The oncogene KRAS promotes cancer cell dissemination by stabilizing spheroid formation via the MEK pathway.

BACKGROUND: Peritoneal dissemination is a critical prognostic factor in ovarian cancer. Although stabilized spheroid formation promotes cancer cell peritoneal dissemination in ovarian cancer, the associated oncogenes are unknown. In this study, we assessed the role of the KRAS oncogene in ovarian cancer cell dissemination, focusing on the stability of cells in spheroid condition, as well as the modulation of intracellular signaling following spheroid transformation. METHODS: We used ID8, a murine ovarian cancer cell line, and ID8-KRAS, an oncogenic KRAS (G12 V)-transduced ID8 cell line in this study. Spheroid-forming (3D) culture and cell proliferation assays were performed to evaluate the growth characteristics of these cells. cDNA microarray analysis was performed to identify genes involved in KRAS-associated signal transduction in floating condition. A MEK inhibitor was used to evaluate the effect on cancer peritoneal dissemination. RESULTS: Cell viability and proliferation in monolayer (2D) cultures did not differ between ID8 and ID8-KRAS cells. However, the proportions of viable and proliferating ID8-KRAS cells in 3D culture were approximately 2-fold and 5-fold higher than that of ID8, respectively. Spheroid-formation was increased in ID8-KRAS cells. Analysis of peritoneal floating cells obtained from mice intra-peritoneally injected with cancer cells revealed that the proportion of proliferating cancer cells was approximately 2-fold higher with ID8-KRAS than with ID8 cells. Comprehensive cDNA microarray analysis revealed that pathways related to cell proliferation, and cell cycle checkpoint and regulation were upregulated specifically in ID8-KRAS cells in 3D culture, and that some genes partially regulated by the MEK-ERK pathway were upregulated only in ID8-KRAS cells in 3D culture. Furthermore, a MEK inhibitor, trametinib, suppressed spheroid formation in 3D culture of ID8-KRAS cells, although trametinib did not affect 2D-culture cell proliferation. Finally, we demonstrated that trametinib dramatically improved the prognosis for mice with ID8-KRAS tumors in an in vivo mouse model. CONCLUSIONS: Our data indicated that KRAS promoted ovarian cancer dissemination by stabilizing spheroid formation and that the MEK pathway is important for stabilized spheroid formation. Disruption of spheroid formation by a MEK inhibitor could be a therapeutic target for cancer peritoneal dissemination.

Blood Vessel Invasion Is a Strong Predictor of Postoperative Recurrence in Endometrial Cancer.

OBJECTIVES: Although lymphovascular space invasion is a prognostic factor for the recurrence of resectable endometrial cancer, the differential impacts of lymphatic vessel invasion (LVI) and blood vessel invasion (BVI) on the recurrence of endometrial cancer are poorly described. We investigated the prognostic significance of LVI and BVI on the recurrence of endometrial cancer and their association with patterns of recurrence. METHODS: We retrospectively reviewed 376 patients with stage I to III endometrial cancer who underwent surgery with curative intent at our institution between 2007 and 2015. The associations of the presence of lymphovascular space invasion or LVI and BVI with recurrence-free survival and patterns of recurrence were evaluated. RESULTS: Lymphovascular space invasion positivity was an independent prognostic factor for recurrence-free survival (hazards ratio [HR], 3.070; 95% confidence interval [CI], 1.404-6.824; P = 0.0048). However, when categorized by LVI versus BVI, the latter was a strong independent prognostic factor (HR, 2.697; CI, 1.288-5.798; P = 0.0081), whereas the former was not (HR, 1.740; CI, 0.795-3.721; P = 0.1637). Hematogenous metastasis was the most prevalent form of recurrence in endometrial cancer (24 [50%] of all 48 recurrent cases). Notably, 17 (19.5%) of 87 patients with BVI developed hematogenous metastases, compared with 7 (2.4%) of 289 without BVI (χ test, P < 0.0001). CONCLUSIONS: Blood vessel invasion rather than LVI was a strong predictor of postoperative recurrence in stage I to III endometrial cancer, probably due to its predisposition to hematogenous metastases.

