Eriko Katsuta

Targeting the SphK1/S1P/S1PR1 Axis That Links Obesity, Chronic Inflammation, and Breast Cancer Metastasis

Although obesity with associated inflammation is now recognized as a risk factor for breast cancer and distant metastases, the functional basis for these connections remain poorly understood. Here, we show that in breast cancer patients and in animal breast cancer models, obesity is a sufficient cause for increased expression of the bioactive sphingolipid mediator sphingosine-1-phosphate (S1P), which mediates cancer pathogenesis. A high-fat diet was sufficient to upregulate expression of sphingosine kinase 1 (SphK1), the enzyme that produces S1P, along with its receptor S1PR1 in syngeneic and spontaneous breast tumors. Targeting the SphK1/S1P/S1PR1 axis with FTY720/fingolimod attenuated key proinflammatory cytokines, macrophage infiltration, and tumor progression induced by obesity. S1P produced in the lung premetastatic niche by tumor-induced SphK1 increased macrophage recruitment into the lung and induced IL6 and signaling pathways important for lung metastatic colonization. Conversely, FTY720 suppressed IL6, macrophage infiltration, and S1P-mediated signaling pathways in the lung induced by a high-fat diet, and it dramatically reduced formation of metastatic foci. In tumor-bearing mice, FTY720 similarly reduced obesity-related inflammation, S1P signaling, and pulmonary metastasis, thereby prolonging survival. Taken together, our results establish a critical role for circulating S1P produced by tumors and the SphK1/S1P/S1PR1 axis in obesity-related inflammation, formation of lung metastatic niches, and breast cancer metastasis, with potential implications for prevention and treatment.Significance: These findings offer a preclinical proof of concept that signaling by a sphingolipid may be an effective target to prevent obesity-related breast cancer metastasis. Cancer Res; 78(7); 1713-25. ©2018 AACR.

High expression of SLCO2B1 is associated with prostate cancer recurrence after radical prostatectomy

Solute carrier organic anion (SLCO) gene families encode organic anion transport proteins, which are transporters that up-take a number of substrates including androgens. Among them, high expression of SLCO2B1 is known to associate with the resistance to androgen deprivation therapy in prostate cancer (PCa). We hypothesized that high expression of SLCO genes enhances PCa progression by promoting the influx of androgen. Here, we demonstrated the impact of the expression levels of SLCO2B1 on prognosis in localized PCa after radical prostatectomy (RP) utilizing 494 PCa cases in The Cancer Genome Atlas (TCGA). SLCO2B1 high expression group showed significantly worse Disease-free survival (DFS) after RP (p = 0.001). The expression level of SLCO2B1 was significantly higher in advanced characteristics including Gleason Score (GS ≤ 6 vs GS = 7; p = 0.047, GS = 7 vs GS ≥ 8; p = 0.002), pathological primary tumor (pT2 vs pT3/4; p < 0.001), and surgical margin status (positive vs negative; p = 0.013), respectively. There was a significant difference in DFS between these two groups only in GS ≥ 8 patients (p = 0.006). Multivariate analysis demonstrated that only SLCO2B1 expression level was an independent predictor for DFS after RP in GS ≥ 8. SLCO2B1 high expressed tumors in GS ≥ 8 not only enriched epithelial mesenchymal transition (EMT) related gene set, (p = 0.027), as well as Hedgehog (p < 0.001), IL-6/JAK/STAT3 (p < 0.001), and K-ras signaling gene sets (p < 0.001), which are known to promote EMT, but also showed higher expression of EMT related genes, including N-cadherin (p = 0.024), SNAIL (p = 0.001), SLUG (p = 0.001), ZEB-1 (p < 0.001) and Vimentin (p < 0.001). In conclusion, PCa with high expression of SLCO2B1 demonstrated worse DFS, which might be due to accelerated EMT.

