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1.
Proc Natl Acad Sci U S A ; 118(41)2021 Oct 12.
Artigo em Inglês | MEDLINE | ID: mdl-34607954

RESUMO

BRCA1 germline mutations are associated with an increased risk of breast and ovarian cancer. Recent findings of others suggest that BRCA1 mutation carriers also bear an increased risk of esophageal and gastric cancer. Here, we employ a Brca1/Trp53 mouse model to show that unresolved replication stress (RS) in BRCA1 heterozygous cells drives esophageal tumorigenesis in a model of the human equivalent. This model employs 4-nitroquinoline-1-oxide (4NQO) as an RS-inducing agent. Upon drinking 4NQO-containing water, Brca1 heterozygous mice formed squamous cell carcinomas of the distal esophagus and forestomach at a much higher frequency and speed (∼90 to 120 d) than did wild-type (WT) mice, which remained largely tumor free. Their esophageal tissue, but not that of WT control mice, revealed evidence of overt RS as reflected by intracellular CHK1 phosphorylation and 53BP1 staining. These Brca1 mutant tumors also revealed higher genome mutation rates than those of control animals; the mutational signature SBS4, which is associated with tobacco-induced tumorigenesis; and a loss of Brca1 heterozygosity (LOH). This uniquely accelerated Brca1 tumor model is also relevant to human esophageal squamous cell carcinoma, an often lethal tumor.

2.
J Immunother Cancer ; 9(9)2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34593617

RESUMO

BACKGROUND: Immune checkpoint inhibitors have revolutionized cancer treatment, but the benefits in refractory patients with esophageal cancer have been modest. Predictors of response as well as new targets for novel therapeutic combinations are needed. In this phase 2 clinical trial, we tested single-agent pembrolizumab in patients with advanced esophageal cancer, who received at least one prior line of therapy. METHODS: Pembrolizumab 200 mg every 3 weeks was tested in 49 patients with refractory esophageal cancer: 39 with adenocarcinoma and 10 with esophageal squamous cell carcinoma. Major endpoints were radiological response by Immune-related Response Evaluation Criteria In Solid Tumors and survival. Tumor samples were evaluated for programmed cell death ligand 1 (PD-L1) expression, tumor mutational burden (TMB), and immune contexture by both NanoString mRNA expression analysis and flow cytometry. Peripheral blood mononuclear cells and a panel of circulating chemokines were also analyzed. RESULTS: The overall response rate (ORR) was 8% (4 of 49 patients; 95% CI 2.3% to 19.6%). Median overall survival (OS) was 5.8 months (95% CI 4.0 to 9.5). ORR and OS were not associated with histology. For PD-L1-positive patients, ORR was 13.3% (95% CI 1.7% to 40.5%) and median OS was 7.9 months (95% CI 4.7 to 15.5). A trend toward improved OS was observed in seven patients with a TMB ≥10 mut/Mb (p=0.086). Tumors with a PD-L1 Combined Positive Score ≥1 showed enrichment of LAG3 (p=0.005) and IDO1 (p=0.04) gene expression. Baseline levels of circulating CXCL10, interleukin 2 (IL2) receptor α (IL2RA) and IL6 were associated with survival: CXCL10 favorably, (HR 0.37, p=0.002 (progression-free survival); HR 0.55, p=0.018 (OS)); IL2RA and IL6 unfavorably (HR 1.57, p=0.020 for IL6 (OS); HR 2.36, p=0.025 for IL2RA (OS)). CONCLUSIONS: Pembrolizumab monotherapy was modestly effective in refractory esophageal cancer. Circulating CXCL10 at baseline appeared to be a robust predictor of response. Other T cell exhaustion markers are upregulated in PD-L1-positive patients, suggesting that immunotherapy combinations such as anti-LAG3/programmed cell death protein 1 (PD-1) or anti-IDO1/PD-1 may be of promise in refractory esophageal cancer.

