Haruka Nishida

Oral vaccination against HPV E7 for treatment of cervical intraepithelial neoplasia grade 3 (CIN3) elicits E7-specific mucosal immunity in the cervix of CIN3 patients

BACKGROUND Cervical intraepithelial neoplasia grade 3 (CIN3) is a mucosal precancerous lesion caused by high-risk human papillomavirus (HPV). Induction of immunological clearance of CIN3 by targeting HPV antigens is a promising strategy for CIN3 therapy. No successful HPV therapeutic vaccine has been developed. METHODS We evaluated the safety and clinical efficacy of an attenuated Lactobacillus casei expressing modified full-length HPV16 E7 protein in patients with HPV16-associated CIN3. Ten patients were vaccinated orally during dose optimization studies (1, 2, 4, or 6 capsules/day) at weeks 1, 2, 4, and 8 (Step 1). Seven additional participants were only tested using the optimized vaccine formulation (Step 2), giving a total of 10 patients who received optimized vaccination. Cervical lymphocytes (CxLs) and peripheral blood mononuclear cells (PBMCs) were collected and E7 specific interferon-γ-producing cells were counted (E7 cell-mediated immune responses: E7-CMI) by ELISPOT assay. All patients were re-evaluated 9 weeks after initial vaccine exposure using cytology and biopsy to assess pathological efficacy. RESULTS No patient experienced an adverse event. E7-CMI in both CxLs and PBMCs was negligible at baseline. All patients using 4-6 capsules/day showed increased E7-CMI in CxLs, whereas patients using 1-2 capsules/day did not. No patient demonstrated an increase in E7-CMI in their PBMCs. In comparison between patients of cohorts, E7-CMI at week 9 (9 wk) in patients on 4 capsules/day was significantly higher than those in patients on 1, 2, or 6 capsules/day. Most patients (70%) taking the optimized dose experienced a pathological down-grade to CIN2 at week 9 of treatment. E7-CMI in CxLs correlated directly with the pathological down-grade. CONCLUSIONS Oral administration of an E7-expressing Lactobacillus-based vaccine can elicit E7-specific mucosal immunity in the uterine cervical lesions. We are the first to report a correlation between mucosal E7-CMI in the cervix and clinical response after immunotherapy in human mucosal neoplasia.

Matrix Metalloproteinase (MMP)-9 in Cancer-Associated Fibroblasts (CAFs) Is Suppressed by Omega-3 Polyunsaturated Fatty Acids In Vitro and In Vivo

Cancer associated fibroblasts (CAFs) are responsible for tumor growth, angiogenesis, invasion, and metastasis. Matrix metalloproteinase (MMP)-9 secreted from cancer stroma populated by CAFs is a prerequisite for cancer angiogenesis and metastasis. Omega-3 polyunsaturated fatty acids (omega-3 PUFA) have been reported to have anti-tumor effects on diverse types of malignancies. Fat-1 mice, which can convert omega-6 to omega-3 PUFA independent of diet, are useful to investigate the functions of endogenous omega-3 PUFA. To examine the effect of omega-3 PUFA on tumorigenesis, TC-1 cells, a murine epithelial cell line immortalized by human papillomavirus (HPV) oncogenes, were injected subcutaneously into fat-1 or wild type mice. Tumor growth and angiogenesis of the TC-1 tumor were significantly suppressed in fat-1 compared to wild type mice. cDNA microarray of the tumors derived from fat-1 and wild type mice revealed that MMP-9 is downregulated in fat-1 mice. Immunohistochemical study demonstrated immunoreactivity for MMP-9 in the tumor stromal fibroblasts was diffusely positive in wild type whereas focal in fat-1 mice. MMP-9 was expressed in primary cultured fibroblasts isolated from fat-1 and wild type mice but was not expressed in TC-1 cells. Co-culture of fibroblasts with TC-1 cells enhanced the expression and the proteinase activity of MMP-9, although the protease activity of MMP-9 in fat-1-derived fibroblasts was lower than that in wild type fibroblasts. Our data suggests that omega-3 PUFAs suppress MMP-9 induction and tumor angiogenesis. These findings may provide insight into mechanisms by which omega-3 PUFAs exert anti-tumor effects by modulating tumor microenvironment.

