Single-cell RNA-sequencing reveals distinct patterns of cell state heterogeneity in mouse models of breast cancer.

Breast cancer stem cells (BCSCs) contribute to intra-tumoral heterogeneity and therapeutic resistance. However, the binary concept of universal BCSCs co-existing with bulk tumor cells is over-simplified. Through single-cell RNA-sequencing, we found that Neu, PyMT and BRCA1-null mammary tumors each corresponded to a spectrum of minimally overlapping cell differentiation states without a universal BCSC population. Instead, our analyses revealed that these tumors contained distinct lineage-specific tumor propagating cells (TPCs) and this is reflective of the self-sustaining capabilities of lineage-specific stem/progenitor cells in the mammary epithelial hierarchy. By understanding the respective tumor hierarchies, we were able to identify CD14 as a TPC marker in the Neu tumor. Additionally, single-cell breast cancer subtype stratification revealed the co-existence of multiple breast cancer subtypes within tumors. Collectively, our findings emphasize the need to account for lineage-specific TPCs and the hierarchical composition within breast tumors, as these heterogenous sub-populations can have differential therapeutic susceptibilities.

FIP200 Suppresses Immune Checkpoint Therapy Responses in Breast Cancers by Limiting AZI2/TBK1/IRF Signaling Independent of Its Canonical Autophagy Function.

Immune checkpoint inhibitors (ICI) have the potential to induce durable therapeutic responses, yet response rates in breast cancer are modest and limited to particular subtypes. To expand the applicability of ICI, we examined the role of an essential autophagy gene, FIP200, which has been shown to be important for tumor progression in mammary tumors. Specific disruption of the autophagy function of FIP200 or complete ablation of FIP200 in genetic mouse models revealed that FIP200 autophagy function was required for progression of PyMT-driven mammary tumors. However, a noncanonical autophagy function of FIP200 was responsible for limiting T-cell recruitment and activation of the TBK1-IFN signaling axis. FIP200 also interacted with the TBK1 adaptor protein, AZI2, which was crucial for activation of TBK1 following FIP200 ablation. Accordingly, disrupting the noncanonical autophagy function of FIP200 in combination with ICI therapy led to superior, durable responses in immune-competent models of breast cancer. Collectively, these insights could guide future development of therapeutic agents against FIP200 for combinatorial ICI therapies in nonresponsive breast cancers. SIGNIFICANCE: These findings show that deletion of FIP200 enhances immune checkpoint inhibitor efficacy in nonresponsive breast cancer.

Use of dermal fat grafts for treating anetoderma with lipoatrophy following involution of hemangiomas.

Infantile hemangioma (IH) is a benign vascular tumor that gradually involutes over several years. Rapidly involuting congenital hemangioma (RICH) is the relatively rare congenital vascular tumor that is fully grown at birth and does not undergo postnatal growth and involutes during the first year. However, after involution of both IH and RICH, some have severe sequelae, such as redundant skin or conspicuous scarring, requiring additional treatment. We present the case of a 6-year-old girl with a concave deformity due to subcutaneous atrophy, skin darkening, and altered skin texture of her left zygomatic region following involution of a hemangioma. We successfully treated this patient by transferring a dermal fat graft. This technique can be beneficial for atrophic sequelae after regression of a hemangioma and is easy to perform and cosmetically effective.

A Case of Large Sarcoid Choroidal Granuloma Treated with Steroid Pulse Therapy.

PURPOSE: To report a case of large sarcoid choroidal granuloma that was successfully treated with steroid pulse therapy. CASE REPORT: A 38-year-old man presented with the primary complaint of decreased visual acuity (VA) in his left eye. Upon examination, a large white protruding lesion of 10 × 8 papilla diameter in size was observed in the macular region, and slightly temporal to it, in the patient's left eye. Whole-body contrast-enhanced computed tomography performed for differential diagnosis detected numerous enlarged lymph nodes throughout the body, including the bilateral hilar regions. Sarcoidosis was diagnosed by biopsy of the right cervical lymph nodes showing noncaseating epithelioid cell granuloma. The fundus lesion was found to be a choroidal granuloma caused by sarcoidosis, and steroid pulse therapy was started. The granuloma was considerably decreased, and the VA in the left eye improved to 0.7 after 2 months. CONCLUSION: Steroid pulse therapy was found to be effective as an initial treatment for a large sarcoid choroidal granuloma.

