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Supplementary Data from Hepatocyte Nuclear Factor-4–Independent Synthesis of Coagulation Factor VII in Breast Cancer Cells and Its Inhibition by Targeting Selective Histone Acetyltransferases

Voriconazole is a triazole antifungal agent used for the prevention and treatment of fungal infections in immunocompromised patients. Prolonged voriconazole therapy may induce phototoxicity and lead to the development of malignant neoplasms of the epidermis, such as squamous cell carcinoma (SCC), especially in immunocompromised patients. We report a case of voriconazole-induced phototoxicity and SCC occurring after hematopoietic stem cell transplantation (HSCT) in a 56-year-old man with primary myelofibrosis. The patient developed chronic graft-versus-host disease (GVHD) post-transplantation and had been receiving long-term immunosuppressive treatment. A year after the initiation of voriconazole therapy for prophylaxis, he developed keratotic erythema, followed by SCC with vascular invasion after three years. A review of SCC in HSCT recipients suggests that the prolonged use of voriconazole is regarded as a risk for SCC after HSCT in patients with chronic GVHD on immunosuppressive therapy. Moreover, a histological examination of the completely resected tumor revealed vascular invasion in this case, although neither the clinical features nor the histological findings of the preoperative biopsy suggested invasive carcinoma. This case may partially explain why voriconazole-associated SCCs show a more aggressive clinical course than non-voriconazole SCCs do.

Gastric contractions show two specific patterns in many species, migrating motor contractions (MMC) and postprandial contractions (PPCs), that occur in the fasted and fed states, respectively. In this study, we examined the role of somatostatin (SST) in gastric motility both in vivo and in vitro using the Asian house shrew (Suncus murinus). We performed in vivo recordings of gastric motility and in vitro organ bath experiments using S. murinus, which was recently established as a small laboratory animal for use in tests of gastrointestinal motility. SST (1.65 µg kg-1 min-1) was intravenously administered during phase II of MMC and PPCs. Next, the effect of SST on motilin-induced gastric contractions at phase I of MMC was measured. Cyclosomatostatin (CSST), an SST receptor antagonist, was administered at the peak of phase III of MMC. In addition, the effect of SST (10-11-10-9 M) on motilin-induced gastric contractions was evaluated using an organ bath experiment in vitro. In conscious, free-moving S. murinus, the administration of SST decreased the occurrence of the spontaneous phase II of MMC and PPCs. Pretreatment with SST and octreotide suppressed the induction of motilin-induced gastric contractions both in vivo and in vitro. Administration of CSST before the peak of spontaneous phase III contractions had no effect on gastric contractions. Endogenous SST is not involved in the regulation of gastric MMC and PPCs, but exogenous SST suppresses spontaneous gastric contractions. Thus, SST would be good for treating abnormal gastrointestinal motility disorders.

Pyoderma gangrenosum (PG) is a relatively rare neutrophilic dermatosis presenting as a rapidly progressive and painful skin ulcer characterized by undermined borders and peripheral erythema. Immunosuppressive therapy is the first-line treatment for PG; however, large ulcers often take months or years to heal. Surgical treatments, such as negative pressure wound therapy (NPWT) and skin grafting, are still controversial due to the risk of inducing the pathergy phenomenon and eliciting PG development by traumatic factors. Herein, we report on four cases of PG treated with skin grafting, with or without NPWT, under the control of immunosuppressive drugs at our institution. All cases adapted well, but one case showed recurrence at the periphery of the grafted area five months postoperatively. The current patients were treated with the following doses of oral prednisolone (PSL): PSL 10 mg daily, PSL 5 mg daily + adalimumab 40 mg/week, PSL 12 mg + 6 mg of tacrolimus daily, and PSL 20 mg daily during skin grafting. No severe complications, including infections, were observed. Surgical treatments, such as skin grafting with or without NPWT, may accelerate wound healing, shorten the administration of analgesics and long-term immunosuppressive therapy, and reduce the risk of infection.

