CD44v9 is expressed in cancer stem cells (CSC) and stabilizes the glutamate‐cystine transporter xCT on the cytoplasmic membrane, thereby decreasing intracellular levels of reactive oxygen species (ROS). This mechanism confers ROS resistance to CSC and CD44v9‐expressing cancer cells. The aims of the present study were to assess: (i) expression status of CD44v9 and xCT in hepatocellular carcinoma (HCC) tissues, including those derived from patients treated with hepatic arterial infusion chemoembolization (HAIC) therapy with cisplatin (CDDP); and (ii) whether combination of CDDP with sulfasalazine (SASP), an inhibitor of xCT, was more effective on tumor cells than CDDP alone by inducing ROS‐mediated apoptosis. Twenty non‐pretreated HCC tissues and 7 HCC tissues administered HAIC therapy with CDDP before surgical resection were subjected to immunohistochemistry analysis of CD44v9 and xCT expression. Human HCC cell lines HAK‐1A and HAK‐1B were used in this study; the latter was also used for xenograft experiments in nude mice to assess in vivo efficacy of combination treatment. CD44v9 positivity was significantly higher in HAIC‐treated tissues (5/7) than in non‐pretreated tissues (2/30), suggesting the involvement of CD44v9 in the resistance to HAIC. xCT was significantly expressed in poorly differentiated HCC tissues. Combination treatment effectively killed the CD44v9‐harboring HAK‐1B cells through ROS‐mediated apoptosis and significantly decreased xenografted tumor growth. In conclusion, the xCT inhibitor SASP augmented ROS‐mediated apoptosis in CDDP‐treated HCC cells, in which the CD44v9‐xCT system functioned. As CD44v9 is typically expressed in HAIC‐resistant HCC cells, combination treatment with SASP with CDDP may overcome such drug resistance.

Background: Although lenvatinib has become the standard therapy for hepatocellular carcinoma (HCC), the high incidence rate of adverse events (AEs) is an issue. This study aimed to clarify the AEs of lenvatinib and the therapeutic impact of five days-on/two days-off administration (i.e., weekends-off strategy) for lenvatinib. Methods: We retrospectively assessed the therapeutic effects and AEs of 135 patients treated with lenvatinib, and the improvement of tolerability and therapeutic efficacy of 30 patients treated with the weekends-off strategy. We also evaluated lenvatinib-induced vascular changes in tumors and healthy organs using a mouse hepatoma model. Results: The incidence rates of any grade and grade ≥ 3 AEs were 82.1% and 49.6%. Fatigue was the most important AE since it resulted in dose reduction and discontinuation. Of the 30 patients who received weekends-off lenvatinib, 66.7% tolerated the AEs. Although 80.8% of the patients showed progression after dose reduction, the therapeutic response improved in 61.5% of the patients by weekends-off lenvatinib. Notably, weekends-off administration significantly prolonged the administration period and survival (p < 0.001 and p < 0.05). The mouse hepatoma model showed that weekends-off administration contributed to recovery of vascularity in the organs. Conclusion: Weekends-off administration of lenvatinib was useful to recover the therapeutic response and tolerability toward AEs.

Doublecortin-like kinase 1 (DCLK1), a marker for intestinal and pancreatic cancer stem cells, is highly expressed in neuroblastomas. This study was conducted to assess DCLK1 expression levels in pancreatic neuroendocrine tumor (PNET) tissues and to explore the roles of this molecule in clinical tissue from multiple PNET patients, cells (BON1, QGP1, and CM) and tumor xenografts. Immunohistochemically, all PNET tissues highly and diffusely expressed DCLK1 as a full-length isoform, identical to that detected in primary liver NETs. A DCLK1-overexpressing PNET cell line (QGP1-DCLK1) exhibited epithelial-mesenchymal transition (EMT)-related gene signatures, and robust upregulation of Slug (SNAI2), N-Cadherin (CDH2), and Vimentin (VIM) was validated by real-time PCR and immunoblotting. QGP1-DCLK1 cells had increased cell migration in a wound-healing assay and formed significantly larger xenograft tumors in nude mice. The factors involved in the formation of the fast-growing tumors included p-FAK (on Tyr925), p-ERK1/2, p-AKT, Paxillin, and Cyclin D1, which upon knockdown or pharmacologic inhibition of DCLK1 abolished the expression of these molecules. In conclusion, robust and ubiquitous expression of DCLK1 was first demonstrated here in human PNET tissue specimens and cells. DCLK1 characterized the PNET cell behavior, inducing p-FAK/SLUG-mediated EMT. These findings suggest the possibility of developing novel therapeutic strategies against PNETs by targeting DCLK1. Evidence here reveals that human PNETs diffusely and robustly express the cancer stem cell marker DCLK1, which drives SLUG-mediated EMT, and suggests that NETs share biological features for druggable targets with other tumors, including neuroblastoma that also highly expresses DCLK1. .

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The cytokine transforming growth factor-β (TGF-β) serves a key role in hepatic fibrosis and has cytostatic effects on hepatocytes. The present study investigated the anti-fibrogenic and regenerative effects of the TGF-β receptor type I kinase inhibitor galunisertib (LY2157299) in mice with carbon tetrachloride (CCl 4 )-induced liver cirrhosis and in vitro. Mice were intraperitoneally treated with CCl 4 for 8 weeks. At week 5, the mice were divided randomly into four treatment groups: Vehicle-treated; and treated with low-; middle-; and high-dose galunisertib, which was administered from weeks 5-8. The mice were sacrificed after 8 weeks of ccl 4 treatment. Liver fibrosis, as evaluated by histology and determination of hydroxyproline content, progressed during week 4-8 of ccl 4 treatment in the vehicle-treated mice. Galunisertib treatment dose-dependently prevented liver fibrosis, as demonstrated by the direct inhibition of α-smooth muscle actin-positive activated hepatic stellate cells (HSCs) after 8 weeks of ccl 4 treatment. The levels of active matrix metalloproteinase (MMP)-9 in galunisertib-treated livers were significantly increased compared with the vehicle-treated livers. In the high-dose group, the number of PCNA-positive hepatocytes and endothelial cells markedly increased compared with the vehicle group. Reverse transcription-quantitative PCR analysis verified that interleukin-6 and epiregulin expression levels were significantly increased in livers from the group treated with high-dose galunisertib compared with the vehicle-treated group. Galunisertib inhibited the proliferation of activated HSCs and collagen synthesis in addition to restoring MMP activity. Moreover, galunisertib promoted liver remodeling by proliferating hepatocytes and vascular endothelial cells, while significantly increasing liver weight. These results are consistent with the cytostatic action of TGF-β that negatively regulates liver regeneration, and demonstrated that galunisertib inhibited TGF-β signaling, halted liver fibrosis progression and promoted hepatic regeneration. The results of the present study suggest that galunisertib may be an effective treatment for liver cirrhosis.