performed in biological samples from 110 advanced GC patients treated with first-line chemotherapy, either in phase II trials or in routine clinical practice. In 90 patients, this characterization was integrated with targeted ultra-deep sequencing for evaluating the mutational status of TP53 and ARID1A. We recorded a positive association between the investigated biomarkers. The combination of two biomarkers (γ-H2AXhigh /pATMhigh ) was an adverse factor for both progression-free survival (multivariate Cox: HR 2.23, 95%CI: 1.47-3.40) and overall survival (multivariate Cox: HR: 2.07, 95%CI: 1.20-3.58). The relationship between the γ-H2AXhigh /pATMhigh model and progression-free survival was consistent across the different TP53 backgrounds and was maintained in the ARID1A wild-type setting. Conversely, this association was no longer observed in an ARID1A-mutated subgroup. The γ-H2AXhigh /pATMhigh model negatively impacted survival outcomes in GC patients treated with chemotherapy. The mutational status of ARID1A, but apparently not TP53 mutations, affects its predictive significance.
Assuntos
Antineoplásicos/uso terapêutico , Dano ao DNA/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Neoplasias Gástricas/tratamento farmacológico , Idoso , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Biomarcadores Tumorais/metabolismo , Proteínas de Ciclo Celular , Proteínas de Ligação a DNA/metabolismo , Intervalo Livre de Doença , Feminino , Mucosa Gástrica/metabolismo , Histonas/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas Quinases/metabolismo , Transdução de Sinais/efeitos dos fármacos , Estômago/efeitos dos fármacos , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismoRESUMO
Overcoming resistance to standard anticancer treatments represents a significant challenge. The interest regarding cancer stem cells, a cellular population that has the ability to self-renew and to propagate the tumor, was prompted by experimental evidence delineating the molecular mechanisms that are selectively activated in this cellular subset in order to survive chemotherapy. This has also stimulated combination strategies aimed at rendering cancer stem cells vulnerable to anticancer agents. Moreover, cancer stem cells offer a unique opportunity for modeling human cancers in mice, thus emerging as a powerful tool for testing novel drugs and combinations in a simulation of human disease. These novel animal models may lay the foundation for a new generation of clinical trials aimed at anticipating the benefit to patients of anticancer therapies.
Assuntos
Neoplasias/metabolismo , Células-Tronco Neoplásicas/metabolismo , Animais , Apoptose , Diferenciação Celular , Proliferação de Células , Sobrevivência Celular , Dano ao DNA , Reparo do DNA , Resistencia a Medicamentos Antineoplásicos , Humanos , Modelos Biológicos , Neoplasias/genética , Neoplasias/terapia , Tolerância a Radiação , Transdução de Sinais , Falha de Tratamento , Resultado do TratamentoRESUMO
Among soluble actors that have emerged as druggable factors, the chemokine interleukin-8 (IL-8) has emerged as a possible determinant of response to immunotherapy and targeted treatment in several cancer types; however, its prognostic/predictive role in colorectal cancer (CRC) remains to be established. We: (i) conducted a systematic review of published literature on IL-8 expression in CRC; (ii) searched public transcriptomics databases; (iii) investigated IL-8 expression, by tumor and infiltrating cells, in a series of CRC samples; and (iv) carried out a meta-analysis of published literature correlating IL-8 expression and CRC prognosis. IL-8 possesses an important role as a mediator of the bidirectional crosstalk between tumor/stromal cells. Transcriptomic analysis indicated that specific IL-8 transcripts were significantly overexpressed in CRC compared to normal colon mucosa. Moreover, in our series we observed a statistically significant correlation between PTEN-loss and IL-8 expression by infiltrating mononuclear and tumor cells. In total, 12 papers met our meta-analysis inclusion criteria, demonstrating that high IL-8 levels significantly correlated with shorter overall survival and progression-free survival. Sensitivity analysis demonstrated a highly significant correlation with outcome for circulating, but not for tissue-detected, IL-8. IL-8 is overexpressed in CRC tissues and differentially produced by tumor or stromal components depending on CRC genetic background. Moreover, circulating IL-8 represents a strong prognostic factor in CRC, suggesting its use in the refining of prognostic CRC assessment and potentially the tailoring of therapeutic strategies in individual CRC patients.