Radical hysterectomy with or without para-aortic lymphadenectomy for patients with stage IB2, IIA2, and IIB cervical cancer: outcomes for a series of 308 patients.

BACKGROUND: Although many studies have already shown that lymph node metastasis is one of the major prognostic factors for cervical cancer, the therapeutic significance of para-aortic lymphadenectomy for the surgical treatment of cervical cancer remains controversial. METHODS: A total of 308 patients diagnosed with stage IB2, IIA2, or IIB cervical cancer and treated with radical hysterectomy were retrospectively investigated to assess the incidence of para-aortic lymph node metastasis and the clinicopathological factors linked to cervical cancer prognosis. RESULTS: Para-aortic lymph node metastases were pathologically confirmed in 13 of the 136 patients (9.6 %) who underwent para-aortic lymphadenectomy. The incidence of para-aortic lymph node metastasis was significantly higher in the patients who had common iliac lymph node metastases (odds ratio 31.5, p < 0.001) according to logistic regression analysis. Common iliac lymph node metastasis was related to risk of recurrence (hazard ratio 2.43, p = 0.003) and death (hazard ratio 2.62, p = 0.007) in Cox regression analysis. Kaplan-Meier analysis and Cox regression analysis showed that para-aortic lymphadenectomy did not have a positive impact on survival in 308 patients or 140 pN1 patients, but para-aortic lymphadenectomy was related to better overall survival with a marginal trend toward significance (p = 0.053) in 30 patients with common iliac lymph node metastasis. CONCLUSIONS: Indication for para-aortic lymphadenectomy in the surgical treatment of stage IB2, IIA2, or IIB cervical cancer needs to be individualized. Patients with common iliac lymph node metastasis are possible candidates, and a prospective study is needed to clarify this issue.

Positive peritoneal cytology at interval surgery is a poor prognostic factor in patients with stage T3c advanced ovarian carcinoma: A retrospective study.

AIM: The purpose of our study is to investigate clinically significant prognostic factors at the time of interval surgery (IS), comprising interval look surgery and interval debulking surgery, for T3c (International Federation of Gynecology and Obstetrics stage IIIc to IV) advanced ovarian cancer (AOC) patients during primary treatment. METHODS: We reviewed records of patients with T3c AOC who underwent IS following neoadjuvant chemotherapy or up-front primary debulking surgery with adjuvant chemotherapy at our institution between January 1996 and December 2010. For analysis of prognostic factors, cytology of peritoneal exfoliative cells at IS was added to clinicopathological variables. RESULTS: A retrospective analysis was performed on 50 cases. The median age was 61.1 years (range, 38-78), with median follow-up of 45.9 months (range, 12-122). Macroscopic tumors were completely resected in 32 cases (64%) at IS. Univariate analyses of clinicopathological factors for IS identified preoperative serum cancer antigen-125 levels (≥20 IU/mL; P = 0.0539), number of residual lesions at IS (≥20; P = 0.0554), incomplete surgery at IS (P = 0.0171) and positive peritoneal cytology at IS (P = 0.0015) as significant factors for prognosis regarding progression-free survival (PFS). Multivariate analysis identified positive peritoneal cytology (P = 0.0303) as a unique independent predictor of poor prognosis in PFS. CONCLUSION: Positive peritoneal cytology at IS appears to be a significant factor for poor prognosis in PFS, which may provide useful information for post-IS chemotherapy planning. IS in the treatment of AOC may be useful for not only complete resection, but also for identification of patients with poor prognosis.

Proposal for Classifying the Emetogenicity of Oral Anticancer Agents with a Focus on PARP Inhibitors: A Prospective, Observational, Multicenter Study (JASCC-CINV 2002).