3.
Nat Commun ; 12(1): 5386, 2021 09 10.
Artigo em Inglês | MEDLINE | ID: mdl-34508104

RESUMO

Although inhibitors targeting CDK4/6 kinases (CDK4/6i) have shown promising clinical prospect in treating ER+/HER2- breast cancers, acquired drug resistance is frequently observed and mechanistic knowledge is needed to harness their full clinical potential. Here, we report that inhibition of CDK4/6 promotes ßTrCP1-mediated ubiquitination and proteasomal degradation of RB1, and facilitates SP1-mediated CDK6 transcriptional activation. Intriguingly, suppression of CK1ε not only efficiently prevents RB1 from degradation, but also prevents CDK4/6i-induced CDK6 upregulation by modulating SP1 protein stability, thereby enhancing CDK4/6i efficacy and overcoming resistance to CDK4/6i in vitro. Using xenograft and PDX models, we further demonstrate that combined inhibition of CK1ε and CDK4/6 results in marked suppression of tumor growth in vivo. Altogether, these results uncover the molecular mechanisms by which CDK4/6i treatment alters RB1 and CDK6 protein abundance, thereby driving the acquisition of CDK4/6i resistance. Importantly, we identify CK1ε as an effective target for potentiating the therapeutic efficacy of CDK4/6 inhibitors.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Neoplasias da Mama/tratamento farmacológico , Caseína Quinase Iépsilon/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Quinase 4 Dependente de Ciclina/antagonistas & inibidores , Quinase 4 Dependente de Ciclina/metabolismo , Quinase 6 Dependente de Ciclina/antagonistas & inibidores , Quinase 6 Dependente de Ciclina/metabolismo , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Sinergismo Farmacológico , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Inibidores de Proteínas Quinases/uso terapêutico , Estabilidade Proteica/efeitos dos fármacos , Proteólise/efeitos dos fármacos , Proteínas de Ligação a Retinoblastoma/metabolismo , Fator de Transcrição Sp1/metabolismo , Ativação Transcricional/efeitos dos fármacos , Ubiquitina-Proteína Ligases/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
4.
Gastroenterology ; 2021 Sep 24.
Artigo em Inglês | MEDLINE | ID: mdl-34571027

RESUMO

BACKGROUND AND AIMS: Genomic alterations that encourage stem cell activity and hinder proper maturation are central to the development of colorectal cancer (CRC). Key molecular mediators that promote these malignant properties require further elucidation to galvanize translational advances. We therefore aimed to characterize a key factor that blocks intestinal differentiation, define its transcriptional and epigenetic program, and provide preclinical evidence for therapeutic targeting in CRC. METHODS: Intestinal tissue from transgenic mice and patients were analyzed by histopathology and immunostaining. Human CRC cells and neoplastic murine organoids were genetically manipulated for functional studies. Gene expression profiling was obtained through RNA sequencing. Histone modifications and transcription factor binding was determined by ChIP sequencing. RESULTS: We demonstrate that SRY-box transcription factor 9 (SOX9) promotes CRC by activating a stem cell-like program that hinders intestinal differentiation. Intestinal adenomas and colorectal adenocarcinomas from mouse models and patients demonstrate ectopic and elevated expression of SOX9. Functional experiments indicate a requirement for SOX9 in human CRC cell lines and engineered neoplastic organoids. Disrupting SOX9 activity impairs primary CRC tumor growth by inducing intestinal differentiation. By binding to genome wide enhancers, SOX9 directly activates genes associated with Paneth and stem cell activity, including PROM1. SOX9 upregulates PROM1 via a WNT-responsive intronic enhancer. A pentaspan transmembrane protein, PROM1 utilizes its first intracellular domain to support stem cell signaling, at least in part through SOX9, reinforcing a PROM1-SOX9 positive feedback loop. CONCLUSIONS: These studies establish SOX9 as a central regulator of an enhancer-driven stem cell-like program and carry important implications for developing therapeutics directed at overcoming differentiation defects in CRC.

5.
Am J Surg Pathol ; 2021 Aug 31.
Artigo em Inglês | MEDLINE | ID: mdl-34469333

RESUMO

While most resection specimens from patients with neoadjuvantly treated esophageal squamous cell carcinoma show therapy-related changes in the form of inflammation and fibrosis, others harbor a florid foreign body-type giant cell response to keratin debris. The purpose of our study was to perform a detailed clinicopathologic analysis of these histologic types of treatment responses and correlate these findings with patient outcome. Clinical and pathologic parameters from 110 esophagogastrectomies were recorded and analyzed. Two main types of histologic responses were observed: inflammatory-predominant response (59%) and florid foreign body-type giant cell response to keratin (41%). Irrespective of cG, cTNM, and amount of residual cancer, florid foreign body-type giant cell reaction was predominantly noted deep within the esophageal wall, while the inflammatory response was restricted to the mucosa, submucosa, and inner half of muscularis propria. Patients with foreign body-type giant cell response showed significantly better overall survival compared with the inflammatory response group (log-rank test P=0.015). Florid foreign body-type giant cell response was the only factor associated with improved survival in a multivariable analysis for overall survival (hazard ratio=0.5; 95% confidence interval=0.3-1.0; P=0.038), but not in the model for disease-specific survival, whereas ypTNM stage II was the only significant risk factor for disease-specific survival in multivariable analysis (hazard ratio=3.4; 95% confidence interval=1.0-11.2; P=0.047). Our results suggest that in addition to the College of American Pathologists Tumor Regression Score and ypTNM stage, subtype of histologic response to therapy may represent another prognostic marker for neoadjuvantly treated esophageal squamous cell carcinoma.