Spheroid cancer stem cells display reprogrammed metabolism and obtain energy by actively running the tricarboxylic acid (TCA) cycle

The Warburg effect is a metabolic hallmark of cancer cells; cancer cells, unlike normal cells, exclusively activate glycolysis, even in the presence of enough oxygen. On the other hand, intratumoral heterogeneity is currently of interest in cancer research, including that involving cancer stem cells (CSCs). In the present study, we attempted to gain an understanding of metabolism in CSCs that is distinct from that in non-CSCs. After forming spheroids from the OVTOKO (ovarian clear cell adenocarcinoma) and SiHa (cervical squamous cell carcinoma) cell lines, the metabolites of these cells were compared with the metabolites of cancer cells that were cultured in adherent plates. A principle components analysis clearly divided their metabolic features. Amino acids that participate in tricarboxylic acid (TCA) cycle reactions, such as serine and glutamine, were significantly increased in the spheroids. Indeed, spheroids from each cell line contained more total adenylates than did their corresponding cells in adherent cultures. This study demonstrated that cancer metabolism is not limited to aerobic glycolysis (i.e. the Warburg effect), but is flexible and context-dependent. In addition, activation of TCA cycles was suggested to be a metabolic feature of CSCs that was distinct from non-CSCs. The amino acid metabolic pathways discussed here are already considered as targets for cancer therapy, and they are additionally proposed as potential targets for CSC treatment.

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.

Systemic delivery of siRNA by actively targeted polyion complex micelles for silencing the E6 and E7 human papillomavirus oncogenes

Human papillomavirus (HPV) E6 and E7 oncogenes are essential for the immortalization and maintenance of HPV-associated cancer and are ubiquitously expressed in cervical cancer lesions. Small interfering RNA (siRNA) coding for E6 and E7 oncogenes is a promising approach for precise treatment of cervical cancer, yet a delivery system is required for systemic delivery to solid tumors. Here, an actively targeted polyion complex (PIC) micelle was applied to deliver siRNAs coding for HPV E6/E7 to HPV cervical cancer cell tumors in immune-incompetent tumor-bearing mice. A cell viability assay revealed that both HPV type 16 and 18 E6/E7 siRNAs (si16E6/E7 and si18E6/E7, respectively) interfered with proliferation of cervical cancer cell lines in an HPV type-specific manner. A fluorescence imaging biodistribution analysis further revealed that fluorescence dye-labeled siRNA-loaded PIC micelles efficiently accumulated within the tumor mass after systemic administration. Ultimately, intravenous injection of si16E6/E7 and si18E6/E7-loaded PIC micelles was found to significantly suppress the growth of subcutaneous SiHa and HeLa tumors, respectively. The specific activity of siRNA treatment was confirmed by the observation that p53 protein expression was restored in the tumors excised from the mice treated with si16E6/E7- and si18E6/E7-loaded PIC micelles for SiHa and HeLa tumors, respectively. Therefore, the actively targeted PIC micelle incorporating HPV E6/E7-coding siRNAs demonstrated its therapeutic potential against HPV-associated cancer.

Expression of Par3 polarity protein correlates with poor prognosis in ovarian cancer

BackgroundPrevious studies have shown that the cell polarity protein partitioning defective 3 (Par3) plays an essential role in the formation of tight junctions and definition of apical-basal polarity. Aberrant function of this protein has been reported to be involved in epithelial–mesenchymal transition (EMT) and cancer invasion. The aim of this study was to examine the functional mechanism of Par3 in ovarian cancer.MethodsFirst, we investigated the association between Par3 expression level and survival of 50 ovarian cancer patients. Next, we conducted an in vitro analysis of ovarian cancer cell lines, focusing on the cell line JHOC5, to investigate Par3 function. To investigate the function of Par3 in invasion, the IL-6/STAT3 pathway was analyzed upon Par3 knockdown with siRNA. The effect of siRNA treatment was assessed by qPCR, ELISA, and western blotting. Invasiveness and cell proliferation following treatment with siRNA against Par3 were investigated using Matrigel chamber, wound healing, and cell proliferation assays.ResultsExpression array data for ovarian cancer patient samples revealed low Par3 expression was significantly associated with good prognosis. Univariate analysis of clinicopathological factors revealed significant association between high Par3 levels and peritoneal dissemination at the time of diagnosis. Knockdown of Par3 in JHOC5 cells suppressed cell invasiveness, migration, and cell proliferation with deregulation of IL-6/STAT3 activity.ConclusionTaken together, these results suggest that Par3 expression is likely involved in ovarian cancer progression, especially in peritoneal metastasis. The underlying mechanism may be that Par3 modulates IL-6 /STAT3 signaling. Here, we propose that the expression of Par3 in ovarian cancer may control disease outcome.

Resveratrol Protects Against Pathological Preterm Birth by Suppression of Macrophage-Mediated Inflammation.