Background: Olaparib and niraparib (poly adenosine diphosphate [ADP]-ribose polymerase [PARP] inhibitors) have significant antitumor action in patients with ovarian cancer. However, the incidence of nausea and vomiting among patients on these drugs in clinical trials is rather high. There are no guidelines on antiemetic treatment for nausea caused by oral anticancer agents. This study aimed to investigate the incidence of nausea and vomiting caused by PARP inhibitors and the actual situation of antiemetic therapy in patients with gynecologic cancer. Methods: Patients with gynecologic cancer who were scheduled to receive PARP inhibitors were enrolled. Data on PARP inhibitor-induced nausea and vomiting were collected from patient diaries for 21 days. The primary endpoint was the incidence of vomiting during the 21 days after starting olaparib and niraparib. Results: Overall, between January 2020 and March 2023, 134 patients were enrolled. Of the 129 patients who were evaluated, 28 (21.7%) received prophylactic antiemetics for 21 days, and 101 (78.3%) did not. The overall incidence of PARP inhibitor-induced vomiting was 16.3%. The incidence of vomiting in the group that did not receive antiemetic prophylaxis was 13.9%. On dividing the group that did not receive antiemetic prophylaxis into the olaparib and niraparib subgroups, the incidence of vomiting was found to be 18.6% for the olaparib group and 10.3% for the niraparib group. Conclusion: The incidence of emesis without antiemetic prophylaxis among patients on olaparib and niraparib ranged from 10% to 30%. Therefore, olaparib and niraparib can be classified in the low emetogenic risk and prophylactic antiemetic therapy at the time of treatment initiation may be unnecessary.

Laparoscopic hemostasis for abdominal brunt massive hemorrhage due to endometriosis.

In the gynecological literature, a limited number of studies have reported intraperitoneal bleeding due to abdominal blunt trauma. In this report, we describe a rare case of massive intraabdominal hemorrhage from the uterine artery triggered by a fall injury without apparent abdominal bruising in the presence of severe endometriosis and a uterine fibroid. A 28-year-old woman who fell from a railway platform was transported to an emergency hospital. Although she did not sustain abdominal bruising and initially had no abdominal symptoms, she complained of gradually worsening abdominal pain. Abdominal CT identified intraabdominal massive hematoma, and emergency exploratory laparoscopy revealed active bleeding from the right uterine artery eroded by endometriosis, which was treated with laparoscopic electrocoagulation. The cause of the intraabdominal bleeding was associated with avulsion of the endometriosis adhesion between the right perimetrium and the right uterine artery due to inertial forces of the uterus during the fall injury. A uterine fibroid discovered during laparoscopy was suspected to strengthen the inertial forces of the uterus. In the case of hemoperitoneum after trauma, gynecological sources of bleeding must be kept in mind, especially for patients with a known history of fibroids or endometriosis.

The current status of robotic surgery for endometrial cancer in Japan.

The da Vinci® surgical system (Intuitive Surgical Inc., Sunnyvale, CA, USA) was approved in 2009 by the Japanese Ministry of Health, Labor, and Welfare. In gynecology, robotic surgery for hysterectomy for benign indications and early-stage endometrial cancer has been covered by National Health Insurance since 2018. In a context where the da Vinci surgical system has prevailed in urology departments in Japan, gynecological robotic surgery has spread rapidly once it was covered by insurance. Although minimally invasive gynecologic surgery (minimally invasive surgery, or MIS) in Japan has a specific context, there are several problems with its safety, surgeon education, and cost in Japan. To maximize the many advantages of robotic surgery, its effectiveness needs to be carefully evaluated and this new technology needs to be safely incorporated in practice.

CD1d, a sentinel molecule bridging innate and adaptive immunity, is downregulated by the human papillomavirus (HPV) E5 protein: a possible mechanism for immune evasion by HPV.