6.
Nat Genet ; 53(6): 881-894, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33972779

RESUMO

Esophageal squamous cell carcinomas (ESCCs) harbor recurrent chromosome 3q amplifications that target the transcription factor SOX2. Beyond its role as an oncogene in ESCC, SOX2 acts in development of the squamous esophagus and maintenance of adult esophageal precursor cells. To compare Sox2 activity in normal and malignant tissue, we developed engineered murine esophageal organoids spanning normal esophagus to Sox2-induced squamous cell carcinoma and mapped Sox2 binding and the epigenetic and transcriptional landscape with evolution from normal to cancer. While oncogenic Sox2 largely maintains actions observed in normal tissue, Sox2 overexpression with p53 and p16 inactivation promotes chromatin remodeling and evolution of the Sox2 cistrome. With Klf5, oncogenic Sox2 acquires new binding sites and enhances activity of oncogenes such as Stat3. Moreover, oncogenic Sox2 activates endogenous retroviruses, inducing expression of double-stranded RNA and dependence on the RNA editing enzyme ADAR1. These data reveal SOX2 functions in ESCC, defining targetable vulnerabilities.


Assuntos
Adenosina Desaminase/metabolismo , Epigenoma , Neoplasias Esofágicas/genética , Carcinoma de Células Escamosas do Esôfago/genética , Proteínas de Ligação a RNA/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Regiões 3' não Traduzidas/genética , Animais , Sequência de Bases , Carcinogênese/genética , Linhagem Celular Tumoral , Transformação Celular Neoplásica/genética , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Retrovirus Endógenos/genética , Elementos Facilitadores Genéticos/genética , Regulação Neoplásica da Expressão Gênica , Genoma Humano , Humanos , Interferons/metabolismo , Íntrons/genética , Fatores de Transcrição Kruppel-Like/genética , Camundongos , Organoides/patologia , Ligação Proteica , RNA de Cadeia Dupla/metabolismo , Fatores de Transcrição SOXB1/genética , Proteína Supressora de Tumor p53/metabolismo
7.
Genes Dev ; 35(7-8): 528-541, 2021 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-33737385

RESUMO

Esophageal squamous cell carcinoma (ESCC) is one of the most lethal cancers worldwide and evolves often to lung metastasis. P53R175H (homologous to Trp53 R172H in mice) is a common hot spot mutation. How metastasis is regulated by p53R175H in ESCC remains to be investigated. To investigate p53R175H-mediated molecular mechanisms, we used a carcinogen-induced approach in Trp53R172H/- mice to model ESCC. In the primary Trp53R172H/- tumor cell lines, we depleted Trp53R172H (shTrp53) and observed a marked reduction in cell invasion in vitro and lung metastasis burden in a tail-vein injection model in comparing isogenic cells (shCtrl). Furthermore, we performed bulk RNA-seq to compare gene expression profiles of metastatic and primary shCtrl and shTrp53 cells. We identified the YAP-BIRC5 axis as a potential mediator of Trp53 R172H -mediated metastasis. We demonstrate that expression of Survivin, an antiapoptotic protein encoded by BIRC5, increases in the presence of Trp53R172H Furthermore, depletion of Survivin specifically decreases Trp53R172H-driven lung metastasis. Mechanistically, Trp53R172H but not wild-type Trp53, binds with YAP in ESCC cells, suggesting their cooperation to induce Survivin expression. Furthermore, Survivin high expression level is associated with increased metastasis in several GI cancers. Taken together, this study unravels new insights into how mutant p53 mediates metastasis.


