Tomohiko Sakabe

Background/Objectives: Mammary serine protease inhibitor (maspin) was originally identified as a tumor suppressor gene, as loss of its expression in breast cancer cell lines promotes invasiveness and tumorigenicity. In contrast, normal pancreatic ductal epithelium does not express the maspin protein, and its expression frequency increases as pancreatic intraepithelial neoplasia (PanIN) progresses to pancreatic ductal adenocarcinoma (PDAC). We have previously reported that cytoplasmic-only localization of maspin (cytMaspin) is an independent indicator of poor prognosis in patients with PDAC. However, the functional role of maspin in PDAC remains unclear. Methods: We investigated the subcellular localization and biological function of maspin in PDAC cell lines using Western blotting, immunofluorescence, RNA-seq, wound-healing assays, and invasion assays. Results: Endogenous maspin was detected in most PDAC cell lines, and RNA-seq was performed in S2-007 (panMaspin; nuclear and cytoplasmic localization of maspin) and S2-028 (cytMaspin), which were derived from the same parental cell line (SUIT-2). The invasive capability of S2-028 cells (cytMaspin) was higher than that of S2-007 cells (panMaspin), showing upregulation of the ErbB family and axon-guidance pathways. Furthermore, maspin overexpression in PANC-1 and S2-020 cells resulted in panMaspin and cytMaspin, respectively. PANC-1 cells overexpressing maspin showed decreased invasive capability via suppression of HER2 expression and Akt activation. Conclusions: Although maspin expression has generally been considered an unfavorable prognostic indicator in patients with PDAC, our findings suggest that its biological effects may differ depending on its subcellular localization. Specifically, nuclear localization is linked to less aggressive phenotypes, whereas cytoplasmic localization is associated with more malignant behavior.

The limited efficacy of immune checkpoint inhibitors in hepatocellular carcinoma (HCC) can arise from the involvement of immunosuppressive cells, such as macrophages. In the present study, we found that the long noncoding RNA NEAT1, particularly its short isoform (NEAT1v1) induced the expression of CD24, which is known as an immune checkpoint molecule for macrophages. Mechanistically, NEAT1v1 sponged miR-320a-3p to upregulate the transcription factor SP1, which in turn, activated CD24 transcription. A spheroid co-culture of primary or THP-1-derived macrophages with HCC cells revealed that NEAT1v1 suppressed M1 marker expression and phagocytic activity in macrophages. In a syngeneic subcutaneous model of HCC, Neat1v1 increased the tumor infiltration of M2-like macrophages and induced resistance to anti-Pd-1 antibody, while combination of the anti-Pd-1 and anti-Cd24 antibodies significantly suppressed the tumor growth. Finally, NEAT1, SP1, and CD24 expression increased in tumor tissues from patients with HCC, compared to adjacent normal tissues, whereas miR-320a-3p was significantly downregulated. Moreover, plasma NEAT1 cell-free RNA was significantly decreased after therapeutic intervention. Taken together, NEAT1v1 protects HCC cells from macrophages by sending a "Don't Eat Me" signal via CD24, and is therefore, a potential target molecule for the treatment and diagnosis of HCC.

Mammary serine protease inhibitor (maspin) is a tumor suppressor protein downregulated during carcinogenesis and cancer progression; cytoplasmic-only maspin expression is an independent, unfavorable prognostic indicator in patients with lung squamous cell carcinoma (LUSC). We hypothesized that the cytoplasmic-only localization of maspin has tumor-promoting functions in LUSC. The subcellular localization of maspin and the invasive capability of LUSC cell lines were investigated using RNA sequencing (RNA-seq), Western blotting, and siRNA transfection. Maspin mRNA and protein expression were suppressed in LK-2 and RERF-LC-AI cells. Cell invasion significantly increased in response to siRNA-mediated maspin knockdown in KNS-62 cells expressing both nuclear and cytoplasmic maspin. In LK-2 cells, both nuclear and cytoplasmic maspin were re-expressed, and cell invasion and migration were significantly decreased. In contrast, re-expressed maspin in RERF-LC-AI cells was detected only in the cytoplasm (cytMaspin), and cell invasion and migration were significantly promoted. RNA-seq and downstream analyses revealed that increased cytMaspin expression downregulated the genes associated with cell adhesion and activated PYK2 and SRC, which play important roles in cancer progression. Our study demonstrates a novel biological function of cytMaspin in enhancing the invasive capabilities of LUSC cells. Understanding cytoplasm-to-nuclear maspin translocation dysregulation may develop novel therapeutic approaches to improve the prognosis of patients with LUSC.