Determining whether the treatment intensity needs to be increased or can be maintained at a constant level may be suggested after 2–4 weeks of treatment. The use of TNF‐α inhibitor, removal of necrotic tissue, and skin grafting may promote epithelialization.

Cholecystokinin (CCK) is a peptide hormone mainly secreted by small intestinal endocrine I-cells and functions as a regulator of gallbladder contraction, gastric emptying, gastrointestinal (GI) motility, and satiety. The cellular effects of CCK in these peripheral tissues are predominantly mediated via CCK-A receptors which are found in smooth muscles, enteric neurons, and vagal afferent neurons in humans and animal models. Although various functions of CCK have been reported to be neurally mediated, it can also stimulate contraction via the CCK receptor on the smooth muscle. However, the entire underlying neural and cellular mechanisms involved in CCK-induced GI contractions are not clearly understood. Here, we first determined the cDNA and amino acid sequences of CCK and CCK-A receptor along with the distributions of cck mRNA and CCK-producing cells in house musk shrew (Suncus murinus, the laboratory strain named as suncus) and examined the mechanism of CCK-induced contraction in the GI tract. Mature suncus CCK-8 was identical to other mammalian species tested here, and suncus CCK-A receptor presented high nucleotide and amino acid homology with that of human, dog, mouse, and rat, respectively. Suncus CCK mRNA and CCK-producing cells were found mainly in small intestine and colon. In the organ bath study, CCK-8 induced dose-dependent contractions in the suncus stomach, duodenum, and jejunum, and these contractions were inhibited by atropine and CCK-A receptor antagonist. These results suggest that CCK-8-induced contraction is mediated in the myenteric cholinergic neural network and that CCK-A receptor is partly responsible for CCK-8-induced contractions. This study indicates that suncus is a useful animal model to study the functions of CCK involved in GI motility.

Background/Aim: To investigate the role and outcomes of radiation therapy (RT) for stage IVB uterine cervical cancer (UCC) patients with lung (oligo) metastases due to the lack of recent reports on the subject. Patients and Methods: The cohort for this retrospective study comprised 23 consecutive patients with UCC (squamous cell carcinoma, n=13) and lung metastases who had received pelvic RT. Ten had lung metastases only, including 7 with oligometastases (≤4 lung metastases); the remaining 13 also had other distant metastases. Results: Nine (39.1%) of the 22 patients (95.7%) completed RT without interruption. The 1-year primary progression-free rate was 95.2%. The 1-year overall survival rate was 47.2 % (estimated median survival: 9 months). Significant prognostic factors for survival included: i) ≤4 lung metastases (p=0.035), ii) unilateral lung metastases (p=0.039), iii) primary tumor diameter

Background Tissue factor pathway inhibitor 2 (TFPI2) is a novel serum biomarker that discriminates ovarian clear cell carcinoma (CCC) from borderline ovarian tumors (BOTs) and non-clear cell epithelial ovarian cancers (EOCs). Here, we examined the performance of TFPI2 for preoperative diagnosis of CCC. Methods Serum samples were obtained preoperatively from patients with ovarian masses, who needed surgical treatment at five hospitals in Japan. The diagnostic powers of TFPI2 and cancer antigen 125 (CA125) serum levels to discriminate CCC from BOTs, other EOCs, and benign lesions were compared. Results A total of 351 patients including 69 CCCs were analyzed. Serum TFPI2 levels were significantly higher in CCC patients (mean ± SD, 508.2 ± 812.0 pg/mL) than in patients with benign lesions (154.7 ± 46.5), BOTs (181 ± 95.5) and other EOCs (265.4 ± 289.1). TFPI2 had a high diagnostic specificity for CCC (79.5%). In patients with benign ovarian endometriosis, no patient was positive for TFPI2, but 71.4% (15/21) were CA125 positive. TFPI2 showed good performance in discriminating stage II–IV CCC from BOTs and other EOCs (AUC 0.815 for TFPI2 versus 0.505 for CA125) or endometriosis (AUC 0.957 for TFPI2 versus 0.748 for CA125). The diagnostic sensitivity of TFPI2 to discriminate CCC from BOTs and other EOCs was improved from 43.5 to 71.0% when combined with CA125. Conclusions High specificity of TFPI2 for preoperative detection of CCC was verified with the defined cutoff level of TFPI2 in clinical practice. TFPI2 and CA125 may contribute substantially to precise prediction of intractable CCC.