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Homeodomain-interacting protein kinase 2 (HIPK2) is an evolutionary conserved kinase that has gained attention as a fine tuner of multiple signaling pathways, among which those commonly altered in colorectal cancer. The aim of this study was to evaluate the relationship of HIPK2 expression with progression markers and mutational pattern and gain insights into the contribution of HIPK2 activity in colorectal cancer. We evaluated a retrospective cohort of colorectal cancer samples by IHC for HIPK2 expression and by next-generation sequencing (NGS) for the detection of mutations of cancer associated genes. We show that the percentage of HIPK2-positive cells increases with tumor progression, significantly correlates with tumor-node-metastasis (TNM) staging and associates with a worse outcome. In addition, we observed that high HIPK2 expression significantly associates with KRAS mutations but not with other cancer-related genes. Functional characterization of the link between HIPK2 and KRAS show that activation of the RAS pathway either due to KRAS mutation or via upstream receptor stimulation, increases HIPK2 expression at the protein level. Of note, HIPK2 physically participates in the active RAS complex while HIPK2 depletion impairs ERK phosphorylation and the growth of tumors derived from KRAS mutated colorectal cancer cells. Overall, this study identifies HIPK2 as a prognostic biomarker candidate in patients with colorectal cancer and underscores a previously unknown functional link between HIPK2 and the KRAS signaling pathway. IMPLICATIONS: Our data indicate HIPK2 as a new player in the complex picture of the KRAS signaling network, providing rationales for future clinical studies and new treatment strategies for KRAS mutated colorectal cancer.
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Neoplasias Colorretais , Proteínas Proto-Oncogênicas p21(ras) , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Neoplasias Colorretais/patologia , Humanos , Mutação , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Estudos Retrospectivos , Transdução de Sinais/genéticaRESUMO
Colorectal cancer (CRC) is the third most frequently diagnosed type of cancer worldwide. Stage II CRC accounts for ~25% all CRC cases and their management after surgical resection remains a clinical dilemma due to the lack of reliable criteria for identifying patients who may benefit from adjuvant chemotherapy. Homeodomaininteracting protein kinase 2 (HIPK2), a multifunctional kinase involved in numerous signaling pathways, serves several key roles in cell response to different types of stresses, including chemotherapyinduced genotoxic damage. In the present study, immunohistochemistry was performed for HIPK2 on a tissue microarray of primary human tumor samples from 84 patients with stage II CRC, treated (30 patients) or not treated (54 patients) with adjuvant chemotherapy, and sequenced for the TP53 gene, a key HIPK2 target in genotoxic damage response. It was observed that, regardless of the TP53 gene status, a high percentage of HIPK2+ cells was associated with therapeutic vulnerability in stage II CRC, suggesting a contribution of HIPK2 to drugresponse in vivo. For the in vitro characterization, HIPK2 was depleted in human CRC cells by CRISPR/Cas9 or RNA interference. HIPK2proficient and HIPK2defective cells were evaluated for their response to 5fluorouracil (5FU) and oxaliplatin (OXA). The results revealed that HIPK2 depletion induced resistance to 5FU and OXA, and that this resistance was not overcome by brusatol, an inhibitor of the antioxidant response regulator nuclear factor erythroid 2related factor 2 (NRF2), which is frequently overexpressed in CRC. By contrast, cell sensitivity to 5FU and OXA was further induced by brusatol supplementation in HIPK2proficient cells, further supporting the contribution of HIPK2 in chemotherapy response. Overall, the present results suggested that HIPK2 may be a potential predictive marker for adjuvanttreated stage II CRC and for prospective therapy with NRF2 modulators.
Assuntos
Proteínas de Transporte/metabolismo , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Antineoplásicos/farmacologia , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Proteínas de Transporte/genética , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Quimioterapia Adjuvante , Neoplasias Colorretais/genética , Neoplasias Colorretais/mortalidade , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Fluoruracila/farmacologia , Humanos , Fator 2 Relacionado a NF-E2/antagonistas & inibidores , Fator 2 Relacionado a NF-E2/metabolismo , Estadiamento de Neoplasias , Oxaliplatina/farmacologia , Proteínas Serina-Treonina Quinases/genética , Quassinas/farmacologia , Quassinas/uso terapêutico , Análise de Sobrevida , Proteína Supressora de Tumor p53/genéticaRESUMO
Inflammation might substantially contribute to the limited therapeutic success of current systemic therapies in colorectal cancer (CRC). Amongst cytokines involved in CRC biology, the proinflammatory chemokine IL-8 has recently emerged as a potential prognostic/predictive biomarker. Here, we show that BRAF mutations and PTEN-loss are associated with high IL-8 levels in CRC models in vitro and that BRAF/MEK/ERK, but not PI3K/mTOR, targeting controls its production in different genetic contexts. In particular, we identified a BRAF/ERK2/CHOP axis affecting IL-8 transcription, through regulation of CHOP subcellular localization, and response to targeted inhibitors. Moreover, RNA Pol II and an open chromatin status in the CHOP-binding region of the IL-8 gene promoter cooperate towards increased IL-8 expression, after a selective BRAF inhibition. Overall, our data show that IL-8 production is finely and differentially regulated depending on the tumor genetic context and might be targeted for therapeutic purposes in molecularly defined subgroups of CRC patients.