Autophagic lipid metabolism sustains mTORC1 activity in TSC-deficient neural stem cells.

Although mTORC1 negatively regulates autophagy in cultured cells, how autophagy impacts mTORC1 signaling, in particular in vivo, is less clear. Here we show that autophagy supports mTORC1 hyperactivation in NSCs lacking Tsc1, thereby promoting defects in NSC maintenance, differentiation, tumourigenesis, and the formation of the neurodevelopmental lesion of Tuberous Sclerosis Complex (TSC). Analysing mice that lack Tsc1 and the essential autophagy gene Fip200 in NSCs we find that TSC-deficient cells require autophagy to maintain mTORC1 hyperactivation under energy stress conditions, likely to provide lipids via lipophagy to serve as an alternative energy source for OXPHOS. In vivo, inhibition of lipophagy or its downstream catabolic pathway reverses defective phenotypes caused by Tsc1-null NSCs and reduces tumorigenesis in mouse models. These results reveal a cooperative function of selective autophagy in coupling energy availability with TSC pathogenesis and suggest a potential new therapeutic strategy to treat TSC patients.

[A Surgically Resected Case of Lung Metastases and Sister Mary Joseph's Nodule 24 Months after Operation for Pancreatic Cancer].

Metastatic umbilical tumors from internal malignancy, known as Sister Mary Joseph's Nodule(SMJN), are a relatively rare prognostic sign. An 86-year-old woman with pancreatic body carcinoma underwent distal pancreatectomy for D2 lymph node removal in 20XX. No peritoneal dissemination was found at that time. Postoperative chemotherapy was not administered due to her age. Eighteen months postoperatively, tumor marker values increased and chest computed tomography(CT) revealed a single mass in the left lung. We resected the suspected lung metastasis. Positron emission tomography-CT performed 23 months postoperatively for increased tumor marker values after resection showed a 18F-fluorodeoxyglucose accumulation ofapproximately 4 cm in the umbilicus. The diagnosis by biopsy was umbilical metastasis ofthe pancreatic cancer. No recurrence or other metastases were found, so we performed an umbilical tumor resection and abdominoplasty 24 months postoperatively. No peritoneal dissemination was found in her abdomen and the ascites cytology was negative. The tumor was in the subcutaneous tissue; thus, the possibility of infiltration from the primary site or peritoneal dissemination was low. The tumor marker values decreased after the resection. She was followed-up without postoperative anticancer chemotherapy. However, the tumor marker values increased again, so chemotherapy was initiated. We report a case ofresection of pancreatic cancer and operation for lung and umbilical metastases of pancreatic cancer.

A Case of Syphilitic Uveitis in Which Vitreous Surgery Was Useful for the Diagnosis and Treatment.

PURPOSE: To report a case of atypical syphilitic uveitis complicated with retinal vasculitis, proliferative retinopathy, and vitreous hemorrhage in which vitreous surgery was useful for the diagnosis and treatment. CASE REPORT: A 38-year-old female was referred to our hospital after noticing visual disturbance in her right eye. Fundoscopy examination of that eye revealed retinal phlebitis accompanied by retinal hemorrhage and soft exudate, and remarkable exudative changes in the retinal vessels from the upper arcade to the macula region. After a blood examination, a serological test showed positive for syphilis; however, systemic findings were scarce. Syphilitic uveitis was suspected, so we administered treatment for syphilis, anticoagulant treatment for retinal vasculitis, steroids for intraocular inflammation, and photocoagulation for the retinal nonperfusion area. However, her visual acuity (VA) decreased to 30 cm/counting fingers due to vitreous hemorrhage resulting from fibrovascular membrane at the optic disc. Since the vitreous hemorrhage was insufficiently absorbed, vitreous surgery was performed to remove the hemorrhage and fibrovascular tissue. Following surgery, the uveitis and retinal vasculitis subsided, and her corrected VA improved to 0.3. Postoperative examination of a fixed quantity of collected vitreous fluid for syphilis showed a Treponema pallidum hemagglutination value of 5,120 times the normal amount, thus confirming the syphilitic uveitis diagnosis. CONCLUSIONS: Our findings show that when observing patients with obstructive retinal vasculitis of unknown causes, syphilitic uveitis should be considered as a differential diagnosis, and that vitreous surgery is useful for the diagnosis and treatment of atypical syphilitic uveitis which has progressed to proliferative retinopathy.