Assuntos
Neoplasias Pulmonares/fisiopatologia , Survivina/genética , Survivina/metabolismo , Animais , Linhagem Celular Tumoral , Modelos Animais de Doenças , Regulação Neoplásica da Expressão Gênica/genética , Neoplasias Pulmonares/genética , Camundongos , Mutação , Metástase Neoplásica , Transcriptoma , Proteína Supressora de Tumor p53/metabolismo
8.
Mol Oncol ; 15(4): 901-914, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33506581

RESUMO

Identification of molecular predictive markers of response to neoadjuvant chemoradiation could aid clinical decision-making in patients with localized oesophageal cancer. Therefore, we subjected pretreatment biopsies of 75 adenocarcinoma (OAC) and 16 squamous cell carcinoma (OSCC) patients to targeted next-generation DNA sequencing, as well as biopsies of 85 OAC and 20 OSCC patients to promoter methylation analysis of eight GI-specific genes, and subsequently searched for associations with histopathological response and disease-free (DFS) and overall survival (OS). Thereby, we found that in OAC, CSMD1 deletion (8%) and ETV4 amplification (5%) were associated with a favourable histopathological response, whereas SMURF1 amplification (5%) and SMARCA4 mutation (7%) were associated with an unfavourable histopathological response. KRAS (15%) and GATA4 (7%) amplification were associated with shorter OS. In OSCC, TP63 amplification (25%) and TFPI2 (10%) gene promoter methylation were associated with an unfavourable histopathological response and shorter DFS (TP63) and OS (TFPI2), whereas CDKN2A deletion (38%) was associated with prolonged OS. In conclusion, this study identified candidate genetic biomarkers associated with response to neoadjuvant chemoradiotherapy in patients with localized oesophageal cancer.

9.
ACS Med Chem Lett ; 12(1): 30-38, 2021 Jan 14.
Artigo em Inglês | MEDLINE | ID: mdl-33488961

RESUMO

Focal adhesion kinase (FAK) is a tyrosine kinase with prominent roles in protein scaffolding, migration, angiogenesis, and anchorage-independent cell survival and is an attractive target for the development of cancer therapeutics. However, current FAK inhibitors display dual kinase inhibition and/or significant activity on several kinases. Although multitargeted activity is at times therapeutically advantageous, such behavior can also lead to toxicity and confound chemical-biology studies. We report a novel series of small molecules based on a tricyclic pyrimidothiazolodiazepinone core that displays both high potency and selectivity for FAK. Structure-activity relationship (SAR) studies explored modifications to the thiazole, diazepinone, and aniline "tail," which identified lead compound BJG-03-025. BJG-03-025 displays potent biochemical FAK inhibition (IC50 = 20 nM), excellent kinome selectivity, activity in 3D-culture breast and gastric cancer models, and favorable pharmacokinetic properties in mice. BJG-03-025 is a valuable chemical probe for evaluation of FAK-dependent biology.

10.
Nature ; 590(7846): 486-491, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33505028

RESUMO

Selective targeting of aneuploid cells is an attractive strategy for cancer treatment1. However, it is unclear whether aneuploidy generates any clinically relevant vulnerabilities in cancer cells. Here we mapped the aneuploidy landscapes of about 1,000 human cancer cell lines, and analysed genetic and chemical perturbation screens2-9 to identify cellular vulnerabilities associated with aneuploidy. We found that aneuploid cancer cells show increased sensitivity to genetic perturbation of core components of the spindle assembly checkpoint (SAC), which ensures the proper segregation of chromosomes during mitosis10. Unexpectedly, we also found that aneuploid cancer cells were less sensitive than diploid cells to short-term exposure to multiple SAC inhibitors. Indeed, aneuploid cancer cells became increasingly sensitive to inhibition of SAC over time. Aneuploid cells exhibited aberrant spindle geometry and dynamics, and kept dividing when the SAC was inhibited, resulting in the accumulation of mitotic defects, and in unstable and less-fit karyotypes. Therefore, although aneuploid cancer cells could overcome inhibition of SAC more readily than diploid cells, their long-term proliferation was jeopardized. We identified a specific mitotic kinesin, KIF18A, whose activity was perturbed in aneuploid cancer cells. Aneuploid cancer cells were particularly vulnerable to depletion of KIF18A, and KIF18A overexpression restored their response to SAC inhibition. Our results identify a therapeutically relevant, synthetic lethal interaction between aneuploidy and the SAC.