Assuntos
Neoplasias Colorretais/metabolismo , Interleucina-8/metabolismo , Proteínas Proto-Oncogênicas B-raf/metabolismo , Fator de Transcrição CHOP/metabolismo , Western Blotting , Linhagem Celular Tumoral , Imunofluorescência , Regulação Neoplásica da Expressão Gênica , Humanos , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Mutagênese Sítio-Dirigida , Proteínas Proto-Oncogênicas B-raf/fisiologia , RNA Polimerase II/metabolismo , Transdução de SinaisRESUMO
Collecting duct carcinoma (CDC) is rare and aggressive histology of kidney cancers. Although different therapeutic approaches have been tested, the 2-year survival remains very poor. Since CDC exhibits overlapping features with urothelial carcinoma, the analysis of shared molecular alterations could provide new insights into the understanding of this rare disease and also therapeutic options. We collected 26 CDC cases, and we assessed HER2 protein expression by immunohistochemistry (IHC) and gene amplification by fluorescence in-situ hybridization (FISH) according to 2018 ASCO/CAP HER2-testing recommendations. Six out of twenty-six (23%) tumors showed HER2 positive staining. In particular, 3+ score was present in 2/6 cases (33%), 2+ in 3/6 cases (50%) and 1+ in 1/6 cases (17%). The 6 HER2+ tumors were also analyzed by FISH to assess gene copy number. One out of six CDC with IHC 3+ was also HER2 amplified, showing an average HER2 copy number ≥4.0 (10.85) and a HER2/CEP17 ratio ≥ (5.63), while the 5/6 cases were HER2 negative. Based on the 2018 ASCO/CAP guidelines overall, 2/26 CDC cases (8%) were HER2+. The present study provides evidence for testing, in future studies, HER2 to assess its clinical value as a novel target for the treatment of this highly malignant cancer.
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BACKGROUND: Colorectal cancer is one of most common tumors in developed countries and, despite improvements in treatment and diagnosis, mortality rate of patients remains high, evidencing the urgent need of novel biomarkers to properly identify colorectal cancer high-risk patients that would benefit of specific treatments. Recent works have demonstrated that the telomeric protein TRF2 is over-expressed in colorectal cancer and it promotes tumor formation and progression through extra-telomeric functions. Moreover, we and other groups evidenced, both in vitro on established cell lines and in vivo on tumor bearing mice, that TRF2 regulates the vascularization mediated by VEGF-A. In the present paper, our data evidence a tight correlation between TRF2 and VEGF-A with prognostic relevance in colorectal cancer patients. METHODS: For this study we sampled 185 colorectal cancer patients surgically treated and diagnosed at the Regina Elena National Cancer Institute of Rome and investigated the association between the survival outcome and the levels of VEGF-A and TRF2. RESULTS: Tissue microarray immunohistochemical analyses revealed that TRF2 positively correlates with VEGF-A expression in our cohort of patients. Moreover, analysis of patients' survival, confirmed in a larger dataset of patients from TCGA, demonstrated that co-expression of TRF2 and VEGF-A correlate with a poor clinical outcome in stage I-III colorectal cancer patients, regardless the mutational state of driver oncogenes. CONCLUSIONS: Our results permitted to identify the positive correlation between high levels of TRF2 and VEGF-A as a novel prognostic biomarker for identifying the subset of high-risk colorectal cancer patients that could benefit of specific therapeutic regimens.