Inflammatory cytokines play a major role in spontaneous preterm birth. Resveratrol has strong anti-inflammatory effects, but its effect on preterm birth in vivo is unknown. We investigated whether resveratrol protects against preterm birth in the lipopolysaccharide (LPS)-induced preterm mouse model. Twelve-day-old pregnant mice were fed 20 to 40 mg/kg resveratrol daily. On day 15, 10 μg of LPS was injected into uterine cervices. Resveratrol administration significantly decreased the rate of preterm birth. Resveratrol administration abolished LPS-induced elevation of tumor necrosis factor α (TNF-α) and interleukin (IL) 1β but not IL-6 levels. The TNF-α messenger RNA levels were decreased in the cervices of resveratrol-administered mice compared with controls. Resveratrol treatment suppressed the elevation in TNF-α and IL-1β levels in LPS-exposed peritoneal macrophages. Further resveratrol treatment eradicated the proinflammatory cytokine-mediated elevation in cyclooxygenase 2 (COX-2) in peritoneal macrophages. Resveratrol may protect against pathological preterm birth by suppression of elevated proinflammatory cytokines and consequent elevation of COX-2 in macrophages.

Modification of the Tumor Microenvironment in KRAS or c-MYC-Induced Ovarian Cancer-Associated Peritonitis

The most common properties of oncogenes are cell proliferation and the prevention of apoptosis in malignant cells, which, as a consequence, induce tumor formation and dissemination. However, the effects of oncogenes on the tumor microenvironment (TME) have not yet been examined in detail. The accumulation of ascites accompanied by chronic inflammation and elevated concentrations of VEGF is a hallmark of the progression of ovarian cancer. We herein demonstrated the mechanisms by which oncogenes contribute to modulating the ovarian cancer microenvironment. c-MYC and KRAS were transduced into the mouse ovarian cancer cell line ID8. ID8, ID8-c-MYC, or ID8-KRAS cells were then injected into the peritoneal cavities of C57/BL6 mice and the production of ascites was assessed. ID8-c-MYC and ID8-KRAS both markedly accelerated ovarian cancer progression in vivo, whereas no significant differences were observed in proliferative activity in vitro. ID8-KRAS in particular induced the production of ascites, which accumulated between approximately two to three weeks after the injection, more rapidly than ID8 and ID8-c-MYC (between nine and ten weeks and between six and seven weeks, respectively). VEGF concentrations in ascites significantly increased in c-MYC-induced ovarian cancer, whereas the concentrations of inflammatory cytokines in ascites were significantly high in KRAS-induced ovarian cancer and were accompanied by an increased number of neutrophils in ascites. A cytokine array revealed that KRAS markedly induced the expression of granulocyte macrophage colony-stimulating factor (GM-CSF) in ID8 cells. These results suggest that oncogenes promote cancer progression by modulating the TME in favor of cancer progression.

Clinical significance of Gremlin 1 in cervical cancer and its effects on cancer stem cell maintenance.

Gremlin 1 is one of the bone morphogenetic protein (BMP) antagonists and is also related to differentiation in combination with BMPs and is associated with various types of diseases. Gremlin 1 is overexpressed in various types of human cancers and has been reported to play a role in cervical cancer oncogenesis. However, there is no report concerning the relationship between Gremlin 1 and cervical cancer stem cells (CSCs). The objective of the present study was to identify the clinical significance of Gremlin 1 in cervical cancer and its effects on CSC-like properties in vitro. Clinical samples were obtained. Gremlin 1 mRNA expression levels in the cervical cancer tissues were measured by RT-qPCR and assessed for correlation with their clinical prognosis [overall survival (OS), progression-free survival (PFS)] and with other prognostic factors. In vitro, cervical cancer, CaSki cells, exposed to Gremlin 1 (1,000 ng/ml) for 24 h were evaluated for expression of undifferentiated-cell markers (Nanog, Oct3/4, Sox2) by RT-qPCR, the population of ALDH-positive cells by flow cytometry and sphere-forming ability on a ultra-low attachment culture dish. Cervical cancer tissues from 104 patients were collected. A high mRNA expression level of Gremlin 1 was an independent poor prognostic factor of PFS but not of OS. A high mRNA expression level of Gremlin 1 was correlated with bulky (>4 cm) tumors. The Nanog mRNA expression level was significantly increased in the CaSki cells exposed to Gremlin 1 (P=0.0008) but not Oct3/4 and Sox2 mRNA expression levels. The population of ALDH-positive cells in the Gremlin 1-exposed cells was 1.41-fold higher compared with the control (P=0.0184). Sphere-forming ability was increased when 1,000 Gremlin 1-exposed cells were seeded (P=0.0379). In cervical cancer, it is suggested that Gremlin 1 may have a role in clinical recurrence and maintaining CSC-like properties.

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.