Yousuke Ueoka

Sodium butyrate induces growth arrest and senescence‐like phenotypes in gynecologic cancer cells

We demonstrated here the growth‐suppressing effects of sodium butyrate (NaB) on human endometrial and ovarian cancer cells. The arrest of cells at the G1 checkpoint accounted for this effect. NaB‐mediated p21 might arrest endometrial and ovarian cancer cells at the G0/G1 phase by eliciting pRb unphosphorylation. To demonstrate the role of pRb regulation by p21, we measured the sensitivity to NaB of cervical cancer cells in which pRb had been inactivated by HPV E7. The cervical cancer cells displayed a sensitivity in NaB‐mediated G2/M arrest in addition to their sensitivity in G0/G1 arrest. Arrest at G0/G1 and G2/M accompanied induction of senescence‐like phenotypes (SLPs). Most importantly, the effect of NaB on senescence induction was not coupled with the predominance of hypophosphorylated pRb forms in the cervical cancer cells. This suggested that NaB had the potential to elicit SLPs through p21‐mediated withdrawal from cell cycle progression. The consequences of p21 induction were manifold. The effects of NaB on gynecologic cancer cell growth indicated its potential use in cancer treatment. NaB was effective even in the cancer cells with mutant p53 and/or Rb genes by eliciting cell senescence.

Hepatocyte growth factor modulates motility and invasiveness of ovarian carcinomas via Ras-mediated pathway

Hepatocyte growth factor (HGF) is a multifunctional growth factor which has pleiotrophic biological effects on epithelial cells such as proliferation, motogenesis, invasiveness and morphogenesis. Peritoneal dissemination is critical for the progression of ovarian cancer, and our study revealed that HGF induces migration and invasion of ovarian cancer cells. We also demonstrated that HGF stimulates autophosphorylation of its receptor, followed by activation of the Ras-MAP (mitogen-activated peptide) kinase cascade. Moreover, infection of ovarian cancer cells with Ras dominant-negative adenovirus reduced the HGF-induced motogenic and invasive activities. Additionally, both MEK and PI3-kinase pathways downstream of Ras were involved in HGF-stimulated ovarian cancer cell invasiveness.

K-Ras and H-Ras activation promote distinct consequences on endometrial cell survival.

A considerable amount of evidence indicates that Ras signaling contributes to the development of endometrial cancer. We previously demonstrated that endometrial cancer cells carrying oncogenic [(12)Val]K-ras were susceptible to apoptosis. The present study examined the role of K-and H-Ras in the induction of apoptosis using rat endometrial cells (RENT4 cells). We found that constitutively activated K-Ras promoted apoptotic cell death, whereas the H-Ras mutant rescued rat endometrial cells from apoptosis. Expression of a constitutively active form of Raf-1 (Raf-CAAX) promoted apoptosis, whereas expression of a constitutively active catalytic subunit of phosphoinositide 3-kinase, p110K227E, allowed cells to escape from apoptosis. Moreover, inhibition of the MEK-MAPK pathway by the specific inhibitor, UO126, rescued the cells from apoptosis, whereas the inhibition of phosphoinositide 3-kinase by its specific inhibitor, LY294002, promoted apoptosis in RENT4 cells expressing activated K-Ras. However, both inhibitors promoted apoptosis in RENT4 cells expressing activated H-Ras. This difference in the regulation of apoptosis by the MEK inhibitor between K-Ras- and H-Ras-expressing cells depended on the interaction of effector proteins downstream of each Ras isoform. Finally, to elucidate the role of downstream K-Ras signal pathways, we generated K-Ras effector domain mutants (K12V35S, K12V40C). We examined the incidence of apoptotic cell death induced by the K-Ras effector domain mutants (K12V35S, K12V40C). The relative ratio of phospho-MAPK to phospho-Akt compared with that of mock cells was higher in K12V35S cells than in K12V40C cells. Ectopic expression of K12V35S protein increased the proportion of apoptotic cells, and in turn, the expression of K12V40C protein decreased compared with the expression of K12V protein without the effector domain mutant. These results demonstrate that K- and H-Ras-mediated signaling pathways exert distinct effects on apoptosis and that K-Ras downstream Raf/MEK/MAPK pathway is required for the induction of apoptosis in endometrial cells. Coordination of the two pathways contributes to endometrial cell survival.