Background/aim This study aimed to evaluate the outcome of radiation therapy for patients with distant lymph node (LN) metastases, without organ metastases from uterine cervical cancer (UCC). Patients and methods Twenty-six patients with UCC with distant LN metastases received radiotherapy and were retrospectively analyzed. The sites of distant LN metastasis were as follows; Supraclavicular in 19, inguinal in nine, axillary in four, and others in three. The mean dose prescribed for these was 50 (range=40-60) Gy. Results The 2-year overall, cause-specific, and progression-free survival, and local control of primary tumor rates were 51.3%, 51.3%, 46.9%, and 67.9%. In multivariate analysis, performance status ≥1 (p=0.007), para-aortic LN metastases (p=0.001), and lack of high-dose-rate intracavitary brachytherapy (p=0.033) were significantly associated with poor overall survival. Performance status ≥1 (p=0.004), and para-aortic LN metastases (p=0.014) were significantly associated with poor cause-specific survival. Conclusion This study demonstrated favorable local control in patients with UCC with distant LN metastases.

Aim Endocervical curettage (ECC) at the time of conization has been reported to be effective for diagnosing cervical intraepithelial neoplasia and/or early stage cervical cancer. We aimed to verify the accuracy of ECC with conization. Methods We retrospectively analyzed the records of 540 patients with suspected neoplastic cervical lesions who underwent conization at the Yokohama City University Hospital from January 2008 to December 2015. To validate the effectiveness of ECC for evaluating endocervical lesions, histopathologic findings from ECC samples were compared with those from endocervical specimens obtained by conization. In patients who subsequently underwent hysterectomy, specimens of residual endocervical stump lesions were compared with the specimens obtained by ECC. Results ECC was performed in 58.9% of patients who underwent conization. Positive findings were only observed in 7.9%, while negative findings were found in 67.3% of ECC samples; however, 24.8% of the samples were inadequate for diagnosis. None of the patients had an upgraded diagnosis according to ECC results. The sensitivity of ECC in predicting endocervical stump lesions that were identified by conization specimens was 25.0%, the specificity was 94.2% and the positive predictive value was 55.0% (κ = 0.238; P < 0.001). ECC samples yielded a sensitivity of 42.9%, a specificity of 83.9%, and positive predictive value of 54.5% (κ = 0.284; P = 0.053) in predicting residual endocervical lesions in the uterus. Conclusions As it offers low sensitivity and positive predictive value, ECC at the time of conization is of limited benefit for evaluating endocervical lesions.

Background/Aim: The aim of this study was to define the outcome of radiation therapy for vulvar carcinoma, and to investigate the effectiveness of therapeutic and prophylactic inguinal lymph node (ILN) irradiation. Because reports about the treatment of ILN were limited. Patients and Methods: Thirty consecutive vulvar carcinoma patients were treated using external beam radiation therapy (EBRT) for definitive disease (n=25) or postoperatively (n=5). Twenty-four (80%) had squamous cell carcinoma (SCC). Tumor stages (2002 UICC) ranged from 0 to IVB, with no distant metastases. Results: The median total prescribed dose for primary tumor was 64.8 Gy. The 2-year overall survival rate was 25.3%. The outcome was significantly better in patients with ILNs60 Gy (p=0.002). Conclusions: ILN diameters ≤30 mm and prescribed doses over 60 Gy were associated with ILN control in patients with vulvar carcinoma.