Assuntos
Aneuploidia , Pontos de Checagem da Fase M do Ciclo Celular/efeitos dos fármacos , Neoplasias/patologia , Cariótipo Anormal/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Segregação de Cromossomos/efeitos dos fármacos , Diploide , Genes Letais , Humanos , Cinesina/deficiência , Cinesina/genética , Cinesina/metabolismo , Neoplasias/genética , Fuso Acromático/efeitos dos fármacos , Mutações Sintéticas Letais/efeitos dos fármacos , Mutações Sintéticas Letais/genética , Fatores de Tempo
11.
Cancer Cell ; 38(5): 626-628, 2020 11 09.
Artigo em Inglês | MEDLINE | ID: mdl-33171128

RESUMO

Barrett's esophagus (BE) is a precursor to esophageal adenocarinoma, and screening for cancer risk focuses upon histologic assessment of dysplasia within endoscopic biopsies. A recent study in Nature Medicine contributes to growing evidence that genomic assessment of non-dysplastic BE samples can identify patients at greatest risk of progressing to cancer.


Assuntos
Adenocarcinoma/diagnóstico , Esôfago de Barrett/genética , Neoplasias Esofágicas/diagnóstico , Sequenciamento Completo do Genoma/métodos , Adenocarcinoma/genética , Adenocarcinoma/patologia , Esôfago de Barrett/patologia , Biópsia , Metilação de DNA , Progressão da Doença , Detecção Precoce de Câncer , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/patologia , Genômica , Humanos
12.
Nature ; 586(7828): 292-298, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32999459

RESUMO

The RecQ DNA helicase WRN is a synthetic lethal target for cancer cells with microsatellite instability (MSI), a form of genetic hypermutability that arises from impaired mismatch repair1-4. Depletion of WRN induces widespread DNA double-strand breaks in MSI cells, leading to cell cycle arrest and/or apoptosis. However, the mechanism by which WRN protects MSI-associated cancers from double-strand breaks remains unclear. Here we show that TA-dinucleotide repeats are highly unstable in MSI cells and undergo large-scale expansions, distinct from previously described insertion or deletion mutations of a few nucleotides5. Expanded TA repeats form non-B DNA secondary structures that stall replication forks, activate the ATR checkpoint kinase, and require unwinding by the WRN helicase. In the absence of WRN, the expanded TA-dinucleotide repeats are susceptible to cleavage by the MUS81 nuclease, leading to massive chromosome shattering. These findings identify a distinct biomarker that underlies the synthetic lethal dependence on WRN, and support the development of therapeutic agents that target WRN for MSI-associated cancers.


Assuntos
Quebras de DNA de Cadeia Dupla , Expansão das Repetições de DNA/genética , Repetições de Dinucleotídeos/genética , Neoplasias/genética , Helicase da Síndrome de Werner/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Linhagem Celular Tumoral , Cromossomos Humanos/genética , Cromossomos Humanos/metabolismo , Cromotripsia , Clivagem do DNA , Replicação do DNA , Proteínas de Ligação a DNA/metabolismo , Endodesoxirribonucleases/metabolismo , Endonucleases/metabolismo , Instabilidade Genômica , Humanos , Recombinases/metabolismo
13.
Curr Protoc Stem Cell Biol ; 53(1): e109, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32294323

RESUMO

Esophageal cancers comprise adenocarcinoma and squamous cell carcinoma, two distinct histologic subtypes. Both are difficult to treat and among the deadliest human malignancies. We describe protocols to initiate, grow, passage, and characterize patient-derived organoids (PDO) of esophageal cancers, as well as squamous cell carcinomas of oral/head-and-neck and anal origin. Formed rapidly (<14 days) from a single-cell suspension embedded in basement membrane matrix, esophageal cancer PDO recapitulate the histology of the original tumors. Additionally, we provide guidelines for morphological analyses and drug testing coupled with functional assessment of cell response to conventional chemotherapeutics and other pharmacological agents in concert with emerging automated imaging platforms. Predicting drug sensitivity and potential therapy resistance mechanisms in a moderate-to-high throughput manner, esophageal cancer PDO are highly translatable in personalized medicine for customized esophageal cancer treatments. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Generation of esophageal cancer PDO Basic Protocol 2: Propagation and cryopreservation of esophageal cancer PDO Basic Protocol 3: Imaged-based monitoring of organoid size and growth kinetics Basic Protocol 4: Harvesting esophageal cancer PDO for histological analyses Basic Protocol 5: PDO content analysis by flow cytometry Basic Protocol 6: Evaluation of drug response with determination of the half-inhibitory concentration (IC50 ) Support Protocol: Production of RN in HEK293T cell conditioned medium.