Live animals for preclinical medical student surgical training

Aims The use of live animals for surgical training is a well-known, deliberated topic. However, medical students who use live animals rate the experience high not only in improving their surgical techniques, but also positively influencing their confidence levels in the operating room later in their careers. Therefore, we hypothesized that the use of live animal models is a unique and influential component of preclinical medical education. Materials and Methods Medical student performed the following surgical procedures using mice; surgical orthotopic implantation of cancer cells into fat pad and subsequently a radical mastectomy. The improvement of skill was then analyzed. Results All cancer cell inoculations were performed successfully. Improvement of surgical skills during the radical mastectomy procedure was documented in all parameters. All wounds healed without breakdown or dehiscence. The appropriate interval between interrupted sutures was ascertained after fifth wound closure. The speed of interrupted sutures was doubled by last wound closure. The time required to complete a radical mastectomy decreased by almost half. A single animal died immediately following the operation due to inappropriate anesthesia, which was attributed to the lack of understanding of the overall operative management. Conclusion Surgical training using live animals for preclinical medical students provides a unique learning experience, not only in improving surgical skills but also and arguably most importantly, to introduce the student to the complexities of the perioperative environment in a way that most closely resembles the stress and responsibility that the operating room demands.

Generation of sphingosine-1-phosphate is enhanced in biliary tract cancer patients and is associated with lymphatic metastasis

Lymphatic metastasis is known to contribute to worse prognosis of biliary tract cancer (BTC). Recently, sphingosine-1-phosphate (S1P), a bioactive lipid mediator generated by sphingosine kinase 1 (SPHK1), has been shown to play an important role in lymphangiogenesis and lymph node metastasis in several types of cancer. However, the role of the lipid mediator in BTC has never been examined. Here we found that S1P is elevated in BTC with the activation of ceramide-synthetic pathways, suggesting that BTC utilizes SPHK1 to promote lymphatic metastasis. We found that S1P, sphingosine and ceramide precursors such as monohexosyl-ceramide and sphingomyelin, but not ceramide, were significantly increased in BTC compared to normal biliary tract tissue using LC-ESI-MS/MS. Utilizing The Cancer Genome Atlas cohort, we demonstrated that S1P in BTC is generated via de novo pathway and exported via ABCC1. Further, we found that SPHK1 expression positively correlated with factors related to lymphatic metastasis in BTC. Finally, immunohistochemical examination revealed that gallbladder cancer with lymph node metastasis had significantly higher expression of phospho-SPHK1 than that without. Taken together, our data suggest that S1P generated in BTC contributes to lymphatic metastasis.

The Roles of Sphingosine Kinases in Skin Aging

Figure 1. Expression of sphingosine kinases with aging in mouse skin and correlations between expressions of SphK2, RAGE, and collagen types I and III in aged male mouse skin. (a, b) Relative mRNA expression of SphK1 and SphK2 in (a) male and (b) female mouse skin (n 1⁄4 5e6). (cef) Correlation between (c) SphK1 and RAGE, (d) SphK2 and RAGE, (e) Col1a1 and RAGE, and (f) Col3a1 and RAGE expression in aged male mouse skin (n 1⁄4 20). Values are presented as mean standard error. *P < 0.05. D GAPDH, glyceraldehyde-3-phosphate dehydrogenase; RAGE, receptor for advanced glycation end product; SphK1, sphingosine kinase 1; SphK2, sphingosine kinase 2. TO THE EDITOR Sphingosine-1-phosphate (S1P) is a bioactive metabolite of ceramide, which is the basic unit of sphingolipids (Aoki et al., 2016). S1P is involved in cellular survival, apoptosis, vasculogenesis, and immunity (Proia and Hla, 2015). Ceramide is degraded by ceramidases to sphingosine, and sphingosine is phosphorylated by sphingosine kinases 1 (SphK1) and 2 (SphK2) to S1P (Takabe and Spiegel, 2014). SphK1 is in the cytoplasm, and translocates to the plasma membrane to generate S1P, whereas SphK2 acts in the nucleus (Studer et al., 2012). S1P formed by SphK2 binds and inhibits the histone deacetylases, HDAC1 and HDAC2, and regulates inflammatory and metabolic gene expression (Hait et al., 2009). Advanced glycation end products (AGEs) affect extracellular matrix turnover, and the receptor of AGEs (RAGE) evokes chronic inflammation (Serban et al., 2016) by activating NFkB and inducing collagen I (Peng et al., 2016). Collagen I is the most abundant structural protein in the skin and is indicative of skin aging (Varani et al., 2002). It has been shown that RAGE is up-regulated in fibroblasts, dendrocytes, and keratinocytes in sun-exposed skin and that AGEs and tumor necrosis factor-a increase RAGE expression (Lohwasser et al., 2006). Thus, AGE-RAGE interaction is implicated in the process of skin aging. Although the roles of sphingolipid metabolites in the inflammatory process and cancer have been gradually elucidated (Aoki et al., 2016), few studies have focused on their role in matrix turnover or skin aging. Considering the involvement of SphK1 in