CD1d and CD1d-restricted natural killer T (NKT) cells serve as a natural bridge between innate and adaptive immune responses to microbes. CD1d downregulation is utilized by a variety of microbes to evade immune detection. We demonstrate here that CD1d is downregulated in human papillomavirus (HPV)-positive cells in vivo and in vitro. CD1d immunoreactivity was strong in HPV-negative normal cervical epithelium but absent in HPV16-positive CIN1 and HPV6-positive condyloma lesions. We used two cell lines for in vitro assay; one was stably CD1d-transfected cells established from an HPV-negative cervical cancer cell line, C33A (C33A/CD1d), and the other was normal human vaginal keratinocyte bearing endogenous CD1d (Vag). Flow cytometry revealed that cell surface CD1d was downregulated in both C33A/CD1d and Vag cells stably transfected with HPV6 E5 and HPV16 E5. Although the steady-state levels of CD1d protein decreased in both E5-expressing cell lines compared to empty retrovirus-infected cells, CD1d mRNA levels were not affected. Confocal microscopy demonstrated that residual CD1d was not trafficked to the E5-expressing cell surface but colocalized with E5 near the endoplasmic reticulum (ER). In the ER, E5 interacted with calnexin, an ER chaperone known to mediate folding of CD1d. CD1d protein levels were rescued by the proteasome inhibitor, MG132, indicating a role for proteasome-mediated degradation in HPV-associated CD1d downregulation. Taken together, our data suggest that E5 targets CD1d to the cytosolic proteolytic pathway by inhibiting calnexin-related CD1d trafficking. Finally, CD1d-mediated production of interleukin-12 from the C33A/CD1d cells was abrogated in both E5-expressing cell lines. Decreased CD1d expression in the presence of HPV E5 may help HPV-infected cells evade protective immunological surveillance.

A Placebo-Controlled, Double-Blind Randomized (Phase IIB) Trial of Oral Administration with HPV16 E7-Expressing Lactobacillus, GLBL101c, for the Treatment of Cervical Intraepithelial Neoplasia Grade 2 (CIN2).

Cervical intraepithelial neoplasia (CIN), a precursor lesion to cervical cancer, is caused by high-risk human papillomavirus (HPV); high-grade CIN lesions (CIN2-3) are precancerous and require treatment. No globally approved therapy is available for CIN2-3 treatment. This study is a placebo-controlled randomized clinical trial of GLBL101c treatment for CIN2 in 40 patients with HPV16-positive CIN2 who were 1:1 randomized to receive GLBL101c (1 g/daily) or placebo for 5 days at 1, 2, 4, and 8 weeks. No differences were noted between the GLBL101c and placebo groups for patient background and adverse events. Moreover, no statistically significant difference was noted between the two groups at the primary endpoint, pathological regression after 16 weeks of the first oral dose; however, only in the GLBL101c group, two patients had complete regression (CR; regression to normal within 16 weeks). IFNγ production was significantly correlated with the number of spots identified by the interferon gamma enzyme-linked immunospot (IFNγ-ELISPOT) assay using cervical lymphocytes (CxLs) or peripheral blood mononuclear cells. In the two cases of CR, E7-specific Th1 immune responses were observed at week 16. Therefore, we concluded as a novel Lactobacillus-based vaccine with stronger immunogenicity than GLBL101c should be developed.

Application of deep learning to the classification of images from colposcopy.

The objective of the present study was to investigate whether deep learning could be applied successfully to the classification of images from colposcopy. For this purpose, a total of 158 patients who underwent conization were enrolled, and medical records and data from the gynecological oncology database were retrospectively reviewed. Deep learning was performed with the Keras neural network and TensorFlow libraries. Using preoperative images from colposcopy as the input data and deep learning technology, the patients were classified into three groups [severe dysplasia, carcinoma in situ (CIS) and invasive cancer (IC)]. A total of 485 images were obtained for the analysis, of which 142 images were of severe dysplasia (2.9 images/patient), 257 were of CIS (3.3 images/patient), and 86 were of IC (4.1 images/patient). Of these, 233 images were captured with a green filter, and the remaining 252 were captured without a green filter. Following the application of L2 regularization, L1 regularization, dropout and data augmentation, the accuracy of the validation dataset was ~50%. Although the present study is preliminary, the results indicated that deep learning may be applied to classify colposcopy images.

