PRMT1 Sustains De Novo Fatty Acid Synthesis by Methylating PHGDH to Drive Chemoresistance in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) chemoresistance hampers the ability to effectively treat patients. Identification of mechanisms driving chemoresistance can lead to strategies to improve treatment. Here, we revealed that protein arginine methyltransferase-1 (PRMT1) simultaneously methylates D-3-phosphoglycerate dehydrogenase (PHGDH), a critical enzyme in serine synthesis, and the glycolytic enzymes PFKFB3 and PKM2 in TNBC cells. 13C metabolic flux analyses showed that PRMT1-dependent methylation of these three enzymes diverts glucose toward intermediates in the serine-synthesizing and serine/glycine cleavage pathways, thereby accelerating the production of methyl donors in TNBC cells. Mechanistically, PRMT1-dependent methylation of PHGDH at R54 or R20 activated its enzymatic activity by stabilizing 3-phosphoglycerate binding and suppressing polyubiquitination. PRMT1-mediated PHGDH methylation drove chemoresistance independently of glutathione synthesis. Rather, activation of the serine synthesis pathway supplied α-ketoglutarate and citrate to increase palmitate levels through activation of fatty acid synthase (FASN). Increased palmitate induced protein S-palmitoylation of PHGDH and FASN to further enhance fatty acid synthesis in a PRMT1-dependent manner. Loss of PRMT1 or pharmacologic inhibition of FASN or protein S-palmitoyltransferase reversed chemoresistance in TNBC. Furthermore, IHC coupled with imaging MS in clinical TNBC specimens substantiated that PRMT1-mediated methylation of PHGDH, PFKFB3, and PKM2 correlates with chemoresistance and that metabolites required for methylation and fatty acid synthesis are enriched in TNBC. Together, these results suggest that enhanced de novo fatty acid synthesis mediated by coordinated protein arginine methylation and protein S-palmitoylation is a therapeutic target for overcoming chemoresistance in TNBC. SIGNIFICANCE: PRMT1 promotes chemoresistance in TNBC by methylating metabolic enzymes PFKFB3, PKM2, and PHGDH to augment de novo fatty acid synthesis, indicating that targeting this axis is a potential treatment strategy.

Polysulfide Serves as a Hallmark of Desmoplastic Reaction to Differentially Diagnose Ductal Carcinoma In Situ and Invasive Breast Cancer by SERS Imaging.

Pathological examination of formalin-fixed paraffin-embedded (FFPE) needle-biopsied samples by certified pathologists represents the gold standard for differential diagnosis between ductal carcinoma in situ (DCIS) and invasive breast cancers (IBC), while information of marker metabolites in the samples is lost in the samples. Infrared laser-scanning large-area surface-enhanced Raman spectroscopy (SERS) equipped with gold-nanoparticle-based SERS substrate enables us to visualize metabolites in fresh-frozen needle-biopsied samples with spatial matching between SERS and HE staining images with pathological annotations. DCIS (n = 14) and IBC (n = 32) samples generated many different SERS peaks in finger-print regions of SERS spectra among pathologically annotated lesions including cancer cell nests and the surrounding stroma. The results showed that SERS peaks in IBC stroma exhibit significantly increased polysulfide that coincides with decreased hypotaurine as compared with DCIS, suggesting that alterations of these redox metabolites account for fingerprints of desmoplastic reactions to distinguish IBC from DCIS. Furthermore, the application of supervised machine learning to the stroma-specific multiple SERS signals enables us to support automated differential diagnosis with high accuracy. The results suggest that SERS-derived biochemical fingerprints derived from redox metabolites account for a hallmark of desmoplastic reaction of IBC that is absent in DCIS, and thus, they serve as a useful method for precision diagnosis in breast cancer.

Single-step Trypsin Inhibitor Assay on a Microchannel Array Device Immobilizing Enzymes and Fluorescent Substrates by Inkjet Printing.