Abstract 3216: H2AX is a novel prognostic marker of breast cancer

INTRODUCTION: Phosphorylation of histone variant H2AX, termed gamma-H2AX, is well known for its role in DNA damage repair. On the other hand, clinical relevance of expression of H2AX in breast cancer remains to be elucidated. Previously we found that H2AX determines clonogenic cell survival under metabolic oxidative stress and cellular levels of Reactive Oxygen Species (ROS). In this study, we investigated the impact of H2AX expression on breast cancer patients9 survival, as well as underlying mechanisms. METHODS: Breast cancer patients in The Cancer Genome Atlas (TCGA) dataset were classified as either high or low expression of H2AX. Overall Survival (OS) and Disease-Free Survival (DFS) as well as Gene Set Enrichment were compared between these two groups. Gene expression of each cell lines was analyzed by RNA-seq. Radiation sensitivity was quantified using cells from patient-derived xenograft (PDX). Gene expression level after neoadjuvant chemo-radio therapy was compared using publically available database (GSE25113). RESULTS: H2AX expression level is higher in triple negative breast cancers (TNBC) than non-TNBC (p CONCLUSIONS: Advanced Stage breast cancers that express high levels of H2AX associated with worse OS and DFS, but showed higher sensitivity to radiation. H2AX can be a prognostic biomarker for recurrent tumor that may be sensitive to radiation therapy. Citation Format: Eriko Katsuta, Mutsuko Ouchi, Toru Ouchi, Kazuaki Takabe. H2AX is a novel prognostic marker of breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3216.

Abstract 5117: Triplatin preferably suppress lung metastasis of breast cancer, and peritoneal carcinomatosis of colon and pancreatic cancer

Background: Since metastatic spread is often directly associated with the poor outcome, an effective treatment for metastatic lesion is expected to improve overall survival. This is particularly the case in peritoneal carcinomatosis of pancreatic cancer where there is no effective treatment. Recent clinical trials demonstrated that Cisplatin is effective in certain metastatic breast cancer, however with severe side effects. Polynuclear platinum analog, Triplatin, was developed to overcome the severe toxicity. Methods: Murine cancer cell lines, 4T1-luc2 and E0771 (breast), Panc02-luc (pancreas) and CT26-luc (colon) were used. Cell proliferation was quantified by CCK8 assay. Syngeneic orthotopic implantation of 4T1-luc2 cells was used for metastatic breast cancer model. For mastectomy model, primary tumors were surgically removed 8 days after inoculation. Syngeneic intraperitoneal injection of CT26-luc cells and Panc02-luc cells were used for peritoneal carcinomatosis models of colon and pancreatic cancer respectively. Triplatin (0.3 mg/kg) or vehicle was administered intraperitoneally every four days three times and tumor burden was quantified by bioluminescence imaging. Results: Triplatin suppressed cell growth of both breast cancer and pancreatic cancer in a dose dependent manner in vitro (IC50 of 4T1-luc2, E0771 and Panc02-luc cells were 0.08, 0.51 and 0.07 μM, respectively). Triplatin did not suppress the growth of 4T1-luc2 primary breast tumor, however, ex vivo results showed that lung metastases were significantly reduced to 14% of the control (p = 0.034) in orthotopic model. This result was reproduced in post-mastectomy “adjuvant therapy” model where Triplatin treatment was began after primary implanted tumor was removed, which suppressed lung metastases down to 1.9% of control by Day21 (p = 0.038), and significantly prolonged survival (p = 0.007). This result led us to use Triplatin in CT26-luc colon cancer carcinomatosis model, where Triplatin suppressed total tumor burden to 1.9% of control by Day13 (p = 0.029) and significantly prolonged survival (p = 0.001). Finally, Triplatin reduced total tumor burden of Panc02-luc pancreatic cancer peritoneal carcinomatosis model to 35% of the control by Day 9 (p = 0.337) and the survival was significantly prolonged (p = 0.025). Conclusion: Triplatin demonstrated significant suppression lung metastatic tumor of 4T1-luc2 breast cancer, and peritoneal carcinomatosis of CT26-luc colon cancer and Panc02-luc pancreatic cancer. Newer platinum compounds with less toxicity and favorable pharmacokinetics warrant further investigation for advanced metastatic cancer. Citation Format: Eriko Katsuta, Erica J. Peterson, Samantha J. Katner, Nicholas P. Farrell, Kazuaki Takabe. Triplatin preferably suppress lung metastasis of breast cancer, and peritoneal carcinomatosis of colon and pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5117. doi:10.1158/1538-7445.AM2017-5117