Distinct roles of autophagy-dependent and -independent functions of FIP200 revealed by generation and analysis of a mutant knock-in mouse model.

Autophagy is an evolutionarily conserved cellular process controlled through a set of essential autophagy genes (Atgs). However, there is increasing evidence that most, if not all, Atgs also possess functions independent of their requirement in canonical autophagy, making it difficult to distinguish the contributions of autophagy-dependent or -independent functions of a particular Atg to various biological processes. To distinguish these functions for FIP200 (FAK family-interacting protein of 200 kDa), an Atg in autophagy induction, we examined FIP200 interaction with its autophagy partner, Atg13. We found that residues 582-585 (LQFL) in FIP200 are required for interaction with Atg13, and mutation of these residues to AAAA (designated the FIP200-4A mutant) abolished its canonical autophagy function in vitro. Furthermore, we created a FIP200-4A mutant knock-in mouse model and found that specifically blocking FIP200 interaction with Atg13 abolishes autophagy in vivo, providing direct support for the essential role of the ULK1/Atg13/FIP200/Atg101 complex in the process beyond previous studies relying on the complete knockout of individual components. Analysis of the new mouse model showed that nonautophagic functions of FIP200 are sufficient to fully support embryogenesis by maintaining a protective role in TNFα-induced apoptosis. However, FIP200-mediated canonical autophagy is required to support neonatal survival and tumor cell growth. These studies provide the first genetic evidence linking an Atg's autophagy and nonautophagic functions to different biological processes in vivo.

Hepatocyte nuclear factor-1ß (HNF-1ß) promotes glucose uptake and glycolytic activity in ovarian clear cell carcinoma.

Ovarian clear cell carcinoma (OCCC) is a morphologically and biologically distinct subtype of ovarian carcinomas that often arises in ovarian endometriosis. We previously reported that a unique carcinogenic environment, especially iron-induced oxidative stress in endometriotic cysts may promote development of OCCC. We also identified a gene expression profile characteristic of OCCC (the "OCCC signature"). This 320-gene OCCC signature is enriched in genes associated with stress response and sugar metabolism. However, the biological implication of this profile is unclear. In this study, we have focused on the biological role of the HNF-1ß gene within the OCCC signature, which was previously shown to be overexpressed in OCCC. Suppression of HNF-1ß in the HNF-1ß-overexpressing human ovarian cancer cell line RMG2 using short hairpin RNA resulted in a significant increase in proliferation. It also facilitated glucose uptake, glycolytic activity, and lactate secretion along with increased expression of the glucose transporter-1 (GLUT-1) gene and several key enzymes in the glycolytic process. Conversely, forced expression of HNF-1ß in the serous ovarian cancer cell line, Hey, resulted in slowed cellular growth and repressed glycolytic activity. These data suggest that HNF-1ß represses cell growth, and at the same time, it promotes aerobic glycolysis which is known as the "Warburg effect." As the Warburg effect is regarded as a characteristic metabolic process in cancer which may contribute to cell survival under hypoxic conditions or in a stressful environment, overexpression of HNF-1ß may play an inevitable role in the occurrence of OCCC in stressful environment.

Epigenetic determinants of ovarian clear cell carcinoma biology.

