Constitutive activation of STAT3 is one of the major oncogenic pathways involved in the development of various types of malignancies including colorectal cancer (CRC); and thus becomes a promising therapeutic target. Spica Prunellae has long been used as an important component in many traditional Chinese medicine formulas to clinically treat CRC. Previously, we found that Spica Prunellae inhibits CRC cell growth through mitochondrion-mediated apoptosis. Furthermore, we demonstrated its anti-angiogenic activities in vivo and in vitro. To further elucidate the precise mechanism of the potential tumoricidal activity of Spica Prunellae, using a CRC mouse xenograft model, in this study we evaluated its therapeutic efficacy against CRC and investigated the underlying molecular mechanisms. CRC mouse xenograft model was generated by subcutaneous injection of human colon carcinoma HT-29 cells into nude mice. Animals were given intra-gastric administration with 6 g/kg of the ethanol extract of Spica Prunellae (EESP) daily, 5 days a week for 16 days. Body weight and tumor growth were measured every two days. Tumor growth in vivo was determined by measuring the tumor volume and weight. HT-29 cell viability was examined by MTT assay. Cell apoptosis and proliferation in tumors from CRC xenograft mice was evaluated via immunohistochemical staining (IHS) for TUNEL and PCNA, and the intratumoral microvessel density (MVD) was examined by using IHS for the endothelial cell-specific marker CD31. The activation of STAT3 was evaluated by determining its phosphorylation level using IHS. The mRNA and protein expression of Bcl-2, Bax, Cyclin D1, VEGF-A and VEGFR2 was measured by RT-PCR and IHS, respectively. EESP treatment reduced tumor volume and tumor weight but had no effect on body weight change in CRC mice; decreased HT-29 cell viability in a dose-dependent manner, suggesting that EESP displays therapeutic efficacy against colon cancer growth in vivo and in vitro, without apparent toxicity. In addition, EESP significantly inhibited the phosphorylation of STAT3 in tumor tissues, indicating its suppressive action on the activation of STAT3 signaling. Consequently, the inhibitory effect of EESP on STAT3 activation resulted in an increase in the pro-apoptotic Bax/Bcl-2 ratio, decrease in the expression of the pro-proliferative Cyclin D1 and CDK4, as well as down-regulation of pro-angiogenic VEGF-A and VEGFR-2 expression. Finally, these molecular effects led to the induction of apoptosis, the inhibition of cell proliferation and tumor angiogenesis. Spica Prunellae possesses a broad range of anti-cancer activities due to its ability to affect STAT3 pathway, suggesting that Spica Prunellae could be a novel potent therapeutic agent for the treatment of CRC.

Microtubule-associated doublecortin-like kinase 1 (DCLK1) is an accepted marker of tuft cells (TCs) and several kinds of cancer stem cells (CSCs), and emerging evidence suggests that DCLK1-positive TCs participate in the initiation and formation of inflammation-associated cancer. DCLK1-expressing CSCs regulate multiple biological processes in cancer, promote resistance to therapy, and are associated with metastasis. In solid tumor cancers, tumor epithelia, immune cells, cancer-associated fibroblasts, endothelial cells and blood vessels, extracellular matrix, and hypoxia all support a CSC phenotype characterized by drug resistance, recurrence, and metastasis. Recently, studies have shown that DCLK1-positive CSCs are associated with epithelial-mesenchymal transition, angiogenesis, and immune checkpoint. Emerging data concerning targeting DCLK1 with small molecular inhibitors, monoclonal antibodies, and chimeric antigen receptor T-cells shows promising effects on inhibiting tumor growth and regulating the tumor immune microenvironment. Overall, DCLK1 is reaching maturity as an anti-cancer target and therapies directed against it may have potential against CSCs directly, in remodeling the tumor microenvironment, and as immunotherapies.