In this paper, we report a single-step trypsin inhibitor assay on a microchannel array device immobilizing enzymes and substrates by inkjet printing. The microdevice is composed of a poly(dimethylsiloxane) (PDMS) microchannel array that immobilizes trypsin and fluorescent substrates as reactive reagents at the two bottom corners of a microchannel. Inkjet printers allow simple, accurate, and position-selective immobilization of reagents as nanoliter spots. Therefore, plural reactive reagents, such as enzymes and substrates, can be separately immobilized at different positions in the same microchannel without mixing, and thus allowing for single-step operation by simply introducing a sample solution through capillary action. Furthermore, reproducible fabrication and mass production of the device could be expected. In this study, the efficiency of an acidic solution as a spotting agent for protease immobilization to prevent decrease in the fluorescence intensity was confirmed. Additionally, single-step trypsin inhibitor screening was performed using three inhibitors. Finally, we investigated the storage stability of the device and confirmed that it remained stable for at least 10 days.

Comprehensive analysis of the homeobox family genes in breast cancer demonstrates their similar roles in cancer and development.

BACKGROUND: The homeobox (HOX) family consists of 39 genes whose expressions are tightly controlled and coordinated within the family, during development. We performed a comprehensive analysis of this gene family in cancer settings. METHODS: Gene correlation analysis was performed using breast cancer data available in The Cancer Genome Atlas (TCGA) and data from the patients admitted to our hospital. We also analyzed the data of normal breast tissue (GSE20437). We next collected gene expression and prognosis data of breast cancer patients (GSE11121, GSE7390, GSE3494, and GSE2990) and performed unsupervised hierarchal clustering by the HOX gene expression pattern and compared prognosis. We additionally performed this analysis to leukemia (available in TCGA) and sarcoma (GSE20196) data. RESULTS: Gene correlation analysis showed that the proximal HOX genes exhibit strong interactions and are expressed together in breast cancer, similar to the expression observed during development. However, in normal breast tissue, less interactions were observed. Breast cancer microarray meta-data classified by the HOX gene expression pattern predicted the prognosis of luminal B breast cancer patients (p = 0.016). Leukemia (p = 0.00016) and sarcoma (p = 0.018) presented similar results. The Wnt signaling pathway, one of the major upstream signals of HOX genes in development, was activated in the poor prognostic group. Interestingly, poor prognostic cancer presented stronger correlation in the gene family compared to favorable prognostic cancer. CONCLUSION: Comprehensive analysis of the HOX family demonstrated their similar roles in cancer and development, and indicated that the strong interaction of HOX genes might be specific to malignancies, especially in the case of poor prognostic cancer.

Impact of neoadjuvant and adjuvant chemotherapy on invasive lobular carcinoma: A propensity score-matched analysis of SEER data.

Invasive lobular carcinoma (ILC) has a different treatment response from invasive ductal carcinoma (IDC). We assessed whether perioperative chemotherapy was associated with improved prognosis in patients with ILC. Retrospective data of women who underwent surgery for ILC were extracted from the SEER database. Subjects were divided into non-chemotherapy and chemotherapy groups. Overall, 10 537 patients were included, and 2107 patients were stratified into each group after propensity score matching. Perioperative chemotherapy significantly improved 10-year survival rates for ILC, particularly in patients with large tumor size and lymph node metastases. Perioperative chemotherapy is effective for ILC patients with proper selection.

Identification of a Modified HOXB9 mRNA in Breast Cancer.

First identified as a developmental gene, HOXB9 is also known to be involved in tumor biological processes, and its aberrant expression correlates with poor prognosis of various cancers. In this study, we isolated a homeodomain-less, novel HOXB9 variant (HOXB9v) from human breast cancer cell line-derived mRNA. We confirmed that the novel variant was produced from variationless HOXB9 genomic DNA. RT-PCR of mRNA isolated from clinical samples and reanalysis of publicly available RNA-seq data proved that the new transcript is frequently expressed in human breast cancer. Exogenous HOXB9v expression significantly enhanced the proliferation of breast cancer cells, and gene ontology analysis indicated that apoptotic signaling was suppressed in these cells. Considering that HOXB9v lacks key domains of homeobox proteins, its behavior could be completely different from that of the previously described variationless HOXB9. Because none of the previous studies on HOXB9 have considered the presence of HOXB9v, further research analyzing the two transcripts individually is warranted to re-evaluate the true role of HOXB9 in cancer.

Inkjet Printing-Based Immobilization Method for a Single-Step and Homogeneous Competitive Immunoassay in Microchannel Arrays.