Assuntos
Adenocarcinoma/mortalidade , Biomarcadores Tumorais/metabolismo , Neoplasias Colorretais/mortalidade , Cirurgia Colorretal/mortalidade , Recidiva Local de Neoplasia/mortalidade , Proteína 2 de Ligação a Repetições Teloméricas/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Adenocarcinoma/metabolismo , Adenocarcinoma/secundário , Adenocarcinoma/cirurgia , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Neoplasias Colorretais/cirurgia , Feminino , Seguimentos , Regulação Neoplásica da Expressão Gênica , Humanos , Metástase Linfática , Masculino , Pessoa de Meia-Idade , Invasividade Neoplásica , Recidiva Local de Neoplasia/metabolismo , Recidiva Local de Neoplasia/patologia , Recidiva Local de Neoplasia/cirurgia , Prognóstico , Estudos Retrospectivos , Taxa de SobrevidaRESUMO
BACKGROUND: Liquid biopsy (LB) in early-stage, non-metastatic colorectal cancer (CRC) must be sensitive enough to detect extremely low circulating tumor DNA (ctDNA) levels. This challenge has been seldom and non-systematically investigated. METHODS: Next generation sequencing (NGS) and digital PCR (dPCR) were combined to test tumor DNAs (tDNAs) and paired ctDNAs collected at surgery from 39 patients, 12 of whom were also monitored during the immediate post-surgery follow up. Patients treated for metastatic disease (n = 14) were included as controls. RESULTS: NGS and dPCR concordantly (100% agreement) called at least one single nucleotide variant (SNV) in 34 tDNAs, estimated differences in allelic frequencies being negligible (±1.4%). However, despite dPCR testing, SNVs were only detectable in 15/34 (44.1%) ctDNAs from patients at surgery, as opposed to 14/14 (100%) metastatic patients. This was likely due to striking differences (average 10 times, up to 500) in ctDNA levels between groups. NGS revealed blood-only SNVs, suggesting spatial heterogeneity since pre-surgery disease stages, and raising the combined NGS/dPCR sensitivity to 58.8%. ctDNA levels at surgery correlated with neither tumor size, stage, grade, or nodal status, nor with variant abundance in paired tDNA. LB sensitivity reached 63.6% when ctDNA was combined with CEA. Finally, persistence and absence of ctDNA on the first conventional (month 3) post-surgery follow-up were associated with fast relapse and a disease-free status in 3 and 7 patients, respectively. CONCLUSIONS: A simple clinical NGS/dPCR/CEA combination effectively addresses the LB challenge in a fraction of non-metastatic CRC patients.
Assuntos
DNA Tumoral Circulante/metabolismo , Neoplasias Colorretais/cirurgia , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Biópsia Líquida/métodos , Estudos Transversais , Feminino , Humanos , MasculinoRESUMO
Colorectal cancer (CRC) is one of the most common malignant tumors worldwide and understanding its underlying molecular mechanisms is crucial for the development of therapeutic strategies. The mitogen-activated protein kinase-kinase 3 (MKK3) is a specific activator of p38 MAP kinases (p38 MAPKs), which contributes to the regulation of several cellular functions, such as proliferation, differentiation, apoptosis as well as response to drugs. At present, the exact MKK3/p38 MAPK pathway contribution in cancer is heavily debated because of its pleiotropic function. In this work, we retrospectively explored the prognostic and pathobiologic relevance of MKK3 in a cohort of CRC patients and assessed MKK3 molecular functions in a panel of CRC lines and colonocytes primary cultures. We found increased MKK3 levels in late-stage CRC patients which correlated with shorter overall survival. Herein, we report that the MKK3 targeting by inducible RNA interference univocally exerts antitumor effects in CRC lines but not in primary colonocytes. While MKK3 depletion per se affects growth and survival by induction of sustained autophagy and death in some CRC lines, it potentiates response to chemotherapeutic drug 5-fluorouracil (5-FU) in all of the tested CRC lines in vitro. Here, we demonstrate for the first time that in CRC the MKK3 specifically activates p38delta MAPK isoform to sustain prosurvival signaling and that such effect is exacerbated upon 5-FU challenge. Indeed, p38delta MAPK silencing recapitulates MKK3 depletion effects in CRC cells in vitro and in vivo. Overall, our data identified a molecular mechanism through which MKK3 supports proliferation and survival signaling in CRC, further supporting MKK3 as a novel and extremely attractive therapeutic target for the development of promising strategies for the management of CRC patients.