Level of reactive oxygen species induced by p21WAF(1)/CIP(1) is critical for the determination of cell fate

p21WAF(1)/CIP(1) is a well‐known cell cycle regulatory protein which is overexpressed in several cancer cell lines, and known to determine cell fate. We generated three recombinant adenovirus vectors that expressed either the full‐length p21 (Ad‐p21F), a p21 mutant with a deletion of the C‐terminal proliferative cell nuclear antigen (PCNA) binding domain (Ad‐p21N), or a p21 mutant with a deletion of the N‐terminal cyclin‐dependent kinase binding domain (Ad‐p21C). We transfected these vectors into five cancer cell lines. Premature senescence was induced in all of the lines only following transfection with Ad‐p21N and Ad‐p21F. In addition, apoptosis was also induced in LoVo and HCT116 cells that harbored wild‐type p53 and the reactive oxygen species (ROS) level was higher than in senescent cells. Finally, the induction of apoptosis was inhibited by using siRNA to downregulate p53. This observation implies that there is a feedback signaling loop involving p21/ROS/p53 in apoptotic responses. It appears to be, at least in part, driven by high levels of p21 protein. Next, we investigated the cell death effect of endogenous p21 protein on cell fate using sodium butyrate (NaB). Treatment with 1 mM NaB or 2 to 5 mM NaB induced senescence or apoptosis, respectively. The level of intracellular ROS in 5 mM NaB treated cells was 2‐fold higher, compared with that in 1 mM NaB treated cells. We also demonstrated that DNA damage response signals including ataxia telangiectasia mutated, γH2AX, and p38 MAPK were involved in NaB‐induced cell death. The magnitude of intracellular ROS levels in response to p21 elicited either senescence or apoptosis in the cancer cell lines. (Cancer Sci 2009; 100: 1275–1283)

Contribution of enhanced transcriptional activation by ER to [12Val] K-Ras mediated NIH3T3 cell transformation

We investigated the biological significance of estrogen receptors (ER) in NIH3T3 cell transformation by the [12Val] K-Ras mutant. This mutant enhanced the steady state level of ER. Cells expressing mutant K-Ras (K12V cell) were tumorigenic. To determine the role of ER accumulation in Ras-transformed cells, we developed cells (KwtER cells) that overexpressed both wild-type (wt) K-Ras and ER, and found these cells were also tumorigenic. E2 stimulated the transcriptional activity by ER dominantly in K12V cells. However, only partial activation of ER by E2 was seen in KwtER cells. In the presence of 10% serum in media, the activation of ER appeared only in transformed KtwER and K12V cells, suggesting that two independently transmitted signals, the E2-ER binding and the ER-AF1 activation, are necessary for ER activation and that the dominant activation of ER might be involved in Ras-mediated cell transformation. Co-expression of progesterone receptor (PR) with mutant K-Ras led to suppression of tumorigenicity and inhibition of the activation of ER. The antisense oligomers complementary to the ER suppressed proliferation and transformed phenotypes of K12V cells. These observations support the importance of ER in Ras-mediated cell transformation.

Oncogenic ras modulates epidermal growth factor responsiveness in endometrial carcinomas

Since the majority of endometrial carcinomas do not contain any detectable ras mutations, the precise contribution of aberrant Ras function, if any, to endometrial carcinoma development remains to be determined. Since there is considerable evidence that Ras transformation is associated with a decreased requirement for growth factors, we compared the growth response of endometrial carcinoma cells harbouring wild-type (Ishikawa cells) or mutated (HHUA cells) K-ras to epidermal growth factor (EGF). K-ras mutation did not significantly affect the level of the EGF receptor (EGFR) expressed in these carcinoma cells. EGF could stimulate the growth of Ishikawa, but not HHUA cells. Furthermore, EGF caused elevation of Ras-GTP levels in Ishikawa, but not HHUA cells. However, the introduction of mutated, but not normal, K-ras into Ishikawa cells rendered them non-responsive to EGF growth stimulation. Thus, the presence of mutated K-ras alone modulated the growth response of endometrial carcinoma cells to EGF. An inhibitor of the EGFR tyrosine kinase activity could prevent soft agar colony formation of Ishikawa cells, but not HHUA or mutant K-ras(12V)-transfected Ishikawa cells. Taken together, these results suggest that mutated K-ras causes a loss of responsiveness to EGF stimulation and that EGFR function is dispensable for the growth of mutant Ras-positive endometrial carcinoma cells.

Hepatocyte growth factor modulates motility and invasiveness of ovarian carcinomas via ras mediated pathway

Hepatocyte growth factor (HGF) is a multifunctional growth factor which has pleiotrophic biological effects on epitherial cells, such as proliferation, motogenesis, invasiveness and morphogenesis. Peritoneal dissemination is critical for the progression of ovarian cancer and our study revealed that HGF induces migration and invasion of ovarian cancer cells. We also demonstrated that HGF stimulates autophosphorylation of its receptor, followed by activation of the Ras-MAP (mitogen-activated peptide) kinase cascade. Moreover, infection of ovarian cancer cells with Ras dominant-negative adenovirus reduced the HGF-induced motogenic and invasive activities. Additionally, both MEK and PI3-kinase pathways downstream of Ras was involved in HGF-stimulated ovarian cancer cell invasiveness.