Abstract P3-06-07: Combination of doxorubicin with S1P signaling modulator FTY720 significantly suppressed obesity-associated breast cancer

Background: Obesity is one of the biggest health issues in the US. It has been shown that obesity-associated breast cancer is more aggressive with poor prognosis, which is partly explained by the low-grade inflammation caused by obesity. Recently we have published that sphingosine-1-phosphate (S1P), a signaling lipid mediator, link inflammation and cancer in colitis-associated colon cancer model. We hypothesized that addition of S1P modulator that block S1P signaling thus suppress the effect of obesity-mediated inflammation should enhance anti-cancer effect of doxorubicin, which is a typical anti-cancer drug for breast cancer used as a standard of care. Methods: Female B6.cg- Lep ob (OB/OB) mice fed with high fat diet for 2 weeks prior to implantation of cancer cells were used as an obesity model, and litter mate control mice fed with normal diet were used as a control. 1 x 10 6 murine mammary adenocarcinoma E0771 cells were inoculated into #2 rt. fat pads as previously described (Katsuta et al JSR 2016). 9 days after inoculation, both OB/OB and control mice were randomized into 4 groups in each group; vehicle, Doxorubicin, FTY720 and Combination of Doxorubicin and FTY720. Doxorubicin was administrated by i.p. injection at a dose of 5 mg/kg on Day 0 and 3. FTY720 was administered everyday by gavage at a dose of 1 mg/kg during the entire course. Tumor growths were measured daily by caliper measurements. Tumor weights were measured on 21 days after cell inoculation. Results: The body weight of obesity model was significantly heavier than control mice at the time of cancer cell inoculation (44.1 g vs 19.4 g; p Conclusion: Modification of S1P signaling by FTY720 was shown to enhance the effect of doxorubicin particularly in obese mice, which implicate a novel approach to treat obesity-associated breast cancer. Citation Format: Katsuta E, Takabe K. Combination of doxorubicin with S1P signaling modulator FTY720 significantly suppressed obesity-associated breast cancer [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P3-06-07.

Abstract P4-06-12: Murine radical mastectomy model for preclinical study of adjuvant systemic therapies