Assuntos
Neoplasias Esofágicas/patologia , Carcinoma de Células Escamosas do Esôfago/patologia , Organoides/patologia , Medicina de Precisão/métodos , Cultura Primária de Células/métodos , Células Cultivadas , Humanos
14.
Cell Stem Cell ; 26(5): 739-754.e8, 2020 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-32142681

RESUMO

Cancer is believed to arise from stem cells, but mechanisms that limit the acquisition of mutations and tumor development have not been well defined. We show that a +4 stem cell (SC) in the gastric antrum, marked by expression of Cck2r (a GPCR) and Delta-like ligand 1 (DLL1), is a label-retaining cell that undergoes predominant asymmetric cell division. This +4 antral SC is Notch1low/ Numb+ and repressed by signaling from gastrin-expressing endocrine (G) cells. Chemical carcinogenesis of the stomach is associated with loss of G cells, increased symmetric stem cell division, glandular fission, and more rapid stem cell lineage tracing, a process that can be suppressed by exogenous gastrin treatment. This hormonal suppression is associated with a marked reduction in gastric cancer mutational load, as revealed by exomic sequencing. Taken together, our results show that gastric tumorigenesis is associated with increased symmetric cell division that facilitates mutation and is suppressed by GPCR signaling.


Assuntos
Carcinogênese , Células-Tronco , Divisão Celular , Humanos , Transdução de Sinais , Estômago
15.
Clin Cancer Res ; 26(13): 3431-3442, 2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32209571

RESUMO

PURPOSE: Lung squamous cell carcinoma (LSCC) is a deadly disease for which only a subset of patients responds to immune checkpoint blockade (ICB) therapy. Therefore, preclinical mouse models that recapitulate the complex genetic profile found in patients are urgently needed. EXPERIMENTAL DESIGN: We used CRISPR genome editing to delete multiple tumor suppressors in lung organoids derived from Cre-dependent SOX2 knock-in mice. We investigated both the therapeutic efficacy and immunologic effects accompanying combination PD-1 blockade and WEE1 inhibition in both mouse models and LSCC patient-derived cell lines. RESULTS: We show that multiplex gene editing of mouse lung organoids using the CRISPR-Cas9 system allows for efficient and rapid means to generate LSCCs that closely mimic the human disease at the genomic and phenotypic level. Using this genetically defined mouse model and three-dimensional tumoroid culture system, we show that WEE1 inhibition induces DNA damage that primes the endogenous type I IFN and antigen presentation system in primary LSCC tumor cells. These events promote cytotoxic T-cell-mediated clearance of tumor cells and reduce the accumulation of tumor-infiltrating neutrophils. Beneficial immunologic features of WEE1 inhibition are further enhanced by the addition of anti-PD-1 therapy. CONCLUSIONS: We developed a mouse model system to investigate a novel combinatory approach that illuminates a clinical path hypothesis for combining ICB with DNA damage-inducing therapies in the treatment of LSCC.

16.
Nat Genet ; 52(2): 219-230, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32025000

RESUMO

Somatic alterations in cancer genes are being detected in normal and premalignant tissue, thus placing greater emphasis on gene-environment interactions that enable disease phenotypes. By combining early genetic alterations with disease-relevant exposures, we developed an integrative mouse model to study gastric premalignancy. Deletion of Trp53 in gastric cells confers a selective advantage and promotes the development of dysplasia in the setting of dietary carcinogens. Organoid derivation from dysplastic lesions facilitated genomic, transcriptional and functional evaluation of gastric premalignancy. Cell cycle regulators, most notably Cdkn2a, were upregulated by p53 inactivation in gastric premalignancy, serving as a barrier to disease progression. Co-deletion of Cdkn2a and Trp53 in dysplastic gastric organoids promoted cancer phenotypes but also induced replication stress, exposing a susceptibility to DNA damage response inhibitors. These findings demonstrate the utility of mouse models that integrate genomic alterations with relevant exposures and highlight the importance of gene-environment interactions in shaping the premalignant state.


