RESUMO
Chronic liver diseases, fibrosis, cirrhosis, and HCC are often a consequence of persistent inflammation. However, the transition mechanisms from a normal liver to fibrosis, then cirrhosis, and further to HCC are not well understood. This study focused on the role of the tumor stem cell protein doublecortin-like kinase 1 (DCLK1) in the modulation of molecular factors in fibrosis, cirrhosis, or HCC. Serum samples from patients with hepatic fibrosis, cirrhosis, and HCC were analyzed via ELISA or NextGen sequencing and were compared with control samples. Differentially expressed (DE) microRNAs (miRNA) identified from these patient sera were correlated with DCLK1 expression. We observed elevated serum DCLK1 levels in fibrosis, cirrhosis, and HCC patients; however, TGF-ß levels were only elevated in fibrosis and cirrhosis. While DE miRNAs were identified for all three disease states, miR-12136 was elevated in fibrosis but was significantly increased further in cirrhosis. Additionally, miR-1246 and miR-184 were upregulated when DCLK1 was high, while miR-206 was downregulated. This work distinguishes DCLK1 and miRNAs' potential role in different axes promoting inflammation to tumor progression and may serve to identify biomarkers for tracking the progression from pre-neoplastic states to HCC in chronic liver disease patients as well as provide targets for treatment.
Assuntos
Quinases Semelhantes a Duplacortina , Inflamação , Peptídeos e Proteínas de Sinalização Intracelular , Cirrose Hepática , Neoplasias Hepáticas , MicroRNAs , Proteínas Serina-Treonina Quinases , Humanos , MicroRNAs/sangue , MicroRNAs/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/sangue , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/sangue , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/sangue , Cirrose Hepática/genética , Cirrose Hepática/sangue , Inflamação/genética , Inflamação/sangue , Masculino , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/sangue , Feminino , Doença Crônica , Hepatopatias/sangue , Hepatopatias/genética , Pessoa de Meia-Idade , Carcinogênese/genética , Idoso , Biomarcadores Tumorais/sangue , Biomarcadores Tumorais/genéticaRESUMO
Ovarian cancer (OvCa) has a dismal prognosis because of its late-stage diagnosis and the emergence of chemoresistance. Doublecortin-like kinase 1 (DCLK1) is a serine/threonine kinase known to regulate cancer cell "stemness", epithelial-mesenchymal transition (EMT), and drug resistance. Here we show that DCLK1 is a druggable target that promotes chemoresistance and tumor progression of high-grade serous OvCa (HGSOC). Importantly, high DCLK1 expression significantly correlates with poor overall and progression-free survival in OvCa patients treated with platinum chemotherapy. DCLK1 expression was elevated in a subset of HGSOC cell lines in adherent (2D) and spheroid (3D) cultures, and the expression was further increased in cisplatin-resistant (CPR) spheroids relative to their sensitive controls. Using cisplatin-sensitive and resistant isogenic cell lines, pharmacologic inhibition (DCLK1-IN-1), and genetic manipulation, we demonstrate that DCLK1 inhibition was effective at re-sensitizing cells to cisplatin, reducing cell proliferation, migration, and invasion. Using kinase domain mutants, we demonstrate that DCLK1 kinase activity is critical for mediating CPR. The combination of cisplatin and DCLK1-IN-1 showed a synergistic cytotoxic effect against OvCa cells in 3D conditions. Targeted gene expression profiling revealed that DCLK1 inhibition in CPR OvCa spheroids significantly reduced TGFß signaling, and EMT. We show in vivo efficacy of combined DCLK1 inhibition and cisplatin in significantly reducing tumor metastases. Our study shows that DCLK1 is a relevant target in OvCa and combined targeting of DCLK1 in combination with existing chemotherapy could be a novel therapeutic approach to overcome resistance and prevent OvCa recurrence.
Assuntos
Quinases Semelhantes a Duplacortina , Neoplasias Ovarianas , Humanos , Feminino , Cisplatino/farmacologia , Resistencia a Medicamentos Antineoplásicos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Linhagem Celular Tumoral , Recidiva Local de Neoplasia , Proteínas Serina-Treonina Quinases/metabolismo , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologiaRESUMO
Chemoresistance cells have features similar to cancer stem cells. Elimination of these cells is an effective therapeutic strategy to clinically combat chemoresistance non-small cell lung cancer (NSCLC). Here, we demonstrate that Doublecortin-like kinase1 (DCLK1) is the key to developing chemoresistance and associated stemness in NSCLC. DCLK1 is highly expressed in human lung adenocarcinoma and strongly correlated with stemness. Silencing DCLK1 inhibits NSCLC cell primary and secondary spheroid formation, which is the prerequisite feature of tumor stem cells. DCLK1 inhibition reduced NSCLC cell migration/invasion in vitro and induced tumor growth inhibition in vivo. NSCLC cells responded differently to cisplatin treatment; indeed, the clonogenic ability of all NSCLC cells was reduced. We found that the cisplatin-resistant NSCLC cells gain the expression of DCLK1 compared with their parental control. However, DCLK1 inhibition in cisplatin-resistance NSCLC cells reverses the tumor cell resistance to cisplatin and reduced tumor self-renewal ability. Specifically, we found that DCLK1-mediated cisplatin resistance in NSCLC is via an ABC subfamily member 4 (ABCD4)-dependent mechanism. Our data demonstrate that increased expression of DCLK1 is associated with chemoresistance and enhanced cancer stem cell-like features in NSCLC. Targeting DCLK1 using gene knockdown/knockout strategies alone or in combination with cisplatin may represent a novel therapeutic strategy to treat NSCLC.