Targeted approaches have revealed frequent epigenetic alterations in ovarian cancer, but the scope and relation of these changes to histologic subtype of disease is unclear. Genome-wide methylation and expression data for 14 clear cell carcinoma (CCC), 32 non-CCC and four corresponding normal cell lines were generated to determine how methylation profiles differ between cells of different histological derivations of ovarian cancer. Consensus clustering showed that CCC is epigenetically distinct. Inverse relationships between expression and methylation in CCC were identified, suggesting functional regulation by methylation, and included 22 hypomethylated (UM) genes and 276 hypermethylated (HM) genes. Categorical and pathway analyses indicated that the CCC-specific UM genes were involved in response to stress and many contain hepatocyte nuclear factor (HNF) 1-binding sites, while the CCC-specific HM genes included members of the estrogen receptor alpha (ERalpha) network and genes involved in tumor development. We independently validated the methylation status of 17 of these pathway-specific genes, and confirmed increased expression of HNF1 network genes and repression of ERalpha pathway genes in CCC cell lines and primary cancer tissues relative to non-CCC specimens. Treatment of three CCC cell lines with the demethylating agent Decitabine significantly induced expression for all five genes analyzed. Coordinate changes in pathway expression were confirmed using two primary ovarian cancer datasets (p < 0.0001 for both). Our results suggest that methylation regulates specific pathways and biological functions in CCC, with hypomethylation influencing the characteristic biology of the disease while hypermethylation contributes to the carcinogenic process.

Laninamivir octanoate for post-exposure prophylaxis of influenza in household contacts: a randomized double blind placebo controlled trial.

Laninamivir octanoate, a long-acting neuraminidase inhibitor, is an effective treatment for influenza. However, its effectiveness for the prevention of influenza has not yet been demonstrated. We conducted a double-blind, multicenter, randomized, placebo-controlled trial to determine whether laninamivir octanoate was superior to a placebo for post-exposure prophylaxis of influenza in household contacts. Eligible participants, who were household members who did not have influenza and were in contact with an influenza-infected index patient, were randomly assigned (1:1:1) to one of three groups: 20 mg of laninamivir octanoate once daily for 2 days (LO-2), 20 mg of laninamivir octanoate once daily for 3 days (LO-3), or a placebo. The primary endpoint was the proportion of participants who developed clinical influenza during a 10-day period. A total of 1711 participants were enrolled, and 1451 participants were included in the primary analysis. The proportion of participants with clinical influenza was 3.9 % (19/487) in the LO-2 group, 3.7 % (18/486) in the LO-3 group, and 16.9 % (81/478) in the placebo group (P < 0.001 for each of the laninamivir octanoate group). The relative risk reductions, compared with the placebo group, were 77.0 % [95 % confidence interval (CI) 62.7-85.8] and 78.1 % (95 % CI 64.1-86.7 %) for the LO-2 and LO-3 groups, respectively. The incidences of adverse events in the laninamivir octanoate groups were similar to that in the placebo group. The inhalation of 20 mg of laninamivir octanoate once daily for 2 or 3 days was well tolerated and effectively prevented the development of influenza in household contacts.

The activated transforming growth factor-beta signaling pathway in peritoneal metastases is a potential therapeutic target in ovarian cancer.

Peritoneal dissemination including omental metastasis is the most frequent route of metastasis and an important prognostic factor in advanced ovarian cancer. We analyzed the publicly available microarray dataset (GSE2109) using binary regression and found that the transforming growth factor (TGF)-beta signaling pathway was activated in omental metastases as compared to primary sites of disease. Immunohistochemical analysis of TGF-beta receptor type 2 and phosphorylated SMAD2 indicated that both were upregulated in omental metastases as compared to primary disease sites. Treatment of the mouse ovarian cancer cell line HM-1 with recombinant TGF-ß1 promoted invasiveness, cell motility and cell attachment while these were suppressed by treatment with A-83-01, an inhibitor of the TGF-ß signaling pathway. Microarray analysis of HM-1 cells treated with TGF-ß1 and/or A-83-01 revealed that A-83-01 efficiently inhibited transcriptional changes that are induced by TGF-ß1. Using gene set enrichment analysis, we found that genes upregulated by TGF-ß1 in HM-1 cells were also significantly upregulated in omental metastases compared to primary sites in the human ovarian cancer dataset, GSE2109 (false discovery rate (FDR) q = 0.086). Therapeutic effects of A-83-01 in a mouse model of peritoneal dissemination were examined. Intraperitoneal injection of A-83-01 (150 µg given three times weekly) significantly improved survival (p = 0.015). In summary, these results show that the activated TGF-ß signaling pathway in peritoneal metastases is a potential therapeutic target in ovarian cancer.