Assuntos
Lesões Pré-Cancerosas/patologia , Neoplasias Gástricas/etiologia , Proteína Supressora de Tumor p53/genética , Adenocarcinoma/genética , Adenocarcinoma/patologia , Animais , Esôfago de Barrett/genética , Esôfago de Barrett/patologia , Linhagem Celular Tumoral , Inibidor p16 de Quinase Dependente de Ciclina/genética , Exposição Ambiental/efeitos adversos , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/patologia , Humanos , Metilnitrosoureia/toxicidade , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Mutação , Neoplasias Experimentais/induzido quimicamente , Neoplasias Experimentais/genética , Neoplasias Experimentais/patologia , Organoides/patologia , Lesões Pré-Cancerosas/genética , Neoplasias Gástricas/tratamento farmacológico , Neoplasias Gástricas/patologia
17.
Cancer Discov ; 10(2): 288-305, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31771969

RESUMO

Diffuse gastric cancer (DGC) is a lethal malignancy lacking effective systemic therapy. Among the most provocative recent results in DGC has been that of highly recurrent missense mutations in the GTPase RHOA. The function of these mutations has remained unresolved. We demonstrate that RHOAY42C, the most common RHOA mutation in DGC, is a gain-of-function oncogenic mutant, and that expression of RHOAY42C with inactivation of the canonical tumor suppressor Cdh1 induces metastatic DGC in a mouse model. Biochemically, RHOAY42C exhibits impaired GTP hydrolysis and enhances interaction with its effector ROCK. RHOA Y42C mutation and Cdh1 loss induce actin/cytoskeletal rearrangements and activity of focal adhesion kinase (FAK), which activates YAP-TAZ, PI3K-AKT, and ß-catenin. RHOAY42C murine models were sensitive to FAK inhibition and to combined YAP and PI3K pathway blockade. These results, coupled with sensitivity to FAK inhibition in patient-derived DGC cell lines, nominate FAK as a novel target for these cancers. SIGNIFICANCE: The functional significance of recurrent RHOA mutations in DGC has remained unresolved. Through biochemical studies and mouse modeling of the hotspot RHOAY42C mutation, we establish that these mutations are activating, detail their effects upon cell signaling, and define how RHOA-mediated FAK activation imparts sensitivity to pharmacologic FAK inhibitors.See related commentary by Benton and Chernoff, p. 182.This article is highlighted in the In This Issue feature, p. 161.


Assuntos
Quinase 1 de Adesão Focal/metabolismo , Inibidores de Proteínas Quinases/administração & dosagem , Quinolonas/administração & dosagem , Neoplasias Gástricas/genética , Sulfonas/administração & dosagem , Proteína rhoA de Ligação ao GTP/genética , Células 3T3 , Animais , Antígenos CD/metabolismo , Células COS , Caderinas/metabolismo , Chlorocebus aethiops , Quinase 1 de Adesão Focal/antagonistas & inibidores , Mutação com Ganho de Função , Mucosa Gástrica/patologia , Células HEK293 , Humanos , Camundongos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Neoplasias Gástricas/tratamento farmacológico , Neoplasias Gástricas/patologia , Ensaios Antitumorais Modelo de Xenoenxerto , Proteína rhoA de Ligação ao GTP/metabolismo
18.
Cancer Discov ; 9(12): 1656-1672, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31727671

RESUMO

Gastroesophageal adenocarcinomas (GEA) are devastating diseases with stark global presence. Over the past 10 years, there have been minimal improvements in treatment approach despite numerous clinical trials. Here, we review recent progress toward understanding the molecular features of these cancers and the diagnostic and therapeutic challenges posed by their intrinsic genomic instability and heterogeneity. We highlight the potential of genomic heterogeneity to influence clinical trial outcomes for targeted therapies and emphasize the need for comprehensive molecular profiling to guide treatment selection and adapt treatment to resistance and genomic evolution. Revising our clinical approach to GEA by leveraging genomic advances will be integral to the success of current and future treatments, especially as novel targets become therapeutically tractable. SIGNIFICANCE: GEAs are deadly cancers with few treatment options. Characterization of the genomic landscape of these cancers has revealed considerable genetic diversity and spatial heterogeneity. Understanding these fundamental properties of GEA will be critical for overcoming barriers to the development of novel, more effective therapeutic strategies.