The approval of immune checkpoint inhibitors has expanded treatment options for renal cell carcinoma (RCC), but new therapies that target RCC stemness and promote anti-tumor immunity are needed. Previous findings demonstrate that doublecortin-like kinase 1 (DCLK1) regulates stemness and is associated with RCC disease progression. Herein, we demonstrate that small-molecule kinase inhibitor DCLK1-IN-1 strongly inhibits DCLK1 phosphorylation and downregulates pluripotency factors and cancer stem cell (CSC) or epithelial-mesenchymal transition (EMT)-associated markers including c-MET, c-MYC, and N-Cadherin in RCC cell lines. Functionally, DCLK1-IN-1 treatment resulted in significantly reduced colony formation, migration, and invasion. Additionally, assays using floating or Matrigel spheroid protocols demonstrated potent inhibition of stemness. An analysis of clinical populations showed that DCLK1 predicts RCC survival and that its expression is correlated with reduced CD8+ cytotoxic T-cell infiltration and increases in M2 immunosuppressive macrophage populations. The treatment of RCC cells with DCLK1-IN-1 significantly reduced the expression of immune checkpoint ligand PD-L1, and co-culture assays using peripheral blood monocytes (PBMCs) or T-cell expanded PBMCs demonstrated a significant increase in immune-mediated cytotoxicity alone or in combination with anti-PD1 therapy. Together, these findings demonstrate broad susceptibility to DCLK1 kinase inhibition in RCC using DCLK1-IN-1 and provide the first direct evidence for DCLK1-IN-1 as an immuno-oncology agent.

Colorectal cancer (CRC) is a major source of morbidity and mortality, characterized by intratumoral heterogeneity and the presence of cancer stem cells (CSCs). Bufalin has potent activity against many tumors, but studies of its effect on CRC stemness are limited. We explored bufalin’s function and mechanism using CRC patient-derived organoids (PDOs) and cell lines. In CRC cells, bufalin prevented nuclear translocation of β-catenin and down-regulated CSC markers (CD44, CD133, LGR5), pluripotency factors, and epithelial–mesenchymal transition (EMT) markers (N-Cadherin, Slug, ZEB1). Functionally, bufalin inhibited CRC spheroid formation, aldehyde dehydrogenase activity, migration, and invasion. Network analysis identified a C-Kit/Slug signaling axis accounting for bufalin’s anti-stemness activity. Bufalin treatment significantly downregulated C-Kit, as predicted. Furthermore, overexpression of C-Kit induced Slug expression, spheroid formation, and bufalin resistance. Similarly, overexpression of Slug resulted in increased expression of C-Kit and identical functional effects, demonstrating a pro-stemness feedback loop. For further study, we established PDOs from diagnostic colonoscopy. Bufalin differentially inhibited PDO growth and proliferation, induced apoptosis, restored E-cadherin, and downregulated CSC markers CD133 and C-Myc, dependent on C-Kit/Slug. These findings suggest that the C-Kit/Slug axis plays a pivotal role in regulating CRC stemness, and reveal that targeting this axis can inhibit CRC growth and progression.

Doublecortin‑like kinase 1 (DCLK1) has been identified as a novel biomarker of cancer stem cells among several different cancer types, including colon, breast, pancreas, kidney, liver, stomach and esophageal cancers. Studies have demonstrated that DCLK1 regulates tumorigenesis and epithelial‑mesenchymal transformation via several important pathways, such as Notch, Wnt/β‑catenin, RAS and multiple microRNAs. The function and biological mechanisms, including their association with the molecular structure and isoforms of DCLK1, are gradually being elucidated. However, the currently available knowledge regarding DCLK1 in terms of developing effective anti‑cancer drugs remains incomplete. In the present review, the molecular characteristics, biomarker function and biological mechanisms of DCLK1 are summarized and DCLK1 is proposed as a potential anti‑tumor target via the glucose metabolism pathway.

Signal transducer and activator of transcription 3 (STAT3) plays a critical role in cell survival and proliferation and is constitutively activated in many types of human cancers including hepatocellular carcinoma (HCC). Therefore, it is a major focus in the development of anticancer agents. Rubus aleaefolius Poir. has been demonstrated to be effective in the treatment of HCC. However, the precise mechanism of its anticancer activity remains largely unknown. Using HepG2 cells and a HCC mouse xenograft model, in the present study we evaluated the effect of the total alkaloids of Rubus aleaefolius Poir. (TARAP) on tumor growth in vitro and in vivo and investigated the underlying molecular mechanisms. We found that TARAP inhibited the proliferation of HepG2 human HCC cells and blocked G1/S cell cycle progression. In addition, TARAP treatment suppressed STAT3 phosphorylation in tumor tissues. Consequently, the inhibitory effect of TARAP on STAT3 activation resulted in the inhibition of proliferation. Moreover, TARAP altered the expression of several important target genes of the STAT3 signaling pathway, such as decreased expression of cyclinD1, cyclinE, cyclin-dependent kinase (CDK) 4 and CDK2 as well as upregulated p21. These results suggest that suppression of the STAT3 signaling pathway leading to inhibition of proliferation and cell cycle arrest may be one of the mechanisms of the anticancer activity of TARAP against HCC.