Background: Current standard of care of breast cancer is removal of primary tumor followed by systemic adjuvant therapy to reduce recurrence and to prolong survival. However, vast majority of the preclinical studies that use murine models evaluate the drug response of primary tumors either in mammary pads or subcutaneous tissues. Lately it has been shown that the genetic profiles of metastatic lung tumors are significantly different from that of their primary mammary tumors, let alone subcutaneous tumors. Therefore we hypothesized that the responses of metastatic tumors rather than primary mammary tumors need to be evaluated for a systemic therapy. However there are few reports of murine mastectomy models used for preclinical study. Methods: Murine mammary adenocarcinoma 4T1-luc2 cells were inoculated into #2 right mammary fat pad under direct vision as previously described (Katsuta et al, JSR 2016). The tumor burden was quantified by bioluminescence IVIS imaging system. Novel platinum drug, Triplatin, or Vehicle was administrated every 4 days for 3 times from the day after inoculation. Amount of lung metastases were quantified ex vivo by IVIS imaging. Then we compared the growth of metastatic tumors between two methods of radical mastectomy; midline incision method and Halsted incision method, which were performed 8 days after inoculation. Triplatin or Vehicle was administered 2 days after mastectomies. Results: First we compared the two methods of chest mammary tumor removal; midline incision method and Halsted incision method. There was no significant difference in weight of resected tumors between these two techniques (p=0.751), however, the bioluminescence in midline incision model was significantly higher than Halsted incision model at the first day after operations (p=0.003). Only 1 out of 7 cases (14%) after Halsted incision method developed local recurrence, whereas all (100%) the animals that underwent midline incision method developed recurrence within 30 days after operation (p Conclusion: We have established an improved murine chest mammary tumor resection model. Effects on metastases, as opposed to primary tumor should be evaluated for the preclinical study of adjuvant systemic therapy, since they may not be the same. Citation Format: Katsuta E, Takabe K. Murine radical mastectomy model for preclinical study of adjuvant systemic therapies [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P4-06-12.

Abstract 1694: Conjugated bile acids aggravate metastatic pancreatic cancer via sphingosine-1-phosphate receptor 2

Introduction: “Painless jaundice” is a classic sign of advanced pancreatic cancer, which is often times lethal. Bile drainage for obstructive jaundice by pancreatic cancer has been known to be clinically beneficial, however, the role of bile acids on pancreatic cancer biology has not been thoroughly investigated. We published that conjugated bile acids bind to sphingosine 1-phosphate receptor 2 (S1PR2) (Hepatology 2012) and regulate lipid metabolism in hepatocytes through SphK2 (Hepatology 2015). SphK1 and K2 are the enzymes that generate sphingosine-1-phosphate (S1P), a lipid mediator, which cause cell proliferation and migration via S1P receptors 1-5. We hypothesize that bile acid from obstructive jaundice aggravate the pancreatic cancer progression via S1PR2. Method: Murine and human pancreatic cancer cell lines were used to examine growth response to CYM5520 (S1PR2 agonist), JTE-013 (S1PR2 antagonist), or FTY720 (functional antagonist of all S1P receptors except S1PR2). Obstructive jaundice model was made by total ligation of middle and left bile ducts with cholecystectomy, and Panc02-luc cells were implanted in the left lobe for liver metastasis, and intraperitoneally injected for carcinomatosis model. Result: Stimulation by TCA or CYM5520 promoted Panc02-luc and AsPC-1 cell proliferation, which dominantly express S1PR2 among S1P receptors in dose dependent manner, but that was not the case in other cells (PANC-1, BxPC-3 and Miapaca-2) that express other S1P receptors. JTE-013 inhibited Panc02-luc and AsPC-1 cell growth, regardless of additional TCA stimulation. Treatment with FTY720 had no effect on S1P mediated Panc02-luc cell proliferation. Neither TCA nor CYM5520 was able to stimulate growth of Panc-1, BxPC-3, nor Miapaca-2. Obstructive jaundice significantly increased liver metastasis and carcinomatosis tumor burden, and significantly shortened survival. The role of S1P from the host was investigated by using Panc02-luc carcinomatosis model on SphK1 or SphK2 knockout mice. Unexpectedly, carcinomatosis progressed slower with less tumor burden in both SphK1 and K2 knockout mice and significantly prolonged survival compared with their littermate control wildtype. Significantly less proliferation, but no difference in apoptosis was seen in tumors in SphK1 knockout mice. Treatment by FTY720 to carcinomatosis of wildtype mice did not suppress cancer progression nor tumor burden. Conclusion: Conjugated bile acids from obstructive jaundice aggravated metastatic pancreatic cancer via S1PR2, which is independent from host S1P. Citation Format: Hiroaki Aoki, Masayo Aoki, Eriko Katsuta, Partha Mukhopadhyay, Jing Yang, Huiping Zhou, Sarah Spiegel, Kazuaki Takabe. Conjugated bile acids aggravate metastatic pancreatic cancer via sphingosine-1-phosphate receptor 2. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1694.