RESUMO
Tumor-associated M2-macrophages are one of the most abundant immunosuppressive cell types in the pancreatic ductal adenocarcinoma (PDAC) tumor microenvironment (TME). However, the molecular mechanisms responsible for the generation of M2-macrophages are unclear. Here, we demonstrated that overexpression of DCLK1-isoform2 in AsPC1 and MIA PaCa2 cells resulted in the polarization of M1-macrophages toward an M2 phenotype via secreted chemokines/cytokines. These M2-macrophages enhanced parental PDAC cell migration, invasion, and self-renewal, and this was associated with increased expression of Snail and Slug. We observed distinct expression of Dclk-isoform2, marked infiltration of M2-macrophages, and a marginal increase of CD8+ T cells in 20-week-old KPCY mice pancreas compared with 5 weeks old. Utilizing an autochthonous mouse model of pancreatic adenocarcinoma, we observed distinct immunoreactive Dclk1 and arginase1 in tissues where CD8+ T-cell infiltration was low and observed a paucity of DCLK1 and arginase1 staining where CD8+ T-cell infiltration was high. Finally, we found that DCLK1-isoform2 tumor-educated M2-macrophages inhibit CD8+ T-cell proliferation and granzyme-B activation. Inhibition of DCLK1 in an organoid coculture system enhanced CD8+ T-cell activation and associated organoid death. We conclude that DCLK1-isoform2 is a novel initiator of alternate macrophage activation that contributes to the immunosuppression observed in the PDAC TME. These data suggest that tumor DCLK1-isoform2 may be an attractive target for PDAC therapy, either alone or in conjunction with immunotherapeutic strategies.
Assuntos
Processamento Alternativo , Carcinoma Ductal Pancreático/imunologia , Regulação Neoplásica da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intracelular/genética , Neoplasias Pancreáticas/imunologia , Proteínas Serina-Treonina Quinases/genética , Animais , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patologia , Carcinoma Ductal Pancreático/prevenção & controle , Movimento Celular , Proliferação de Células , Quinases Semelhantes a Duplacortina , Humanos , Ativação de Macrófagos , Camundongos , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/prevenção & controle , Isoformas de Proteínas , Microambiente TumoralRESUMO
Oncogenic KRAS mutation plays a key role in pancreatic ductal adenocarcinoma (PDAC) tumorigenesis with nearly 95% of PDAC harboring mutation-activated KRAS, which has been considered an undruggable target. Doublecortin-like kinase 1 (DCLK1) is often overexpressed in pancreatic cancer, and recent studies indicate that DCLK1+ PDAC cells can initiate pancreatic tumorigenesis. In this study, we investigate whether overexpressing DCLK1 activates RAS and promotes tumorigenesis, metastasis, and drug resistance. Human pancreatic cancer cells (AsPC-1 and MiaPaCa-2) were infected with lentivirus and selected to create stable DCLK1 isoform 2 (alpha-long, AL) overexpressing lines. The invasive potential of these cells relative to vector control was compared using Matrigel coated transwell assay. KRAS activation and interaction were determined by a pull-down assay and coimmunoprecipitation. Gemcitabine, mTOR (Everolimus), PI3K (LY-294002), and BCL-2 (ABT-199) inhibitors were used to evaluate drug resistance downstream of KRAS activation. Immunostaining of a PDAC tissue microarray was performed to detect DCLK1 alpha- and beta-long expression. Analysis of gene expression in human PDAC was performed using the TCGA PAAD dataset. The effects of targeting DCLK1 were studied using xenograft and Pdx1CreKrasG12DTrp53R172H/+ (KPC) mouse models. Overexpression of DCLK1-AL drives a more than 2-fold increase in invasion and drug resistance and increased the activation of KRAS. Evidence from TCGA PAAD demonstrated that human PDACs expressing high levels of DCLK1 correlate with activated PI3K/AKT/MTOR-pathway signaling suggesting greater KRAS activity. High DCLK1 expression in normal adjacent tissue of PDAC correlated with poor survival and anti-DCLK1 mAb inhibited pancreatic tumor growth in vivo in mouse models.