In this study, we report an inkjet printing-based method for the immobilization of different reactive analytical reagents on a single microchannel for a single-step and homogeneous solution-based competitive immunoassay. The immunoassay microdevice is composed of a poly(dimethylsiloxane) microchannel that is patterned using inkjet printing by two types of reactive reagents as dissolvable spots, namely, antibody-immobilized graphene oxide and a fluorescently labeled antigen. Since nanoliter-sized droplets of the reagents could be accurately and position-selectively spotted on the microchannel, different reactive reagents were simultaneously immobilized onto the same microchannel, which was difficult to achieve in previously reported capillary-based single-step bioassay devices. In the present study, the positions of the reagent spots and amount of reagent matrix were investigated to demonstrate the stable and reproducible immobilization and a uniform dissolution. Finally, a preliminary application to a single-step immunoassay of C-reactive protein was demonstrated as a proof of concept.

Optimal use of anthracycline-free perioperative chemotherapy in HER2-positive breast cancer patients.

PURPOSE: In adjuvant settings of human epidermal growth factor receptor 2 (HER2)-positive breast cancer, anthracycline-based chemotherapy followed by taxane and trastuzumab is a standard regimen. Recent studies have reported the use of anthracycline-free adjuvant chemotherapy in selected HER2-positive breast cancer patients. We conducted a single-center retrospective study to identify the characteristics of HER2-positive breast cancer patients for whom anthracyclines can be safely omitted. METHODS: A total of 238 women were diagnosed with HER2-positive breast cancer and treated with neoadjuvant and/or adjuvant chemotherapy between January 1, 2008 and December 31, 2015 at Keio University Hospital. They were divided in two cohorts: an "anthracycline" cohort of 112 anthracycline-treated women and a "no anthracycline" cohort of 126 anthracycline-untreated women. Survival outcomes were estimated by Kaplan-Meier method. RESULTS: The 3-year disease-free survival rates in the no-anthracycline and anthracycline cohorts were 91.3% and 93.1%, respectively (P = 0.692). After using a statistical method with inverse probability of treatment weighting to minimize the selection bias, no significant differences were observed between the two cohorts (adjusted hazard ratio for disease-free survival: 1.042; P = 0.909). Stratified by tumor size, no significant differences were observed between the two cohorts in the cT1N0 and cT2N0 subsets (P = 0.516 and P = 0.579, respectively). The recurrence rate was low among patients who achieved pathological complete response after receiving neoadjuvant chemotherapy with or without anthracyclines. CONCLUSION: Our study suggests that anthracyclines can be safely omitted in selected patients with HER2-positive breast cancer, who have cT1N0 or cT2N0 and achieved pathological complete response after receiving neoadjuvant chemotherapy.

Prediction of lowest nocturnal blood glucose level based on self-monitoring of blood glucose in Japanese patients with type 2 diabetes.

AIMS: Continuous glucose monitoring (CGM) is not available for all patients with type 2 diabetes (T2D) at risk of nocturnal hypoglycemia (NH). This study was performed to predict the lowest nocturnal blood glucose (LNBG) levels. METHODS: An LNBG prediction formula was developed by multivariate analysis using the data including self-monitoring of blood glucose from a formula making (FM) group of 29 insulin-treated T2D patients with CGM. The validity of the formula was assessed by nonparametric regression analysis of actual and predicted values in a formula validation group consisting of 21 other insulin-treated patients. The clinical impact on prediction was evaluated using a Parkes error grid. RESULTS: In the FM group with a median age of 64.0, the following formula was established: Predicted LNBG (mg/dL) = 127.4-0.836 × Age (y) + 0.119 × Self-monitored fasting blood glucose (mg/dL) + 0.717 × Basal insulin dose (U/day) (standard error of calibration 17.2 mg/dL). Based on the validation results, standard error of prediction was 31.0 mg/dL. All predicted values fell within zones A (no effect on clinical action) and B (little or no effect on clinical outcome) on the grid. CONCLUSIONS: LNBG could be predicted, and may be helpful for NH prevention.

Hereditary breast cancer associated with Cowden syndrome-related PTEN mutation with Lhermitte-Duclos disease.