Distinguishing primary from secondary mucinous ovarian tumors: an algorithm using the novel marker DPEP1.

Distinguishing primary mucinous ovarian cancers from ovarian metastases of digestive organ cancers is often challenging. Dipeptidase 1 was selected as the candidate novel marker of colorectal cancer based on an analysis of a gene expression microarray. Immunohistochemical analysis indicated that 13/16 ovarian metastases of colorectal cancers, but only 1/58 primary mucinous ovarian cancers, were dipeptidase 1-positive (threshold; ≧25% expression, P<0.0001). Next, five immunohistochemical markers (dipeptidase 1, estrogen receptor-α, cytokeratin 7, cytokeratin 20, and caudal type homeobox 2) were analyzed in combination. In a hierarchical clustering analysis, the mutually exclusive expression of cytokeratin 7 and dipeptidase 1 specifically identified the ovarian metastases of colorectal cancers (P<0.0001). In a decision tree analysis, cytokeratin 7, caudal type homeobox 2, and dipeptidase 1 classified primary mucinous ovarian cancers and ovarian metastases of digestive organ cancers with 90% accuracy. Finally, the five immunohistochemical markers were combined with six preoperative factors (patient's age, tumor size, laterality, serum CEA, CA19-9, and CA125) and combinations were analyzed. Of the 11 factors, 4 (dipeptidase 1, cytokeratin 7, caudal type homeobox 2, and tumor size) were used to generate a decision tree to classify primary mucinous ovarian cancers and metastases of digestive organ cancers with 93% accuracy. In conclusion, we identified a novel immunohistochemical marker, dipeptidase 1, to distinguish primary mucinous ovarian cancers from ovarian metastasis of colorectal cancers. The algorithm using immunohistochemical and clinical factors to distinguish metastases of digestive organ cancers from primary mucinous ovarian cancers will be useful to establish a protocol for the diagnosis of ovarian metastasis.

Sorafenib efficacy in ovarian clear cell carcinoma revealed by transcriptome profiling.

The purpose of this study was to investigate a new modality of therapy against ovarian clear cell carcinoma (OCCC), a chemoresistant subtype of ovarian cancer. Microarray datasets of ovarian cancer cell lines and cancer tissues were analyzed using bioinformatic tools. The gene expression profile of OCCC was similar to that of renal cell carcinoma (RCC). This similarity was at least partially due to hepatocyte nuclear factor 1 pathway activation common to both malignancies. In addition, oncogenic pathway alterations were characteristic of OCCC including hypoxia inducible factor 1 alpha subunit and relatively high Ras activities. Therefore, we predicted that the multi-kinase inhibitor sorafenib, which is approved for RCC and suppresses Ras activity, would also be effective against OCCC. Orally administered sorafenib (40 mg/kg per day) significantly inhibited tumor growth in nude mice when it was given after inoculation with the OCCC cell line RMG-2 (P = 0.002). Furthermore, sorafenib significantly reduced tumor size when it was administered to established RMG-2 tumors (P = 0.0002), while intraperitoneal injection of cisplatin (5 mg/kg per week) did not. In conclusion, the prominent anti-tumor effect of sorafenib against OCCC indicates that sorafenib is a promising candidate drug and supports the need for clinical trials using sorafenib against OCCC. This report demonstrates a method to utilize genome-wide information to facilitate translational research for treatments against less common subtypes of cancers.

Stress affects uterine receptivity through an ovarian-independent pathway.