Background Mammary serine protease inhibitor (maspin) is well known as a tumor suppressor gene in several types of cancers and its nuclear localization is essential for its tumor-suppressive function. We previously reported that the cytoplasmic-only localization of maspin is significantly correlated with unfavorable prognosis in patients with lung adenocarcinoma (LUAD). To clarify whether maspin in LUAD acts as a tumor promoter or suppressor, we examined the subcellular localization-dependent biological functions of maspin in human LUAD cell lines. Methods The expression levels and subcellular localization of maspin were investigated by performing immunoblotting and immunofluorescence in human LUAD cell lines (PC-9, A549, NCI-H23, RERF-LC-KJ) and human bronchial epithelial cell line (BEAS-2B). We then established stable cell lines overexpressing maspin (A549-maspin and RERF-LC-KJ-maspin) and investigated their subcellular localization. Cell invasion assays of these cell lines were performed to examine their invasiveness. Moreover, the mRNA expression levels between epithelial cell markers (E-cadherin) and mesenchymal cell markers (N-cadherin and vimentin) were compared. Results The expression of maspin in PC-9 cells was comparable to that in BEAS-2B cells, whereas its expression in A549, NCI-H23, and RERF-LC-KJ cells was decreased. The cell invasion capability of A549-maspin cells showing pancellular expression was significantly decreased compared with that of A549-control cells. By contrast, the cell invasion capability of RERF-LC-KJ-maspin cells showing cytoplasmic-only expression was significantly increased compared with that of RERF-LC-KJ-control cells. The mRNA expression levels of N-cadherin, but not E-cadherin and vimentin, in A549-maspin cells was significantly downregulated compared with that in A549-control cells. No significant differences in these markers were observed between RERF-LC-KJ-maspin and RERF-LC-KJ-control cells. Conclusion The invasive capability of LUAD cells is regulated by the intracellular localization of maspin. Clarification of the molecular mechanism underlying the subcellular localization-dependent function of maspin will promote a deeper understanding of LUAD development and progression.

Aims: Podoplanin expression in cancer‐associated fibroblasts (CAFs) has been proposed as an unfavourable indicator in squamous cell carcinoma of the lung, but little is known about its clinical significance in early‐stage lung adenocarcinoma. We evaluated the prognostic impact of podoplanin expression in patients with pathological stage (p‐stage) IA lung adenocarcinoma as categorised by the 8th edition of the tumour–node–metastasis classification for lung cancer.Methods and results: Immunohistochemical analyses using anti‐podoplanin antibody were performed on resected specimens from 158 patients with p‐stage IA lung adenocarcinoma. When more than 10% of cancer cells or CAFs showed immunoreactivity with podoplanin, the specimens were classified as podoplanin‐positive. Podoplanin‐positive status in cancer cells (n = 8) was not correlated with clinicopathological factors or with patient prognosis. Podoplanin‐positive status in CAFs (n = 41) was correlated significantly with poorer tumour differentiation (P < 0.001), the presence of lymphatic invasion (P < 0.001) and high‐grade (solid and/or micropapillary) components constituting ≥1% of the entire tumour (P < 0.001). The log‐rank test showed that podoplanin‐positive status in CAFs was associated significantly with shorter disease‐free survival (DFS) (P < 0.001) and disease‐specific survival (P = 0.015). In Cox's multivariate analysis, podoplanin‐positive status in CAFs had the most significant effect on shorter DFS [hazard ratio (HR) = 4.411, P = 0.004], followed by the presence of high‐grade components (HR = 3.581, P = 0.013).Conclusions: Podoplanin expression in CAFs could be an independent predictor of increased risk of recurrence in patients with p‐stage IA lung adenocarcinoma.