BACKGROUND: Cowden syndrome is characterized by multiple hamartomas in various tissues, including the skin, brain, breast, thyroid, mucous membrane, and gastrointestinal tract, and is reported to increase the risk of malignant disease. CASE PRESENTATION: We describe the case of a 52-year-old woman in whom a tumor was diagnosed in the left cerebellar hemisphere and treated by surgical resection. Phosphatase and tensin homolog (PTEN) mutation in exon 8 insertion was found in the brain tumor tissue and leukocytes. This finding supported the diagnosis of Cowden syndrome. She consequently developed endometrial cancer and underwent abdominal total hysterectomy with bilateral salpingo-oophorectomy. Four years later, hormone receptor-positive breast cancer was found in the right breast, and breast-conserving surgery with radiation therapy and sentinel lymph node biopsy was performed. CONCLUSIONS: Herein, we describe a patient who was diagnosed as having familial breast cancer associated with PTEN mutation-related Cowden syndrome. We also reviewed reports of this syndrome in the literature for disease appraisal.

Effects of the phosphodiesterase 5 inhibitor Tadalafil on bladder function in a rat model of partial bladder outlet obstruction.

AIMS: To investigate the effect of the phosphodiesterase 5 (PDE5) inhibitor Tadalafil on bladder blood flow and bladder function in a rat model of partial bladder outlet obstruction (BOO). METHODS: Female 14-15-week-old Sprague-Dawley rats were divided into three groups. BOO was surgically induced in rats by placing a rubber ring around the urethra. BOO rats were administered daily oral Tadalafil (BOO-Tadalafil) or vehicle (BOO-Vehicle), while sham-operated animals were treated with vehicle (Sham). On the 14th day after surgery, micturition behavior was recorded for 24 hr by using a metabolic cage. On the 15th day after surgery, bladder blood flow and bladder weight were measured. The expression of PDE5 mRNA in the vesical and iliac arteries of intact rats was measured by reverse transcriptase-polymerase chain reaction. RESULTS: BOO led to a significant decrease in bladder blood flow and a significant increase in bladder weight. These changes were partially suppressed by Tadalafil treatment. The number of micturitions in the BOO group was significantly increased and the average micturition volume was significantly decreased, without affecting the total micturition volume. Repeated Tadalafil treatment markedly inhibited the increase in micturition frequency and the decrease in average micturition volume. PDE5 mRNA was expressed in the vesical and iliac arteries. CONCLUSION: Tadalafil suppressed the reduction in bladder blood flow caused by BOO in rats and improved urinary function. This action of Tadalafil may contribute to its amelioration of bladder function. Neurourol. Urodynam. 35:444-449, 2016. © 2015 Wiley Periodicals, Inc.

Efficacy of oral glucocorticoid and cyclosporine in a case of rituximab-refractory type B insulin resistance syndrome.

We describe a case of type B insulin resistance syndrome associated with systemic lupus erythematosus (SLE) that was refractory to rituximab and successfully treated with a combination of oral glucocorticoids and cyclosporine. Prior to treatment, insulin resistance was severe, and application of a hyperinsulinemic euglycemic clamp was not possible despite the continuous intravenous infusion of insulin at a maximum rate of 9.0 mU/kg/min. The addition of cyclosporine to oral glucocorticoid therapy resulted in remission of insulin resistance. The combination of oral prednisolone and cyclosporine might be effective in treating type B insulin resistance syndrome, particularly in rituximab-resistant cases. However, nephrotoxicity is a particular concern for patients receiving long-term cyclosporine therapy.

Effect of a single treatment with tadalafil on blood flow in lower urinary tract tissues in rat models of bladder overdistension/emptying and abdominal aorta clamping/release.