A 41-year-old female smoker presented with a vaginal mass. Gynecological examination showed a mass filling the uterine corpus, cervix, and vagina. A total abdominal hysterectomy was performed. Macroscopic findings included a large fragile mass involving the uterine cavity, cervix, and vagina. Histology revealed atypical ducts admixed with solid components consisting of large atypical cells. The initial pathological diagnosis was grade 3 endometrioid adenocarcinoma. The patient was designated as stage II according to the 2008 International Federation of Gynecology and Obstetrics (FIGO) staging. Two years later, two nodules were found in the upper lobe of the left lung, and the patient underwent an upper lobectomy. The masses, which exhibited solid and organoid growth patterns of large atypical cells, had histological characteristics of large cell neuroendocrine carcinoma (LCNEC) of the lung. However, the tumor was immunohistochemically positive for neuroendocrine markers, such as synaptophysin in addition to estrogen receptor and progesterone receptor, and the tumor was negative for thyroid transcription factor-1. These immunohistochemical results were almost identical to those of the solid portions of the uterine carcinoma. The final diagnosis was LCNEC combined with endometrioid adenocarcinoma of the uterine corpus and lung metastasis of the LCNEC component of the endometrial carcinoma. LCNEC often arises in the lung, but it rarely arises in other organs. Some patients with metastatic components exhibited only a LCNEC pattern although the primary tumor was a mixed carcinoma consisting of LCNEC and other histology, like the present case. LCNEC is often poorly differentiated, especially in extrapulmonary primary organ LCNEC. Therefore, pathologists should consider metastatic carcinoma when they encounter lung LCNEC in a patient with a preceding extrapulmonary carcinoma composed of a poorly differentiated component or LCNEC component, and they should clarify tumor immunohistochemical characteristics to confirm the diagnosis.

e18077 Background: Tissue factor pathway inhibitor 2 (TFPI2) is a novel serum biomarker that discriminates ovarian clear cell carcinomas (CCC) from borderline ovarian tumors (BD) and non-CCC epithelial ovarian cancers (EOC). We examined the prediction performances of TFPI2, including high specificity, for preoperative diagnosis of CCC in multi-center settings. Methods: We collected serum samples obtained preoperatively from patients with ovarian masses who needed surgical treatment to confirm pathological diagnoses in five hospitals in Japan. The diagnostic powers of serum levels of TFPI2 (cutoff value 270 pg/mL) and CA125 (cutoff value 35 U/mL) to discriminate CCC from BD, non-CCC-EOC, and benign lesions were compared. Results: A total of 351 patients (77 ovarian benign lesions, 65 BD and 209 EOC including 69 CCC) were analyzed. Serum TFPI2 levels were significantly higher in CCC patients (mean±SD, 508.2±812.0 pg/mL) than in patients with benign lesions (154.7±46.5), BD (181±95.5) and non-CCC EOC (265.4±289.1). TFPI2 had a high diagnostic specificity to CCC (sensitivity and specificity: 43.5% and 79.5%, respectively). The sensitivity of TFPI2 was improved to 71.1% when combined with CA125, and it was increased in Stage II–IV CCC (66.7%) compared with Stage I CCC (33.3%). In patients with benign endometriotic cysts (BEM), no patient was positive for TFPI2 but 71.4% (15/21) were CA125 positive. TFPI2 showed good performance in discriminating Stage II–IV CCC from BD and non-CCC EOC patients (AUC 0.815 for TFPI2 versus 0.505 for CA125) or from BEM (AUC 0.957 for TFPI2 versus 0.748 for CA125). Conclusions: High specificity of TFPI2 for the preoperative detection of CCC was re-verified with the defined cutoff level of TFPI2 in practice. TFPI2 and CA125 may have a substantial contribution to the precise prediction of intractable CCC. [Table: see text]