Assuntos
Adenocarcinoma/genética , Antineoplásicos/uso terapêutico , Neoplasias Esofágicas/genética , Neoplasias Gástricas/genética , Adenocarcinoma/tratamento farmacológico , Antineoplásicos/farmacologia , Ensaios Clínicos como Assunto , Neoplasias Esofágicas/tratamento farmacológico , Heterogeneidade Genética , Humanos , Terapia de Alvo Molecular , Medicina de Precisão , Neoplasias Gástricas/tratamento farmacológico
19.
Cancer Res ; 79(22): 5884-5896, 2019 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-31585941

RESUMO

Gastric cancer is the world's third leading cause of cancer mortality. In spite of significant therapeutic improvements, the clinical outcome for patients with advanced gastric cancer is poor; thus, the identification and validation of novel targets is extremely important from a clinical point of view. We generated a wide, multilevel platform of gastric cancer models, comprising 100 patient-derived xenografts (PDX), primary cell lines, and organoids. Samples were classified according to their histology, microsatellite stability, Epstein-Barr virus status, and molecular profile. This PDX platform is the widest in an academic institution, and it includes all the gastric cancer histologic and molecular types identified by The Cancer Genome Atlas. PDX histopathologic features were consistent with those of patients' primary tumors and were maintained throughout passages in mice. Factors modulating grafting rate were histology, TNM stage, copy number gain of tyrosine kinases/KRAS genes, and microsatellite stability status. PDX and PDX-derived cells/organoids demonstrated potential usefulness to study targeted therapy response. Finally, PDX transcriptomic analysis identified a cancer cell-intrinsic microsatellite instability (MSI) signature, which was efficiently exported to gastric cancer, allowing the identification, among microsatellite stable (MSS) patients, of a subset of MSI-like tumors with common molecular aspects and significant better prognosis. In conclusion, we generated a wide gastric cancer PDX platform, whose exploitation will help identify and validate novel "druggable" targets and optimize therapeutic strategies. Moreover, transcriptomic analysis of gastric cancer PDXs allowed the identification of a cancer cell-intrinsic MSI signature, recognizing a subset of MSS patients with MSI transcriptional traits, endowed with better prognosis. SIGNIFICANCE: This study reports a multilevel platform of gastric cancer PDXs and identifies a MSI gastric signature that could contribute to the advancement of precision medicine in gastric cancer.


Assuntos
Neoplasias Gástricas/genética , Transcrição Genética/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Biomarcadores Tumorais/genética , Feminino , Perfilação da Expressão Gênica/métodos , Genes ras/genética , Humanos , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Instabilidade de Microssatélites , Pessoa de Meia-Idade , Estadiamento de Neoplasias/métodos , Fenótipo , Prognóstico , Neoplasias Gástricas/patologia
20.
JCI Insight ; 4(15)2019 08 08.
Artigo em Inglês | MEDLINE | ID: mdl-31391338

RESUMO

Despite the propensity for gastric and esophageal adenocarcinomas to select for recurrent missense mutations in TP53, the precise functional consequence of these mutations remains unclear. Here we report that endogenous mRNA and protein levels of mutant p53 were elevated in cell lines and patients with gastric and esophageal cancer. Functional studies showed that mutant p53 was sufficient, but not necessary, for enhancing primary tumor growth in vivo. Unbiased genome-wide transcriptome analysis revealed that hypoxia signaling was induced by mutant p53 in 2 gastric cancer cell lines. Using real-time in vivo imaging, we confirmed that hypoxia reporter activity was elevated during the initiation of mutant p53 gastric cancer xenografts. Unlike HIF co-factor ARNT, HIF1α was required for primary tumor growth in mutant p53 gastric cancer. These findings elucidate the contribution of missense p53 mutations in gastroesophageal malignancy and indicate that hypoxia signaling rather than mutant p53 itself may serve as a therapeutic vulnerability in these deadly set of cancers.


Assuntos
Adenocarcinoma/genética , Neoplasias Esofágicas/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias Gástricas/genética , Proteína Supressora de Tumor p53/genética , Adenocarcinoma/patologia , Animais , Translocador Nuclear Receptor Aril Hidrocarboneto/metabolismo , Carcinogênese/genética , Hipóxia Celular/genética , Linhagem Celular Tumoral , Análise Mutacional de DNA , Conjuntos de Dados como Assunto , Mucosa Esofágica/patologia , Neoplasias Esofágicas/patologia , Mucosa Gástrica/patologia , Perfilação da Expressão Gênica , Técnicas de Inativação de Genes , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Microscopia Intravital , Camundongos , Mutação de Sentido Incorreto , Transdução de Sinais/genética , Neoplasias Gástricas/patologia , Transcrição Genética , Proteína Supressora de Tumor p53/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
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