Detachment from the primary site and suspension in ascites as the initial step in metabolic reprogramming and metastasis to the omentum in ovarian cancer.

Cancer cell metabolism is currently considered to be context dependent, and metabolic reprogramming is being widely investigated. It is known that ovarian cancer often metastasizes to the omentum. Given that the omentum itself contains a high concentration of adipocytes, ovarian cancer is thought to be a good model for research into metabolic reprogramming (particularly the shift to lipid metabolism). The present study investigated the switch to lipid metabolism in the metabolic reprogramming of ovarian cancer cells. The present study first considered the possibility of epigenetic involvement. Using an open database (GSE 85293 and GSE2109), the methylation status and gene expression patterns of the primary tumor site (ovary) and the metastatic tumor site (omentum) were compared. However, no evidence was obtained regarding the involvement of epigenetics (at least in terms of DNA methylation). The influence of suspension in ascites on metabolism was then considered, and a suspension culture was used as an in vitro model. It was demonstrated that ovarian cancer cells that are detached from the primary site and suspended in ascites have enhanced lipid metabolism. Additionally, it was demonstrated that these cells express high levels of the cancer stem cell (CSC) marker cluster of differentiation 44 and c-kit in a balanced manner as they approach the omentum. Accordingly, these cells activate the mammalian target of rapamycin pathway, which is thought to be advantageous for cancer cell metastasis. In conclusion, the present study proposed one explanation for why ovarian cancer cells are likely to disseminate to the peritoneal cavity, and in particular to the omentum.

Therapeutic significance of targeting survivin in cervical cancer and possibility of combination therapy with TRAIL.

Loss of p53 function due to human papillomavirus (HPV) infection induces resistance to apoptosis in cervical cancer cells. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), which induces apoptosis in a p53-independent manner, may provide an alternative strategy for treating cervical cancer. Survivin, an antiapoptotic protein that is highly expressed in cancer cells, regulates apoptosis and the cell cycle. Here, we investigated the therapeutic potential of targeting survivin, while focusing on the TRAIL-induced apoptosis pathway. The viability and cell cycle of HPV16-positive CaSki and SiHa cells were assessed after survivin knockdown by small interfering RNA (si-survivin). E-cadherin expression was also assessed after si-survivin treatment, using western blotting. SiHa (a TRAIL-resistant cell line) was used for further studies. The small molecule YM155 and resveratrol (RVT; a polyphenol with the potential to suppress survivin expression) were used as survivin inhibitors. The effects of si-survivin and survivin inhibitors on TRAIL- or cisplatin (CDDP)-induced apoptosis were analyzed by annexin-V staining. si-survivin treatment decreased cell viability and led to G2/M arrest, accompanied by morphological changes and E-cadherin upregulation in both CaSki and SiHa cells. si-survivin and YM155 synergistically sensitized TRAIL-resistant SiHa cells to TRAIL-induced apoptosis (p < 0.05). However, si-survivin and YM155 only slightly increased CDDP-induced apoptosis. RVT markedly enhanced TRAIL-induced apoptosis by suppressing survivin expression. Targeting of survivin expression might be an ideal strategy for cervical cancer treatment as it would decrease viable cell number and enhance apoptosis sensitivity. Further, combination therapy with TRAIL, rather than CDDP, may be compatible with the proposed survivin-targeting strategy.

Intraperitoneal neutrophils activated by KRAS-induced ovarian cancer exert antitumor effects by modulating adaptive immunity.