Background Uterine serous endometrial intraepithelial carcinoma (SEIC) is an immediate precursor of invasive carcinoma. The majority of stage IA SEICs are curable, but those with latent peritoneal metastasis and/or capillary lymphatics invasion may have poor prognoses Careful pathologic staging is thus needed to predict the risk of recurrence and to determine postoperative therapeutic strategies. Case Presentation A 71-year-old woman was hospitalized for the treatment of peritoneal carcinoma. She had undergone total hysterectomy and bilateral salpingo-oophorectomy due to SEIC (stage IA) at age 63 years, and had received medical check-ups every year since. Elevated serum CA125 (184 U/mL) was detected for the first time 8 years after surgery. A thorough workup revealed no potential primary lesion other than that in the peritoneum. Tumor reduction surgery was performed. Histologic analysis of the peritoneal lesion was high-grade serous carcinoma. The peritoneal carcinoma was diffusely immunostained for p53; thus, possible recurrence of SEIC was suspected. Tumor DNAs were microdissected from the uterine and peritoneal lesions and p53 mutation analysis was done. SEIC and peritoneal carcinomas had distinct p53 mutations that were mutually exclusive. Conclusions The present case raised a concern about the difficulty of histologic staging for SEICs. Although SEICs confined to the uterine endometrium in most cases predict a good prognosis, microscopic metastasis to the peritoneum may not be detectable at hysterectomy. If secondary malignancies of a serous phenotype develop years later, comprehensive reexamination of SEIC is mandated, with the help of DNA analysis.

Patients with dermatomyositis have a predisposition to the development of malignant tumors. The incidence of ovarian carcinoma, especially, is higher in patients with dermatomyositis than in the general population. Recently, it was reported that serum levels of KL-6 (Krebs von den Lungen-6) may be elevated in patients with dermatomyositis associated with ovarian carcinoma. Herein, we describe two cases of ovarian carcinoma associated with dermatomyositis. The first patient was a 46-year-old woman with stage Ib serous papillary adenocarcinoma of the ovary. At the time of diagnosis, she had a 13-year history of dermatomyositis, and an elevated serum KL-6 level. Reduction of the serum KL-6 level was noted after resection of the tumor. Immunohistochemistry revealed positive staining of the tumor for KL-6. The other case was a 58-year-old woman with stage IV ovarian carcinoma who also had a history of dermatomyositis. She was initially started on chemotherapy, which resulted in shrinkage of the tumor and reduction of the serum KL-6 level. Surgery was then performed for resection of the tumor, and the chemotherapy was continued. However, the patient died of cancer 1 year and 4 months after the start of the treatment. For patients with dermatomyositis and with elevation of the serum KL-6 level in the absence of severe interstitial pneumonia, associated ovarian malignancy should be ruled out.

Constitutive expression of tissue factor (TF) by cancer cells triggers local activation of the coagulation cascade and promotes breast cancer progression through cell signaling involving protease activated receptor (PAR)2. In human breast cancer, TF and PAR2 are up-regulated and TF cytoplasmic domain phosphorylation is correlated with relapse. Here we show that cancer cell PAR2 signaling promotes angiogenesis independent of PAR2 phosphorylation at the recognized beta-arrestin recruitment site. Similar to PAR2(-/-) mice, TF cytoplasmic domain-deleted (TF Delta CT) mice have delayed spontaneous breast cancer development in the polyoma middle T model. Simultaneous deletion of PAR2 in TF Delta CT mice did not further delay tumor appearance, consistent with overlapping roles of TF and PAR2 in promoting the angiogenic switch in early stages of breast cancer. In advanced carcinomas, tumor-associated macrophages were reduced in TF Delta CT and TF Delta CT/PAR2(-/-) mice, and increased tumor vessel diameters of TF Delta CT mice were partially reversed by PAR2-deficiency, indicating that the TF cytoplasmic domain has additional roles that are interdependent with PAR2 signaling in regulating host angiogenic responses. These experiments demonstrate a crosstalk of tumor cell TF cytoplasmic domain and PAR2 signaling and provide a possible mechanism for the close correlation between TF phosphorylation and cancer recurrence of TF and PAR2-positive clinical breast cancer. (Blood. 2010; 116(26):6106-6113)