Impaired blood flow in lower urinary tract (LUT) tissues is a pathophysiological cause of LUT symptoms. We investigated the effects of the phosphodiesterase 5 (PDE5) inhibitor tadalafil on the sustained decrease in bladder blood flow (BBF) and time-dependent changes in BBF and prostate blood flow (PBF) resulting from ischemia/reperfusion in two rat models. In a rat model of bladder overdistension/emptying (O/E), the bladder was overdistended by saline infusion and emptied after 2h. Tadalafil was administered intraduodenally immediately after emptying. In a rat model of clamping/release (C/R), the abdominal aorta was clamped for 2h after a single oral dose of tadalafil and then the clamp was released. BBF in O/E and C/R rats and PBF in C/R rats were measured by laser Doppler flow imaging. BBF decreased on overdistension and partially recovered after emptying. A progressive decrease in BBF was observed after O/E, and this was prevented by tadalafil treatment. Both BBF and PBF decreased during clamping of the abdominal aorta and partially recovered after clamp removal. Oral pretreatment with tadalafil partially or completely prevented the decreases in BBF and PBF not only after clamp removal but also during clamping. PDE5 mRNA was highly expressed in the bladder and the supporting vasculature. Tadalafil inhibited the O/E-induced decrease in BBF and the C/R-induced time-dependent decreases in BBF and PBF. PDE5 inhibition by tadalafil may improve both BBF and PBF.

Diffusion-weighted line-scan echo-planar spectroscopic imaging technique to reduce motion artifacts in metabolite diffusion imaging.

Metabolite diffusion is expected to provide more specific microstructural and functional information than water diffusion. However, highly accurate measurement techniques have still not been developed, especially for reducing motion artifacts caused by cardiac pulsation and respiration. We developed a diffusion-weighted line-scan echo-planar spectroscopic imaging (DW-LSEPSI) technique to reduce such motion artifacts in measuring diffusion-weighted images (DWI) of metabolites. Our technique uses line-scan and echo-planar techniques to reduce phase errors induced by such motion during diffusion time. The phase errors are corrected using residual water signals in water suppression for each acquisition and at each spatial pixel specified by combining the line-scan and echo-planar techniques. We apply this technique to a moving phantom and a rat brain in vivo to demonstrate the reduction of motion artifacts in DWI and apparent diffusion coefficient (ADC) maps of metabolites. DW-LSEPSI will be useful for investigating a cellular diffusion environment using metabolites as probes.

Accuracy and validity of sentinel lymph node biopsy for breast cancer using a photosensitizer: 8-year follow-up.

BACKGROUND AND OBJECTIVE: We evaluated an alternative procedure for sentinel lymph node biopsy (SLNB) for breast cancer after approval of the study by the Ethics Committee of Tokyo Medical University Hospital in 2004. We examined the efficacy and safety of SLNB using the photosensitizer talaporfin sodium (Laserphyrin®, Meiji Seika Pharma, Tokoyo, Japan), compared with current methods. STUDY DESIGN/PATIENTS AND METHODS: The study included 21 breast cancer patients (Japanese women; median age, 54 years; range, 35-75). All patients received a breast cancer operation combined with SLNB between June 2004 and May 2005. Three milliliters of talaporfin solution was locally injected into the subareolar region just before the operation. We attempted to identify a sentinel lymph node (SLN) that exhibited fluorescence and was consistent with a radioisotope (RI) localization technique. Our purpose was to verify the accuracy and validity of the talaporfin fluorescence imaging method after 8 years of application. RESULTS: There was no consistent correlation between fluorescence and pathological SLN metastasis, although all four cases of pathological SLN metastasis revealed positive fluorescence. In some cases in which we could not identify SLNs by the RI technique, we could identify SLNs using talaporfin. The method using talaporfin did not adversely affect the patients after the operation, even the chronic renal failure patient. After 8 years, all patients are alive, and none had lymph node recurrence. Side effects were not observed. CONCLUSION: SLNB using the photosensitizer talaporfin sodium in breast cancer patients is considered to be useful as complementary to other current methods. We could evaluate the accuracy and validity of this method 8 years after all of the procedures were performed. In the future, a large-scale clinical study with statistical analyses should be conducted.

Recruited brown adipose tissue as an antiobesity agent in humans.

Brown adipose tissue (BAT) burns fat to produce heat when the body is exposed to cold and plays a role in energy metabolism. Using fluorodeoxyglucose-positron emission tomography and computed tomography, we previously reported that BAT decreases with age and thereby accelerates age-related accumulation of body fat in humans. Thus, the recruitment of BAT may be effective for body fat reduction. In this study, we examined the effects of repeated stimulation by cold and capsinoids (nonpungent capsaicin analogs) in healthy human subjects with low BAT activity. Acute cold exposure at 19°C for 2 hours increased energy expenditure (EE). Cold-induced increments of EE (CIT) strongly correlated with BAT activity independently of age and fat-free mass. Daily 2-hour cold exposure at 17°C for 6 weeks resulted in a parallel increase in BAT activity and CIT and a concomitant decrease in body fat mass. Changes in BAT activity and body fat mass were negatively correlated. Similarly, daily ingestion of capsinoids for 6 weeks increased CIT. These results demonstrate that human BAT can be recruited even in individuals with decreased BAT activity, thereby contributing to body fat reduction.