Increased neutrophil counts are a hallmark of a poor prognosis for cancer. We previously reported that KRAS promoted tumorigenesis and increased neutrophil counts in a mouse peritoneal cancer model. In the current study, we evaluated the role of increased neutrophils in cancer progression, as well as their influence on the intraperitoneal microenvironment. A mouse peritoneal cancer model was established using the KRAS-transduced mouse ovarian cancer cell line, ID8-KRAS. Neutrophil function was assessed by neutrophil depletion in ID8-KRAS mice. Neutrophil depletion markedly accelerated tumor formation; this was accompanied by an increase in interleukin-6 concentrations in ascites. Neutrophil depletion significantly decreased the amount of local and systemic CD8+ T cells, while increasing the amount of local CD4+ T cells, accompanied by an increased amount of monocytic myeloid-derived suppressor cells (M-MDSCs) and regulatory T cells (Tregs) (P<0.05). The roles of peritoneal neutrophils (PENs) in CD8+ T cell activation were assessed in vitro. PENs of ID8-KRAS mice had a strong potential to enhance T cell proliferation with a higher expression of the T cell costimulatory molecules OX40 ligand (OX40L) and 4-1BB ligand (4-1BBL), as compared with peripheral blood neutrophils (PBNs). These findings suggest that neutrophils recruited into the KRAS-induced tumor microenvironment (TME) have antitumor properties with the potential to modulate the numbers of M-MDSCs and Tregs and activate CD8+ T cells through T cell costimulatory molecules.

Targeting glutamine metabolism and the focal adhesion kinase additively inhibits the mammalian target of the rapamycin pathway in spheroid cancer stem-like properties of ovarian clear cell carcinoma in vitro.

Ovarian cancer is one of the leading causes of death in the world, which is linked to its resistance to chemotherapy. Strategies to overcome chemoresistance have been keenly investigated. Culturing cancer cells in suspension, which results in formation of spheroids, is a more accurate reflection of clinical cancer behavior in vitro than conventional adherent cultures. By performing RNA-seq analysis, we found that the focal adhesion pathway was essential in spheroids. The phosphorylation of focal adhesion kinase (FAK) was increased in spheroids compared to adherent cells, and inhibition of FAK in spheroids resulted in inhibition of the downstream mammalian target of the rapamycin (mTOR) pathway in ovarian clear cell carcinomas. This result also suggested that only using a FAK inhibitor might have limitations because the phosphorylation level of FAK could not be reduced to the level in adherent cells, and it appeared that some combination therapies might be necessary. We previously reported that glutamine and glutamate concentrations were higher in spheroids than adherent cells, and we investigated a synergistic effect targeting glutamine metabolism with FAK inhibition on the mTOR pathway. The combination of AOA, a pan-transaminase inhibitor, and PF 573228, a FAK inhibitor, additively inhibited the mTOR pathway in spheroids from ovarian clear cell carcinomas. Our in vitro study proposed a rationale for the positive and negative effects of using FAK inhibitors in ovarian clear cell carcinomas and suggested that targeting glutamine metabolism could overcome the limitation of FAK inhibitors by additively inhibiting the mTOR pathway.

Intracellular signaling entropy can be a biomarker for predicting the development of cervical intraepithelial neoplasia.

While the mortality rates for cervical cancer have been drastically reduced after the introduction of the Pap smear test, it still is one of the leading causes of death in women worldwide. Additionally, studies that appropriately evaluate the risk of developing cervical lesions are needed. Therefore, we investigated whether intracellular signaling entropy, which is measured with microarray data, could be useful for predicting the risks of developing cervical lesions. We used three datasets, GSE63514 (histology), GSE27678 (cytology) and GSE75132 (cytology, a prospective study). From the data in GSE63514, the entropy rate was significantly increased with disease progression (normal < cervical intraepithelial neoplasia, CIN < cancer) (Kruskal-Wallis test, p < 0.0001). From the data in GSE27678, similar results (normal < low-grade squamous intraepithelial lesions, LSILs < high-grade squamous intraepithelial lesions, HSILs ≤ cancer) were obtained (Kruskal-Wallis test, p < 0.001). From the data in GSE75132, the entropy rate tended to be higher in the HPV-persistent groups than the HPV-negative group. The group that was destined to progress to CIN 3 or higher had a tendency to have a higher entropy rate than the HPV16-positive without progression group. In conclusion, signaling entropy was suggested to be different for different lesion statuses and could be a useful biomarker for predicting the development of cervical intraepithelial neoplasia.

Regeneration of cervical reserve cell-like cells from human induced pluripotent stem cells (iPSCs): A new approach to finding targets for cervical cancer stem cell treatment.