Background/Aim: The presence of cancer stem cells (CSCs) contributes to metastasis, recurrence, and resistance to chemo/radiotherapy in hepatocellular carcinoma (HCC). The WNT signaling pathway is reportedly linked to the maintenance of stemness of CSCs. In the present study, in order to eliminate liver CSCs and improve the prognosis of patients with HCC, we explored whether small-molecule compounds targeting WNT signaling pathway suppress liver CSCs. Materials and Methods: The screening was performed using cell proliferation assay and reporter assay. We next investigated whether these compounds suppress liver CSC properties by using flow cytometric analysis and sphere-formation assays. A mouse xenograft model transplanted with CD44-positive HuH7 cells was used to examine the in vivo antitumor effect of IC-2. Results: In HuH7 human HCC cells, 10 small-molecule compounds including novel derivatives, IC-2 and PN-3-13, suppressed cell viability and WNT signaling activity. Among them, IC-2 significantly reduced the CD44-positive population, also known as liver CSCs, and dramatically reduced the sphere-forming ability of both CD44-positive and CD44-negative HuH7 cells. Moreover, CSC marker-positive populations, namely CD90-positive HLF cells, CD133-positive HepG2 cells, and epithelial cell adhesion molecule-positive cells, were also reduced by IC-2 treatment. Finally, suppressive effects of IC-2 on liver CSCs were also observed in a xenograft model using CD44-positive HuH7 cells. Conclusion: The novel derivative of small-molecule WNT inhibitor, IC-2, has the potential to suppress liver CSCs and can serve as a promising therapeutic agent to improve the prognosis of patients with HCC.

Non-small cell lung cancer (NSCLC) accounts for >85% of incidences of lung cancer, for which the predicted 5-year survival rates are low and recurrence rates remain high. Although it has been reported that the patients with SCLC cells that possess the cluster of differentiation (CD) 117 marker exhibited poor prognosis and poor response to chemotherapy, no studies concerning the association of CD117 expression with prognosis of the patients with NSCLC have been reported. An in vitro study reportedly revealed that CD117-positive cell populations in NSCLC cell lines exhibited cancer stem cell (CSC) phenotypes including self-renewal and chemoresistance. Therefore, the present study hypothesized that if CD117-positive cells are CSC-like cells, CD117 positivity may be associated with the prognosis of patients with NSCLC. To confirm this hypothesis, the association between CD117 expression in patients with NSCLC and clinicopathological characteristics was investigated. CD177 expression was examined by immunohistochemistry in 99 patients with NSCLC who underwent curative surgical resection. Tumor samples in the present study included 73 samples of adenocarcinoma and 26 of squamous carcinoma. The associations of CD177 expression with clinicopathological features and prognosis were examined. The lymph node metastasis and rates of recurrence were significantly associated with overall survival rates through multivariate analysis (P<0.001 and P<0.001), respectively. A Kaplan-Meier analysis for relapse-free survival and the log-rank test revealed that the patients with CD117-positive cell populations exhibited shorter relapse-free survival rates compared with patients whose cells were CD117-negative (P=0.014). The multivariate analysis demonstrated that venous invasion, pathological stage, and CD117 expression were independent prognostic parameters for relapse-free survival in patients with NSCLC (P=0.001, P=0.001 and P=0.002), respectively. In conclusion, these data suggest that CD117 expression in NSCLC may serve as a useful marker for predicting the prognosis of patients with NSCLC.