BACKGROUND: Although stress is known to disturb natural fertility through the inhibition of the hypothalamic-pituitary-gonadal (HPG) axis, the impact of stress on infertile women who receive exogenous gonadal hormones is not well defined. This is probably due to lack of experimental models for evaluating the impacts of stress through an ovarian-independent pathway. The objective of this study was to investigate the possible impact of stress on uterine receptivity, independent of HPG axis dysfunction, using a mouse implantation model maintained with hormone supplementation. METHODS: Blastocysts from donor mice were transferred into the uterine lumen of ovariectomized (OVX) Balb/c female recipient mice following supplementation with estradiol and progesterone. The recipients were divided into two groups: those exposed (stress group) or not exposed (control group) to intermittent sonic exposure prior to embryo transfer (ET). The number of implantation sites (IS) was compared between these groups. Microarray analysis was performed to elucidate stress-induced molecular alterations in uteri during the implantation period. Sequential gene expression of leukemia inhibitory factor (Lif), an estradiol-inducible gene, was also analyzed using real-time PCR. RESULTS: A non-mating OVX model with satisfactory implantation rates was established. The number of IS in the stress group (n = 20) was significantly less than that in the control group (n = 18) (Mann-Whitney test, P = 0.0375). Implantation-related genes and ovarian-hormone-responsive genes were repressed in the stress group despite ovarian hormone supplementation. The expression of Lif was suppressed in the stress group. CONCLUSIONS: Stress can cause decreased uterine receptivity through an ovarian-independent pathway.

[Bilateral massive ovarian edema: a case report including MR findings and etiology].

A 42-year-old woman with bilateral massive ovarian edema (MOE) is presented. MOE is usually seen in women 6 to 33 years of age. Therefore, accurate re-operative assessment of MOE is important to avoid unnecessary oophorectomy procedures. MR findings of MOE are characteristic and reflect very well the diffuse stromal edema noted on microscopy. The etiology and MR findings of MOE are discussed.

Rapidly growing leiomyoma in a postmenopausal woman.

We present here a case of a rapidly growing leiomyoma occurring after menopause. The tumor weighed 4329 g, suggesting the rapid accumulation of 'hyaline fibrosis'. A small amount of proliferative activity was detected as evidenced by Ki-67 antigen immunoreactivity.

The relationship between endometrial carcinoma and coexistent adenomyosis uteri, endometriosis externa and myoma uteri.

The relationship between endometrial carcinoma and coexistent adenomyosis uteri, endometriosis externa and myoma uteri has been reported in only a few studies. We studied the characteristics of the endometrial carcinomas accompanied by these benign diseases. The total number of endometrial carcinoma cases was 179, consisting of 29 (16%) endometrial carcinomas with adenomyosis uteri, 12 (7%) with endometriosis externa, 51 (28%) with myoma uteri, and 87 controls (49%) without these benign diseases. Seventy-nine, 75, and 65% of the endometrial carcinomas with adenomyosis uteri, endometriosis externa and myoma uteri, respectively, showed a low histologic grade (G1). In particular, the patients with adenomyosis uteri and endometriosis externa were relatively younger than the control patients (54.2, 54.1 years old versus 57.7 years old). Furthermore, these patients were all treated at stage 1 and had a good prognosis. In brief, there are some clinicopathologic differences between the endometrial carcinoma cases with benign hormone-dependent disease and the cases without these disease.

Treatment of huge uterine tumors thought to be benign in post-menopausal women.

BACKGROUND: When we find a huge uterine myoma-like tumor after menopause, we are hesitant whether to recommend surgery or not. MATERIAL/METHODS: In order to treat huge uterine tumors after menopause, we surgically removed a total of 25 uterine tumors over 10 cm in diameter from 25 post-menopausal women, and examined these tumors microscopically. RESULTS: Clinical assessments of the 25 tumors before surgery indicated that 24 were myomas and one was a sarcoma, based on cytology-, sonography- and MR imaging-examinations. However, the postoperative histological results revealed 22 leiomyomas, one leiomyosarcoma, one endometrial stromal sarcoma and one endometrial carcinoma at Ic stage complicated by multiple leiomyomas. Overall, 12% of the tumors were malignant (3/25 cases) and 8.3% were false negative cases (2/24 cases; diagnosed as myoma uteri) among the cases of huge uterine tumors after CONCLUSIONS: When a huge uterine tumor after menopause is diagnosed clinically as myoma uteri, surgery should be recommended since there ia a significant chance of malignancy.