Cervical reserve cells are epithelial progenitor cells that are pathologically evident as the origin of cervical cancer. Thus, investigating the characteristics of cervical reserve cells could yield insight into the features of cervical cancer stem cells (CSCs). In this study, we established a method for the regeneration of cervical reserve cell-like properties from human induced pluripotent stem cells (iPSCs) and named these cells induced reserve cell-like cells (iRCs). Approximately 70% of iRCs were positive for the reserve cell markers p63, CK5 and CK8. iRCs also expressed the SC junction markers CK7, AGR2, CD63, MMP7 and GDA. While iRCs expressed neither ERα nor ERß, they expressed CA125. These data indicated that iRCs possessed characteristics of cervical epithelial progenitor cells. iRCs secreted higher levels of several inflammatory cytokines such as macrophage migration inhibitory factor (MIF), soluble intercellular adhesion molecule 1 (sICAM-1) and C-X-C motif ligand 10 (CXCL-10) compared with normal cervical epithelial cells. iRCs also expressed human leukocyte antigen-G (HLA-G), which is an important cell-surface antigen for immune tolerance and carcinogenesis. Together with the fact that cervical CSCs can originate from reserve cells, our data suggested that iRCs were potent immune modulators that might favor cervical cancer cell survival. In conclusion, by generating reserve cell-like properties from iPSCs, we provide a new approach that may yield new insight into cervical cancer stem cells and help find new oncogenic targets.

Low uptake of fluorodeoxyglucose in positron emission tomography/computed tomography in ovarian clear cell carcinoma may reflect glutaminolysis of its cancer stem cell-like properties.

The characteristics of ovarian cancers that showed low activation of glycolysis were investigated. Using medical records of patients with ovarian cancers who had undergone fluorodeoxyglucose positron emission tomography/computed tomography (FDG-PET/CT) prior to their primary surgery at the University of Tokyo Hospital between 2010 and 2015, we identified cases with a low uptake of FDG in PET/CT. We considered the maximum standardized uptake value (SUVmax) as the degree of glucose uptake. We investigated the properties which may account for the low activation of glycolysis in vitro. The expression level of alanine, serine, cysteine-preferring transporter 2 (ASCT2, a glutamine influx transporter), system L-type amino acid transporter 1 (LAT1, a glutamine efflux transporter) and glucose transporter 1 (GLUT1, a glucose influx transporter) were investigated by western blotting. The phosphorylation level of AMP-activated protein kinase (AMPK), which is one of the metabolic sensors, was also investigated. Most of the cases with a low uptake SUVmax were limited to patients with ovarian clear cell carcinoma (CCC). We obtained cancer stem cell (CSC)-like properties from CCC cell lines, and compared the expression levels of transporters between non-CSCs and CSCs. Whereas the expression level of ASCT2 was nearly unchanged between non-CSCs and CSCs, the expression levels of LAT1 and GLUT1 were decreased in CSCs compared to non-CSCs. The phosphorylation level of AMPK was reduced in CSCs compared to non-CSCs. In conclusion, we suggested that ovarian CCC showed low activation of glycolysis, and this may reflect glutaminolysis of its CSC-like properties.

Measurement of endometrial thickness by transvaginal ultrasonography to predict pathological response to medroxyprogesterone acetate in patients with grade 1 endometrioid adenocarcinoma.

The aim of the present study was to evaluate whether measuring endometrial thickness during fertility-sparing treatment with medroxyprogesterone acetate (MPA) can be a predictive marker for effectiveness in women with endometrioid adenocarcinoma, grade 1 (EmCa, G1). A total of 32 patients with stage IA EmCa, G1 underwent treatment with MPA. Patients were <40 years of age and preferred fertility-sparing treatment. MPA (600 mg/day) with low-dose aspirin was administered orally for 26 weeks. Pathological evaluation was performed by total curettage at weeks 8 and 16 and by fractional curettage at week 26. Patients underwent curative surgery in case of disease progression. Endometrial thickness was measured by transvaginal ultrasonography at weeks 8 and 16. Patients who showed non-complete response (non-CR) had thicker endometrium than that of CR patients at weeks 8 and 16. Receiver operating characteristic analysis revealed cut-off values of 8.3 and 4.7 mm endometrial thickness at weeks 8 and 16, respectively, for non-CR. Endometrial thickness >5 mm at week 16 was an independent factor for prediction of non-CR. Measurement of endometrial thickness during MPA treatment may be useful as a predictive marker for pathological response to MPA in patients with EmCa, G1.