The increasing numbers of patients with hepatocellular carcinoma (HCC), which is characterized by an extremely poor prognosis, are causing serious problems worldwide. To improve their prognosis, the development of a more effective therapy is required. Combination therapy with interferon-α (IFN-α) and 5-fluorouracil (5-FU) was reported to provide benefits to patients with advanced HCC, although its efficacy is still unsatisfactory. In this study, taking advantage of functional screening, three chemosensitizing genes for this therapy against HCC cells were identified. Identification of chemosensitizing genes was performed using ribozyme-based functional screening. We constructed a plasmid DNA library expressing random ribozyme genes with ∼6.0 × 10⁶ target recognition sequences. We performed a positive-selection strategy based on the ability of 5-FU to eliminate cells expressing a ribozyme that targets chemosensitivity-unrelated genes. The ribozyme library was transfected into HepG2 cells, which were subsequently treated with the IC50 dose of 5-FU. This cycle was repeated 10 times to fully concentrate the ribozymes targeting those genes involved in the sensitivity of 5-FU. Consequently, 100 target ribozyme recognition sequences that were recovered before screening and after 10 cycles of screening were subjected to a BLAST search to retrieve genes with homologous sequences. This analysis indicated 3 genes (PRKAG2, TGFBR2, and EXT1) as candidate genes. The chemosensitizing effects of these genes on IFN-α/5-FU as well as 5-FU treatment were confirmed by adenovirus-mediated gene transfer. In addition, the mechanisms of effects of these genes on enhancing effects on combination therapy with IFN-α/5-FU were also evaluated. Furthermore, to evaluate the relationship between their expression levels in tumor tissues and the clinical outcome of IFN-α/5-FU combination therapy in advanced HCC patients, mRNA expression was measured using real-time reverse transcription-polymerase chain reaction. As a result, PRKAG2, TGFBR2, and EXT1 were identified as novel chemosensitizing genes for the combination therapy against HCC. TGFBR2 and EXT1 induced transforming growth factor-β signal activation and endoplasmic reticulum stress, respectively. Moreover, PRKAG2 and TGFBR2 were suggested to be promising predictive markers for the clinical outcome of this therapy. In conclusion, the identified genes are promising candidates as targets to enhance the therapeutic effect of IFN-α/5-FU combination therapy in advanced HCC and could be used as predictive markers for response to this therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5131. doi:1538-7445.AM2012-5131

Aim: Bone marrow-derived mesenchymal stem cells (MSC) are expected to be an excellent source of cells for transplantation. We aimed to study the culture conditions and involved genes to differentiate MSC into hepatocytes. Methods: The culture conditions to induce the efficient differentiation of human bone marrow-derived UE7T-13 cells were examined using cytokines, hormones, 5-azacytidine and type IV collagen. Results: We found that combination of acidic fibroblast growth factor (aFGF), basic fibroblast growth factor (bFGF) and hepatocyte growth factor (HGF) with type IV collagen coating induced hepatic differentiation of UE7T-13 cells at over 30% frequency, where expression of albumin mRNA was increased over 20-fold. The differentiated cells had functions of albumin production, glycogen synthesis and urea secretion as well as expressing hepatocyte-specific genes. In addition, these cellshave binuclear and cuboidal morphology, which is a characteristic feature of hepatocytes. During hepatic differentiation, UE7T-13 cells showed depressed expression of WISP1 and WISP2 genes, members of the CCN family. Conversely, knockdown of WISP1 or WISP2 gene by siRNA stimulated hepatic differentiation. The effect of aFGF/bFGF/HGF/type IV collagen coating and WISP1-siRNA on hepatic differentiation was additive. Conclusion: The present study suggests that aFGF/bFGF/HGF/type IV collagen coating is the efficient condition for hepatic differentiation of UE7T-13 cells, and that WISP1 and WISP2 play an important role in hepatic transdifferentiation of these cells.