Assuntos
Neoplasias Colorretais/tratamento farmacológico , MAP Quinase Quinase 3/genética , Proteína Quinase 13 Ativada por Mitógeno/genética , Autofagia/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Intervalo Livre de Doença , Feminino , Fluoruracila/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células HT29 , Xenoenxertos , Humanos , Masculino , Transdução de Sinais/efeitos dos fármacos , Ativação Transcricional/efeitos dos fármacosRESUMO
BACKGROUND: Human microsatellite-stable (MSS) colorectal cancers (CRCs) are immunologically "cold" tumour subtypes characterized by reduced immune cytotoxicity. The molecular linkages between immune-resistance and human MSS CRC is not clear. METHODS: We used transcriptome profiling, in silico analysis, immunohistochemistry, western blot, RT-qPCR and immunofluorescence staining to characterize novel CRC immune biomarkers. The effects of selective antagonists were tested by in vitro assays of long term viability and analysis of kinase active forms using anti-phospho antibodies. RESULTS: We identified the lymphocyte antigen 6 complex, locus G6D (LY6G6D) as significantly overexpressed (around 15-fold) in CRC when compared with its relatively low expression in other human solid tumours. LY6G6D up-regulation was predominant in MSS CRCs characterized by an enrichment of immune suppressive regulatory T-cells and a limited repertoire of PD-1/PD-L1 immune checkpoint receptors. Coexpression of LY6G6D and CD15 increases the risk of metastatic relapse in response to therapy. Both JAK-STAT5 and RAS-MEK-ERK cascades act in concert as key regulators of LY6G6D and Fucosyltransferase 4 (FUT4), which direct CD15-mediated immune-resistance. Momelotinib, an inhibitor of JAK1/JAK2, consistently abrogated the STAT5/LY6G6D axis in vitro, sensitizing MSS cancer cells with an intact JAK-STAT signaling, to efficiently respond to trametinib, a MEK inhibitor used in clinical setting. Notably, colon cancer cells can evade JAK2/JAK1-targeted therapy by a reversible shift of the RAS-MEK-ERK pathway activity, which explains the treatment failure of JAK1/2 inhibitors in refractory CRC. CONCLUSIONS: Combined targeting of STAT5 and MAPK pathways has superior therapeutic effects on immune resistance. In addition, the new identified LY6G6D antigen is a promising molecular target for human MSS CRC.
Assuntos
Neoplasias Colorretais/genética , Reparo de Erro de Pareamento de DNA/genética , Imunoglobulinas/genética , Fator de Transcrição STAT5/genética , Antígeno B7-H1/genética , Antígeno B7-H1/imunologia , Benzamidas/farmacologia , Linhagem Celular Tumoral , Neoplasias Colorretais/imunologia , Neoplasias Colorretais/patologia , Feminino , Fucosiltransferases/genética , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Janus Quinase 1/antagonistas & inibidores , Janus Quinase 1/genética , Janus Quinase 2/antagonistas & inibidores , Janus Quinase 2/genética , Antígenos CD15/genética , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , Instabilidade de Microssatélites , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Receptor de Morte Celular Programada 1/genética , Receptor de Morte Celular Programada 1/imunologia , Pirimidinas/farmacologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologiaRESUMO
Adaptive resistance to therapy is a hallmark of cancer progression. To date, it is not entirely clear how microenvironmental stimuli would mediate emergence of therapy-resistant cell subpopulations, although a rearrangement of the cancer cell secretome following therapy-induced stress can be pivotal for such a process. Here, by using the highly chemoresistant malignant pleural mesothelioma (MPM) as an experimental model, we unveiled a key contribution of the chaperone HSP90 at assisting a chemotherapy-instigated Senescence-Associated-Secretory-Phenotype (SASP). Thus, administration of a clinical trial grade, HSP90, inhibitor blunted the release of several cytokines by the chemotherapy-treated MPM cells, including interleukin (IL)-8. Reduction of IL-8 levels hampered the FAK-AKT signaling and inhibited 3D growth and migration. This correlated with downregulation of key EMT and chemoresistance genes and affected the survival of chemoresistant ALDHbright cell subpopulations. Altogether, inhibition of HSP90 provoked a switch from a pro-tumorigenic SASP to a pro-apoptotic senescence status, thus resulting in chemosensitizing effects. In mouse xenografts treated with first-line agents, inhibiting HSP90 blunted FAK activation and reduced the expression of ALDH1A3 and the levels of circulating human IL-8, these latter strongly correlating with the effect on tumor growth. We validated the above findings in primary mesothelioma cultures, a more clinically relevant model. We unveiled here a key contribution of the chaperone HSP90 at assisting the secretory stress in chemotherapy-treated cells, which may warrant further investigation in combinatorial therapeutic settings.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Neoplasias Pulmonares/metabolismo , Mesotelioma/metabolismo , Via Secretória/efeitos dos fármacos , Triazóis/farmacologia , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Linhagem Celular Tumoral , Senescência Celular/efeitos dos fármacos , Cisplatino/farmacologia , Resistencia a Medicamentos Antineoplásicos , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Masculino , Mesotelioma/tratamento farmacológico , Mesotelioma/genética , Mesotelioma Maligno , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Pemetrexede/farmacologia , Via Secretória/genéticaRESUMO