Background: Tumor cell tissue factor (TF)-initiated coagulation supports hematogenous metastasis by fibrin formation, platelet activation and monocyte/macrophage recruitment. Recent studies identified host anticoagulant mechanisms as a major impediment to successful hematogenous tumor cell metastasis. Objective: Here we address mechanisms that contribute to enhanced metastasis in hyperthrombotic mice with functional thrombomodulin deficiency (TMPromice). Methods: Pharmacological and genetic approaches were combined to characterize relevant thrombin targets in a mouse model of experimental hematogenous metastasis. Results: TF-dependent, but contact pathway-independent, syngeneic breast cancer metastasis was associated with marked platelet hyperreactivity and formation of leukocyte-platelet aggregates in immune-competent TMPromice. Blockade of CD11b or genetic deletion of platelet glycoprotein Ibα excluded contributions of these receptors to enhanced platelet-dependent metastasis in hyperthrombotic mice. Mice with very low levels of the endothelial protein C receptor (EPCR) did not phenocopy the enhanced metastasis seen in TMPromice. Genetic deletion of the thrombin receptor PAR1 or endothelial thrombin signaling targets alone did not diminish enhanced metastasis in TMPromice. Combined deficiency of PAR1 on tumor cells and the host reduced metastasis in TMPromice. Conclusions: Metastasis in the hyperthrombotic TMPromouse model is mediated by platelet hyperreactivity and contributions of PAR1 signaling on tumor and host cells. © 2013 International Society on Thrombosis and Haemostasis.

Ovarian clear cell adenocarcinoma (CCC) is the second most common subtype of ovarian cancer after high-grade serous adenocarcinomas. CCC tends to develop resistance to the standard platinum-based chemotherapy, and has a poor prognosis when diagnosed in advanced stages. The ANXA4 gene, along with its product, a Ca(++)-binding annexin A4 (ANXA4) protein, has been identified as the CCC signature gene. We reported two subtypes of ANXA4 with different isoelectric points (IEPs) that are upregulated in CCC cell lines. Although several in vitro investigations have shown ANXA4 to be involved in cancer cell proliferation, chemoresistance, and migration, these studies were generally based on its overexpression in cells other than CCC. To elucidate the function of the ANXA4 in CCC cells, we established CCC cell lines whose ANXA4 expressions are stably knocked down. Two parental cells were used: OVTOKO contains almost exclusively an acidic subtype of ANXA4, and OVISE contains predominantly a basic subtype but also a detectable acidic subtype. ANXA4 knockdown (KO) resulted in significant growth retardation and greater sensitivity to carboplatin in OVTOKO cells. ANXA4-KO caused significant loss of migration and invasion capability in OVISE cells, but this effect was not seen in OVTOKO cells. We failed to find the cause of the different IEPs of ANXA4, but confirmed that the two subtypes are found in clinical CCC samples in ratios that vary by patient. Further investigation to clarify the mechanism that produces the subtypes is needed to clarify the function of ANXA4 in CCC, and might allow stratification and improved treatment strategies for patients with CCC.

Cancer progression from a dormant, non-vascularized benign tumor to metastatic disease is a multiple steps process that critically depends on contributions from the hemostatic system. Tissue factor (TF), protease activated receptors (PARs), factor VIIa, and the endothelial protein C receptor (EPCR) are expressed by tumor cells as well as the host compartment. These components of the hemostatic system regulate tumor growth, angiogenesis and metastasis. Here we review the evidence that TF-dependent signaling is the major driver of primary tumor growth, whereas TF-initiated coagulation and interactions of procoagulant tumor cells with the host compartments initiate multiple pathways that support and regulate the efficiency of metastatic tumor dissemination.

The close link between coagulation activation and clinical cancer is well established and recent progress has defined underlying molecular pathways by which tumour cells interact with the haemostatic system to promote cancer progression. Tumour type-specific oncogenic transformations cause constitutive and hypoxia-dependent upregulation of tissue factor (TF) in cancer cells, but TF expressed by vascular, stromal and inflammatory cells also contributes to the procoagulant character of the tumour microenvironment. A growing body of genetic and pharmacological evidence implicates signalling by protease activated receptors (PARs) and specifically by tumour cell-expressed TF-VIIa-PAR2 in the induction of an array of proangiogenic and immune modulating cytokines, chemokines and growth factors. Specific inhibition of this pathway results in attenuated tumour growth and angiogenesis. PARs are increasingly recognised as targets for proteases outside the coagulation system and emerging evidence indicates that alternative protease signalling pathways synergise with the coagulation system to promote tumour growth, angiogenesis and metastasis. The elucidation of new therapeutic targets in tumour-promoting protease signalling pathways requires new diagnostic approaches to identify patients that will benefit from tailored therapy targeting procoagulant or signalling aspects of the TF pathway.

