Hepatocellular carcinomas (HCCs) mainly develop from liver cirrhosis and severe liver fibrosis that are established with long-lasting inflammation of the liver. Silencing of the suppressor of the cytokine signaling-1 (SOCS1) gene, a negative regulator of cytokine signaling, by DNA methylation has been implicated in development or progress of HCC. However, how SOCS1 contributes to HCC is unknown. We examined SOCS1 gene methylation in >200 patients with chronic liver disease and found that the severity of liver fibrosis is strongly correlated with SOCS1 gene methylation. In murine liver fibrosis models using dimethylnitrosamine, mice with haploinsufficiency of the SOCS1 gene (SOCS1−/+ mice) developed more severe liver fibrosis than did wild-type littermates (SOCS1+/+ mice). Moreover, carcinogen-induced HCC development was also enhanced by heterozygous deletion of the SOCS1 gene. These findings suggest that SOCS1 contributes to protection against hepatic injury and fibrosis, and may also protect against hepatocarcinogenesis.

Sprouty and the Sprouty-related protein, Spred (Sprouty-related Ena/vasodilator-stimulated phosphoprotein homology-1 (EVH1) domain-containing protein), inhibit Ras-dependent extracellular signal-regulated kinase (ERK) signaling induced by a variety of growth factors. Since Sprouty proteins have been shown to inhibit not only ERK activation but also cell migration, we postulated that Spreds also inhibit cellular migration. Using stably highly metastatic LM8 cells infected with the Spred1-Sendai virus vector, we demonstrated that Spred1 inhibits the metastasis of LM8 cells in nude mice. Spred1 overexpression also inhibited migration of cells in vitro in response to chemokines, CCL19 and CCL21. We also found that Spred1 overexpression dissolved actin-stress fibers. Both EVH1 domain and C-terminal Sprouty-related domain were required for actin reassembly. Spred1 and Spred2 suppressed constitutively activated RhoA (V14RhoA)-induced stress fiber formation and serum response factor activation. Spred1 bound to activated RhoA, but not cdc42 and Rac. Spred1 also inhibited chemokine-induced RhoA activation and active RhoA-induced Rho-kinase activation. These data suggest that Spreds are key regulators of RhoA-mediated cell motility and signal transduction. Furthermore, our study suggests that the induction of Spreds could be a novel strategy for preventing cancer cell metastasis.

Human papilloma viruses (HPVs) are small doublestranded DNA viruses that infect mucosal and cutaneous epithelium and induce cervical cancer. It has been shown that interferon (IFN)c suppresses proliferation of HPVinfected cells by suppressing expression of HPV E7. Here, we found that IFNc induces not only suppression of E7 transcription but also proteasome-dependent degradation. Suppressor of cytokine signaling-1 (SOCS1)/JAB, a suppressor of cytokine signaling, is known to be induced by IFNc, and functions as an antioncogene against various hematopoietic oncogenic proteins. SOCS1 contains the SOCS-box, which is shown to recruit ubiquitin transferase to the molecules that interact with SOCS1. We found that SOCS1 interacted with HPV E7 protein and induced ubiquitination and degradation of E7 in a SOCS-boxdependent manner. SOCS1 overexpression also increased Rb protein levels and suppressed proliferation of cervical cancer cell lines infected with HPV. Moreover, E7 protein levels were higher and Rb protein levels were lower in SOCS1-deficient fibroblasts infected with retrovirus vector carrying E7 gene than in wild-type fibroblasts. E7 induced anchorage-independent growth in SOCS1-deficient fibroblasts, but not in wild-type cells. These data suggested that SOCS1 plays an important role in regulating the levels of E7 protein and their transforming potential, and could be a new therapeutic tool for HPVmediated tumors.

<Introduction> Cancer stem cells (CSCs) have been defined by the potential to self-renew and to differentiate. CSCs pose a major hurdle in the treatment of cancer. However, the mechanisms by which cells acquire CSC properties such as drug resistance remain unclear. Dual-specificity tyrosine-regulated kinase 2 (DYRK2) is a protein kinase that phosphorylates its substrates on serine/threonine. Initially, we found that DYRK2 phosphorylates p53 at Ser 46 to regulate apoptotic cell death in response to DNA damage. Recently, we have shown that DYRK2 controls Snail degradation in breast cancer and ovarian serous adenocarcinoma. We also found that knockdown of DYRK2 in luminal-type breast cancer MCF-7 cells increased the cancer stem cell population. Kruppel-like factor 4 (KLF4) is one of the Yamanaka factors. It has been reported that pluripotent stem cells from mouse embryonic or adult fibroblasts are induced by introducing four factors, Oct3/4, Sox2, c-Myc, and Klf4. This finding led us to determine if KLF4 is indispensable for the maintenance of CSCs. The aim of this study is to clarify whether DYRK2 regulates CSCs through KLF4 in breast cancer. <Methods> Cell lines: MCF-7 (human mammary carcinoma: ATCC) cells were grown according to standard protocols. We established stable DYRK2-depleted cells. MCF-7 cells were transfected with pSuper-puro vector (pSuper control) or pSuper-puro DYRK2 shRNAs (shDYRK2) with puromycin to isolate stable cell lines. In turn, we established both stable DYRK2- and KLF4-depleted cells. shDYRK2 cells were transfected with pSuper-neo vector (pSuper-neo control) or pSuper-neo KLF4 shRNAs (shKLF4) with puromycin and G418. Knockdown of DYRK2 or KLF4 was confirmed by real-time RT-PCR and immunoblotting. The depleted cells were compared with the control cells using real-time RT-PCR, immunoblotting, flow cytometric analysis, mammosphere assay, xenograft models and immunohistological staining. <Results> We analyzed the population of breast cancer stem cells by flow cytometric analysis and in vitro mammosphere assay. The results showed that knockdown of DYRK2 was associated with the increase of CD44+/CD24- cells. While pSuper control cells formed mammospheres, they did in a lesser extent compared to shDYRK2 cells. In real-time RT-PCR and immunoblotting analysis, stable DYRK2 depletion in MCF-7 cells induced KLF4 accumulation. We then investigated the effect of KLF4 on stemness by flow cytometric analysis and in vitro mammosphere assay. The results showed that knockdown of KLF4 in shDYRK2 cells reduced the proportion of CD44+/CD24- cells. Whereas shDYRK2/shKLF4 cells formed mammospheres, they did in a lesser extent compared to shDYRK2/pSuper-neo control cells. Moreover, the scale of the mammospheres formed in shDYRK2/shKLF4 cells was significantly smaller, as compared with that in shDYRK2/pSuper-neo control cells. In xenograft models, the loss of KLF4 protein expression significantly decreased tumor formation. Immunohistological staining of fifty-nine samples from surgically treated breast cancer patients showed an inverse correlation between DYRK2 and KLF4 expression. <Conclusion> These findings revealed that DYRK2 contributes to the generation of breast cancer stem cells through KLF4. Citation Format: Imawari Y, Mimoto RK, Yamaguchi N, Kamio M, Kato K, Nogi H, Toriumi Y, Uchida K, Takeyama H, Yoshida K. DYRK2 contributes to the generation of breast cancer stem cells through KLF4 [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P5-07-07.

Owing to a typesetting error, the title of the above article was published incorrectly. The correct title is 'SOCS1 inhibits HPV-E7-mediated transformation by inducing degradation of E7 protein.'