BACKGROUND: Circulating tumor DNA (ctDNA) and miRNA (ctmiRNA) are promising biomarkers for early tumor diagnosis, prognosis and monitoring, and to predict therapeutic response. However, a clear understanding of the fine control on their circulating levels is still lacking. METHODS: Three human colorectal carcinoma cell lines were grown in culture and as tumor xenograft models in nude mice. Chip-based and droplet digital PCR platforms were used to systematically and quantitatively assess the levels of DNAs and miRNAs released into the culture supernatants and mouse blood plasma. RESULTS: Strikingly, mutated DNAs from the same (KRAS) and different (PIK3CA and FBWX7) genomic loci were differentially detected in culture supernatants and blood, with LS174T releasing 25 to 60 times less DNA in culture, but giving rise to 7 to 8 times more DNA in blood than LoVo cells. Greater LS174T ctDNA accumulation occurred in spite of similar CD31 immunostaining (micro-vascularization) and lesser proliferation and tissue necrosis as compared to LoVo. As to the three selected miRNAs (miR-221, miR-222 and miR-141), all of them were constitutively present in the plasma of tumor-free mice. Micro-RNA miR-141 was released into HT-29 cell supernatants 10 and 6.5 times less abundantly with respect to LoVo and LS174T, respectively; on the contrary, release of miR-141 in blood of HT-29 xenografted mice was found similar to that observed in LoVo and LS174T mice. CONCLUSIONS: Taken together, our results support the existence of multiple, finely tuned (non-housekeeping) control gateways that selectively regulate the release/accumulation of distinct ctDNA and miRNA species in culture and tumor xenograft models. Different xenografts (proxies of different patients) considerably differ in gateway usage, adding several layers of complexity to the well-known idea of molecular heterogeneity. We predict that even high tissue representation of mutated DNA and miRNA may result in insufficient diagnostic analyte representation in blood. In this respect, our data show that careful modeling in mice may considerably help to alleviate complexity, for instance by pre-screening for the most abundant circulating analytes in enlarged sets of tumor xenografts.
Assuntos
MicroRNA Circulante , DNA Tumoral Circulante , Neoplasias Colorretais/genética , Biópsia Líquida , Animais , Biomarcadores Tumorais , Linhagem Celular Tumoral , Neoplasias Colorretais/sangue , Neoplasias Colorretais/diagnóstico , Modelos Animais de Doenças , Xenoenxertos , Humanos , Camundongos , Reação em Cadeia da Polimerase , Fluxo de TrabalhoRESUMO
Although the medical treatments of sarcoma have evolved in the last years, a significant portion of patients develops recurrence after therapies suggesting the need to identify novel targets to improve the treatments. By the use of patient-derived and established cell lines from liposarcoma, as well as specimens from patient biopsies, we found that HMGA1 is involved in the progression of dedifferentiated and myxoid liposarcoma. The immunohistochemical and RT-PCR analyses of 68 liposarcoma specimens revealed a significant high expression of HMGA1, at the protein and RNA levels, both in myxoid and dedifferentiated liposarcoma subtypes compared with differentiated ones. Loss- and gain-of-function experiments by HMGA1-specific depletion and overexpression in dedifferentiated and myxoid liposarcoma cells showed the contribution of this oncogenic factor in cell proliferation, motility, invasion, and drug resistance. The in vitro and in vivo treatment of myxoid liposarcoma with trabectedin, a drug with a potent anti-tumor activity, revealed downregulation of HMGA1, E2F1, and its-downstream targets, vimentin and ZEB1, indicating a critical role of trabectedin in inhibiting the mesenchymal markers of these tumors through the HMGA1/E2F1 axis. These data were also confirmed in patients' tumor biopsies being HMGA1, E2F1, and vimentin expression significantly reduced upon trabectedin therapy, administered as neo-adjuvant chemotherapy. Furthermore, trabectedin treatment inhibits in vitro NFkB pathway in mixoyd liposarcoma sensitive but not in resistant counterparts, and the inhibition of NFkB pathway re-sensitizes the resistant cells to trabectedin treatment. These data support the rational for combining NFkB inhibitors with trabectedin in liposarcoma patients, who have become resistant to the drug.