Constitutive expression of tissue factor (TF) by cancer cells triggers local and systemic activation of the coagulation cascade and is a major cause of cancer-associated thrombosis. Primary breast cancer biopsies show a marked upregulation of TF and protease activated receptor (PAR) 2, as well as increased TF cytoplasmic domain phosphorylation that is correlated with cancer relapse. TF signaling involving PAR2 and integrins has multiple effects on angiogenesis and tumor progression. The non-coagulant, alternatively spliced form of TF retains an integrin-binding site and, upon deposition into the tumor stroma, stimulates angiogenesis by ligating endothelial integrins alpha(v)beta(3) and alpha(6)beta(1). On tumor cells, full-length TF is constitutively associated with laminin-binding beta(1) integrins that support TF-VIIa-PAR2 signaling leading to upregulation of pro-angiogenic and immune modulatory cytokines and growth factors. Deficiency of PAR2, but not of the thrombin receptor PAR1, delays spontaneous breast cancer development and the angiogenic switch in mice. In addition, human xenograft breast cancer growth and angiogenesis is suppressed by selective antibody inhibition of TF-VIIa-PAR2 signaling, but not by blocking TF initiated coagulation. Thus, interruption of TF signaling represents a potential anti-angiogenic strategy that does not carry an increased risk of bleeding associated with prolonged inhibition of the TF coagulation pathway.

Tissue factor/coagulation factor VII (fVII) complex formation on the surface of cancer cells plays important roles in cancer biology, such as cell migration and invasion, angiogenesis, and antiapoptotic effects. We recently found that various cancer cells ectopically synthesize fVII, resulting in activation of cell motility and invasion. Here, we characterized mechanisms of hepatic and ectopic fVII (FVII) gene expression to identify molecular targets enabling selective inhibition of the ectopic expression. Unlike hepatic expression, hepatocyte nuclear factor-4 binding to the promoter is not required for ectopic FVII expression, although Sp1 binding is essential. Furthermore, we found novel nuclear targets of basal hepatocytic and ectopic FVII expression. Notably, histone acetyltransferases p300 and cyclic AMP–responsive element binding protein–binding protein (CBP) are exclusively recruited to the promoter region of the FVII gene specifically in breast cancer cells. We further show that curcumin, a dietary compound, can selectively inhibit ectopic fVII expression by targeting p300/CBP activity. These results suggest a strategy to inhibit ectopic fVII-induced tumor progression without impairment of the physiologic hemostatic process. (Mol Cancer Res 2009;7(12):1928–36)

Background: Thromboembolic events are a major complication in ovarian cancer patients. Tissue factor (TF) is frequently overexpressed in ovarian cancer tissue and correlates with intravascular thrombosis. TF binds to coagulation factor VII (fVII), changing it to its active form, fVIIa. This leads to activation of the extrinsic coagulation cascade. fVII is produced by the liver and believed to be supplied from blood plasma at the site of coagulation. However, we recently showed that ovarian cancer cells express fVII transcripts under normoxia and that this transcription is inducible under hypoxia. These findings led us to hypothesise that ovarian cancer cells are intrinsically associated with TF-fVIIa coagulation activity, which could result in thrombosis. Methods: In this study, we examined whether ectopically expressed fVII could cause thrombosis by means of immunohistochemistry, RT–PCR, western blotting and flow cytometry. Results: Ectopic fVII expression occurs frequently in ovarian cancers, particularly in clear cell carcinoma. We further showed that ovarian cancer cells express TF-fVIIa on the cell surface under normoxia and that this procoagulant activity is enhanced by hypoxic stimuli. Moreover, we showed that ovarian cancer cells secrete microparticles (MPs) with TF-fVIIa activity. Production of this procoagulant secretion is enhanced under hypoxia. Conclusion: These results raise the possibility that cancer cell-derived TF-fVIIa could cause thrombotic events in ovarian cancer patients.