Adenovirus-Mediated Calponin h1 Gene Therapy Directed against Peritoneal Dissemination of Ovarian Cancer: Bifunctional Therapeutic Effects on Peritoneal Cell Layer and Cancer Cells

Purpose: Calponin h1 (CNh1), one of the family of actin-binding proteins, stabilizes the filaments of actin and modulates various cellular biological phenotypes. Recent studies revealed the close correlation between the invasive tumor spread and the reduced expression of CNh1 and α-smooth muscle actin in the surrounding stromal cells. The purpose of this study is to evaluate the efficacy of i.p. CNh1 gene therapy against peritoneal dissemination of ovarian cancer. Experimental Design: We used an adenoviral vector to induce the CNh1 gene into peritoneal cells and ovarian cancer cells as a means of enhancing or inducing the expression of α-smooth muscle actin as well as CNh1. The efficacy of gene transfer was examined by in vitro cell culture and in vivo animal experiments. Results: The formation of longer and thicker actin fibers was observed in each transfected cell line, and the localization of these fibers coincided with that of externally transducted CNh1. With respect to changes in cell behavior, the CNh1-transfected peritoneal cells acquired an ability to resist ovarian cancer-induced shrinkage in cell shape; thus, cancer cell invasion through the monolayer of peritoneal cells was inhibited. In addition, CNh1-transfected ovarian cancer cells showed suppressed anchorage-independent growth and invasiveness, the latter of which accompanied impaired cell motility. The concomitant CNh1 transfection into both peritoneal cells and ovarian cancer cells produced an additive inhibitory effect with respect to cancer cell invasion through the peritoneal cell monolayer. By in vivo experiments designed to treat nude mice that had been i.p. inoculated with ovarian cancer cells, we found that the i.p. injected CNh1 adenovirus successfully blocked cancer-induced morphologic changes in peritoneal cell surface and significantly prolonged the survival time of tumor-bearing mice. Moreover, CNh1 adenovirus could successfully enhance the therapeutic effect of an anticancer drug without increase in side effects. Conclusions: Thus, CNh1 gene therapy against peritoneal dissemination of ovarian cancer is bifunctionally effective (i.e., through inhibitory effects on the infected peritoneal cell layers that suppress cancer invasion and through direct antitumor effects against invasion and growth properties of cancer cells).

Relevance of ER to the Development of Endometrial Hyperplasia and Adenocarcinoma.

Estrogen has an important role in both the etiology and treatment of hormone-dependent endometrial cancers, although the mechanism remains elusive. To define the role of estrogen-mediated signaling we investigated the biological significance of estrogen receptors (ER) in NIH3T3 cell transformation via the [12Val] K-Ras mutant. This mutant enhanced the steady state level and transcriptional activity of ER. In addition, overexpression of both wild type K-Ras and ER transformed NIH3T3 cells. Co-expression of the progesterone receptor (PR) with mutant K-Ras led to suppression of tumorigenicity and inhibition of ER activation. The antisense oligomers complementary to ER suppressed proliferation and transformed phenotypes of K12V cells. These observations support the importance of ER in Ras-mediated cell transformation.To address whether ER activation is also important in the development of human endometrial cancers, we investigated ER and PR expression levels in premalignant and malignant endometrial lesions. The results suggested the implication of ER abundance in endometrial hyperplasias, though modulation of PR expression by ER was retained. Gl adenocarcinoma also expressed higher levels of ER while PR modulation by ER was abrogated. These data implied the importance of ER activities in endometrial hyperplasia and Gl adenocarcinoma development.

Carcinoma of the Endometrium Surveillance Counterpoint: Japan

Endometrial carcinoma is the third most common female genital tract carcinoma in Japan and its incidence is increasing. According to the Center for Cancer Control and Information Services at the National Cancer Center, endometrial cancer accounted for 6,625 new cancer cases in Japan in 2002 [1]. The incidence of endometrial cancer recurrence is also increasing proportionately. In 2006, the number of endometrial cancer-related deaths was 1,481, over three times higher than that reported 20 years ago (Fig. 61.1). Recently, the incidence of endometrial cancer among younger patients, under 39 years of age, has also increased in Japan (from 75 patients in 1975 to 355 patients in 2002 [1]). In younger patients, hormone therapy has been attempted at several institutions in order to preserve fertility. Endometrial cancer generally has a favorable prognosis because about 80 % of cases are diagnosed in stage I. For endometrial cancer, routine surgicopathological staging, including lymph node examination, has been recommended by the Cancer Committee of the International Federation of Gynecology and Obstetrics (FIGO) since 1988. With early-stage endometrial cancer, only approximately 10–15 % of patients will experience recurrence. The primary aim of the follow-up strategy for endometrial cancer patients is thus to facilitate the early detection of recurrent lesions. It is generally assumed that detecting recurrence before symptoms have developed will permit earlier treatment and hence improve prognosis and survival rate. The goals of follow-up are to improve overall survival duration and to improve quality of life. However, a standard program for the follow-up of endometrial cancer patients has not yet been established. Generally, the risk of endometrial cancer recurrence depends on surgicopathological factors, including histological type and grade, depth of myometrial invasion, presence of lymph node metastasis, and the presence of extrauterine disease. Differences in operative technique and adjuvant therapy between Japan and the West may influence the type of recurrence.