Inhibition of endoplasmic reticulum (ER) stress sensors sensitizes cancer stem-like cells to ER stress-mediated apoptosis.

Although cancer stem cells (CSC) have been implicated in the development of resistance to anti-cancer therapy including chemotherapy, the mechanisms underlying chemo-resistance by CSC have not yet been elucidated. We herein isolated sphere-forming (cancer stem-like) cells from the cervical cancer cell line, SiHa, and examined the unfolded protein reaction (UPR) to chemotherapeutic-induced endoplasmic reticulum (ER) stress. We revealed that tunicamycin-induced ER stress-mediated apoptosis occurred in monolayer, but not sphere-forming cells. Biochemical assays demonstrated that sphere-forming cells were shifted to pro-survival signaling through the inactivation of IRE1 (XBP-1 splicing) and activation of PERK (elF2α phosphorylation) branches under tunicamycin-induced ER stress conditions. The proportion of apoptotic cells among sphere-forming cells was markedly increased by the tunicamycin+PERK inhibitor (PERKi) treatment, indicating that PERKi sensitized sphere-forming cells to tunicamycin-induced apoptosis. Cisplatin is also known to induce ER stress-mediated apoptosis. A low concentration of cisplatin failed to shift sphere-forming cells to apoptosis, although IRE1 branch, but not PERK, was activated. ER stress-mediated apoptosis occurred in sphere-forming cells by the cisplatin+IRE1α inhibitor (IRE1i) treatment. IRE1i, synergistic with cisplatin, up-regulated elF2α phosphorylation, and this was followed by the induction of CHOP in sphere-forming cells. The results of the present study demonstrated that the inhibition of ER stress sensors, combined with ER stress-inducible chemotherapy, shifted cancer stem-like cells to ER stress-mediated apoptosis.

STAT3 activity regulates sensitivity to tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis in cervical cancer cells.

In cervical cancer, p53-induced apoptosis is abrogated by human papilloma virus (HPV)-derived oncoprotein E6. Although tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) provides tumor-specific apoptosis in various cancers, including cervical cancer, the sensitivity differs depending on the cell lines. Signal transducer and activator of transcription 3 (STAT3) is a hub molecule that shifts the cellular fate to apoptosis or survival in response to cellular stresses. However, the contribution of STAT3 activity to TRAIL-induced apoptosis in cervical cancer remains unknown. We examined the TRAIL sensitivity in cervical cancer cells, using TRAIL-resistant (SiHa) and -sensitive (CaSki) cervical cancer cell lines and focused on STAT3 function involving the apoptotic pathway. STAT3 was inactivated by TRAIL stimulation in the CaSki cell line, but not in the SiHa cell line. We then inhibited STAT3 expression in the SiHa cell line using siRNA against STAT3 and suppressed STAT3 activity using a STAT3 inhibitor; both these treatments sensitized TRAIL-induced apoptosis in the SiHa cell line. Furthermore, the SiHa cells were exposed to tunicamycin (TM), an endoplasmic reticulum (ER) stress inducer that inactivates STAT3, with or without TRAIL. Accompanied by STAT3 inactivation, TM pretreatment significantly enhanced TRAIL-induced apoptosis. We therefore concluded that TRAIL-induced apoptosis was regulated by STAT3 in response to TRAIL stimulation. Our results also suggest that STAT3 inhibition increases the sensitivity of malignancies, particularly HPV-related cancer, to TRAIL-based therapy.