Here, we demonstrate by chromatin immunoprecipitation that the binding of hypoxia-inducible factors to gene regulatory regions is differentially influenced in cancer cells. Binding of HIF-2alpha varies depending on hypoxic conditions, although HIF-1alpha is constantly bound to these regions. We found by RNA interference experiments that HIF-2alpha plays a minor role in VEGF gene upregulation under hypoxia or CoCl(2) treatment, even when both HIFs are similarly bound to the promoter region. HIF-2alpha activated or suppressed the ENO1 gene under various conditions, irrespective of promoter binding. We additionally found that HIF dependence on EPO gene induction could be altered depending on the conditions, irrespective of the binding pattern of HIFs. These results demonstrate that, unlike HIF-1alpha, HIF-2alpha differentially binds and regulates transcription under hypoxia.

We report a rare case in which a cystic lesion in the cervix, caused by endometriosis, resulted in a massive vaginal hemorrhage. Cervical endometriosis is relatively common and is usually considered a mild condition. However, we report a case in which a severe vaginal bleed originated from an endometrial cyst of the cervix and in which diagnosis was confused by the presence of atypical cells.

Backgrounds: Ovarian carcinoma is the first leading cause of death in gynecologic malignancies. Unlike the histological majority, serous adenocarcinoma, ovarian clear cell adenocarcinoma (OCCA), the second most histology in Japan, is mostly resistant to the conventional chemotherapy. Patients with advanced OCCA have poorer prognosis than serous histology. By searching molecular characteristics for OCCA, we found and have reported annexin A4 as a protein much more expressed in OCCA than in other ovarian malignancies. Moreover, we found two types of annexin A4; the acidic and the basic forms in 2D-PAGE. The objective of this study is to elucidate the still unclear annexin A4 function in relation to the type-difference. Methods: Stable annexin A4 knock-down (KO) clones were established for OCCA cell lines, OVISE and OVTOKO by expression of shRNA targeting annexin A4 mRNA. Changes in several phenotypes in vitro, including cell growth, sensitivity for chemotherapeutics such as paclitaxel (PTX) and carboplatin (CBDCA), and migration/invasion activity were evaluated in these KO clones. Amounts of the two types of annexin A4 were estimated by 2D-PAGE followed by Western blot. In addition, OCCA clinical specimens were collected under the approval of the review board of our institute, and proteins extracted from frozen tumor tissues were subjected to the same 2D-PAGE analysis. Results: Cell growth and IC 50 for CBDCA were significantly decreased in annexin A4-KO-OVTOKO, while migration and invasion were suppressed in annexin A4-KO-OVISE. No other phenotypes were significantly altered by annexin A4-KO. The acidic annexin A4 was predominant in OVTOKO, and the basic in OVISE. As for the clinical tissues, the ratio of the two types of annexin A4 on 2D-PAGE showed diversities as patients. Implications: This is the first report to elucidate the annexin A4 function in OCCA cells by knocking down strategy. The result that annexin A4 may make cells more resistant to CBDCA is comparative to the previous report in which forced overexpression of annexin A4 in ovarian serous adenocarcinoma cells led to resistance to CBDCA. Because the phenotypic changes by annexin A4-KO found in OVTOKO and OVISE were not the same, one could speculate that the two different annexin A4 proteins have somewhat different functions at least in vitro; that is, the acidic form of annexin A4 might have a significant role in proliferation and CBDCA drug resistance, as the basic form might be related to migration / invasion ability. To elucidate the clinicopathological significance of annexin A4 in OCCA, analysis of the subtype difference will be meaningful. 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 1856. doi:1538-7445.AM2012-1856