Bladder function in 17ß-estradiol-induced nonbacterial prostatitis model in Wistar rat.

OBJECTIVES: To investigate bladder function in a model of nonbacterial prostatitis (NBP) induced in castrated rats by 17ß-estradiol injection. METHODS: Ten-month-old male Wistar rats were divided into two groups, sham and NBP (both N = 8). NBP was induced by castration followed by daily subcutaneous injection of 17ß-estradiol for 30 days. On the 31st day after surgery, we investigated (1) voiding behavior, (2) bladder blood flow (BBF), (3) prostate and bladder weight, and proinflammatory cytokines (TNF-α and CXCL1) levels and (4) bladder contractile responses to electrical field stimulation (EFS), carbachol and KCl. RESULTS: (1) Voiding behavior (average micturition volume, total urine volume and number of micturitions) and (2) BBF were not significantly different between the sham and NBP groups. (3) NBP led to a significant decrease in prostatic weight and increase in proinflammatory cytokine levels in the prostate, but NBP did not cause a significant change in bladder weight or proinflammatory cytokine levels in the bladder. (4) Bladder contractile forces in response to EFS, carbachol and KCl were not significantly affected by NBP. CONCLUSIONS: In this rat model, NBP did not cause a significant change in the level of proinflammatory cytokines in the bladder and affect bladder function.

Wide-field fundus imaging using the Optos Optomap and a disposable eyelid speculum.

The EzSpec (a disposable eyelid speculum recently designed for intravitreal injections) is a useful device for removing eyelash artifacts and for potentially increasing the sensitivity of the Optos Optomap system, thereby improving the detection of retinal lesions.

Grains of paradise (Aframomum melegueta) extract activates brown adipose tissue and increases whole-body energy expenditure in men.

Brown adipose tissue (BAT) is responsible for cold- and diet-induced thermogenesis, and thereby contributes to the control of whole-body energy expenditure (EE) and body fat content. BAT activity can be assessed by fluoro-2-deoxyglucose (FDG)-positron emission tomography (PET) in human subjects. Grains of paradise (GP, Aframomum melegueta), a species of the ginger family, contain pungent, aromatic ketones such as 6-paradol, 6-gingerol and 6-shogaol. An alcohol extract of GP seeds and 6-paradol are known to activate BAT thermogenesis in small rodents. The present study aimed to examine the effects of the GP extract on whole-body EE and to analyse its relation to BAT activity in men. A total of nineteen healthy male volunteers aged 20-32 years underwent FDG-PET after 2 h of exposure to cold at 19°C with light clothing. A total of twelve subjects showed marked FDG uptake into the adipose tissue of the supraclavicular and paraspinal regions (BAT positive). The remaining seven showed no detectable uptake (BAT negative). Within 4 weeks after the FDG-PET examination, whole-body EE was measured at 27°C before and after oral ingestion of GP extract (40 mg) in a single-blind, randomised, placebo-controlled, crossover design. The resting EE of the BAT-positive group did not differ from that of the BAT-negative group. After GP extract ingestion, the EE of the BAT-positive group increased within 2 h to a significantly greater (P<0·01) level than that of the BAT-negative group. Placebo ingestion produced no significant change in EE. These results suggest that oral ingestion of GP extract increases whole-body EE through the activation of BAT in human subjects.

Genetic analysis of non-hydrogen sulfide-producing Salmonella enterica serovar typhimurium and S. enterica serovar infantis isolates in Japan.

Whole-genome sequencing of non-H(2)S-producing Salmonella enterica serovar Typhimurium and S. enterica serovar Infantis isolates from poultry meat revealed a nonsense mutation in the phsA thiosulfate reductase gene and carriage of a CMY-2 ß-lactamase. The lack of production of H(2)S might lead to the incorrect identification of S. enterica isolates carrying antimicrobial resistance genes.