細胞激素受體 IL-17RB 與乳腺脂肪細胞在乳癌癌化過程中所扮演之角色

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(1) . 國⽴立台灣⼤大學醫學院⽣生物化學暨分⼦子⽣生物學研究所 Graduate Institute of Biochemistry and Molecular Biology, College of Medicine, National Taiwan University. 博⼠士論⽂文 Ph.D Dissertation. 細胞激素受體 IL-17RB 與乳腺脂肪細胞在乳癌癌化過程中所扮演之⾓角⾊色 The roles of IL-17RB and mammary gland adipocytes in breast cancer tumorigenesis. 研究⽣生：⿈黃俊凱 (Chun-Kai Huang) 指導教授：李⽂文華博⼠士 (Dr. Wen-Hwa Lee) 中華民國 104 年 7 ⽉月 June 2015 .

(2) . 中⽂文摘要. 癌症發展過程中，癌細胞除了⾃自⾝身細胞內調控影響外，也受周遭細胞影響。︒｡以癌細胞本⾝身⽽而⾔言，癌細胞常常利⽤用⼤大量表現表⾯面受體⽅方式來增加⾃自⼰己⽣生⾧長與存活優勢。︒｡乳癌病⼈人中⼤大約有 20~25% 病⼈人會⼤大量表現第⼆二型⼈人類表⽪皮⽣生⾧長因⼦子受體 (HER2)，這類病⼈人可利⽤用針對此受體之單株抗體-trastuzumab 進⾏行有效治療。︒｡然⽽而⽬目前除了 HER2 外，少有其他可應⽤用於乳癌治療之專⼀一標的被證實。︒｡在本論⽂文中，我們證實細胞激素受體 IL-17RB 及其配體 IL-17B 對於乳癌癌化過程是重要的，此路徑活化亦可⼲干擾正常乳腺上⽪皮細胞乳腺發育過程，進⽽而促進乳癌發展。︒｡藉由受體 IL-17RB 及其配體 IL-17B 結合，可活化下游轉錄因⼦子 NF-κB，並進⼀一步促進抑制細胞凋亡分⼦子 Bcl-2 表現，使癌細胞對於 Etoposide 細胞毒殺耐受性增加。︒｡另⼀一⽅方⾯面，在乳癌病⼈人臨床檢體中我們也觀察到 IL-17RB 與 HER2 表現量呈現正相關，同時⼤大量表現 IL-17RB 與 HER2 的乳癌病⼈人具有較差預後。︒｡在具 trastuzumab 抗性之乳癌細胞中，抑制 IL-17RB 的表現亦可有效抑制其致癌性，顯⽰示 IL-17RB 與 HER2 在乳癌細胞中可能扮演不同⾓角⾊色。︒｡當我們以 IL-17RB 與 IL-17B 中和性抗體對乳癌細胞進⾏行處理後發現，無論是中和 IL-17RB 或 IL-17B 皆可有效抑制乳癌細胞致癌性，顯⽰示 IL-17RB 與 IL-17B 在乳癌癌化過程中扮演重要⾓角⾊色，並具有可⽤用來發展相對應治療⽅方式之潛⼒力。︒｡另⼀一⽅方⾯面，癌細胞與其微環境之交互作⽤用對於癌症產⽣生與發展過程亦扮演重要⾓角⾊色。︒｡乳腺組織中，脂肪細胞是間質細胞中⽐比例最⾼高的，臨床研究上也指出較⼤大乳房⼥女性具有較⾼高乳癌危險因⼦子。︒｡然⽽而，⽬目前對於脂肪細胞是如何影響乳癌發. . . I .

(3) . 展則是未有定論。︒｡因此在本論⽂文中，我們⾸首先證實從乳癌病⼈人乳房組織分離之脂肪細胞可促進某類乳癌細胞⽣生⾧長，此類細胞與雌激素受體之表現並無顯著相關性，暗⽰示有其他重要路徑存在。︒｡透過 cDNA 微陣列與 RNA ⼲干擾實驗結果交叉分析，我們發現 monocarboxylate transporter 2 (MCT2) 分⼦子在對於脂肪細胞促進乳癌細胞⽣生⾧長過程中扮演重要⾓角⾊色。︒｡MCT2 已知功能主要參與在酮體之⼀一 β-hydroxybutyrate 由外⽽而內之運輸。︒｡β-hydroxybutyrate 在細胞中可被做為內⽣生性 HDAC 抑制劑，當我們以 β-hydroxybutyrate 處理具有 MCT2 表現之細胞時，可有效增加組蛋⽩白 H3K9 位置的⼄乙醯化，此位置⽬目前被認為與基因表現呈現正相關。︒｡細胞經 βhydroxybutyrate 處理後，可被誘導產⽣生許多與促進癌細胞⽣生⾧長相關之因⼦子表現，例如細胞激素 IL1β 與脂肪素 LCN2，進⽽而促進乳癌細胞致癌性。︒｡同時，我們也觀察到在乳癌臨床檢體中，MCT2 表現量與 IL1β 及 LCN2 表現量具有⾼高度正相關，並顯⽰示較差預後。︒｡總結來說，在本研究中我們闡述了乳癌微環境中脂肪細胞如何與 MCT2 表現之乳癌細胞進⾏行交互作⽤用，進⽽而促進乳癌發展。︒｡. . . II .

(4) . Abstract Cancer cells are not only regulated by autonomous signals, but also influenced by the surrounding cells. In the aspect of cancer cell-autonomous regulation, gain of function of membrane receptor is a good strategy exploited by cancer cells to benefit own growth and survival. Overexpression of HER2 has been served as a target for developing trastuzumab to treat 20~25% of breast cancer. However, little or none of other membrane receptor is found to be useful as potential target for breast cancer treatment since then. Here, we found that amplified signal of Interleukin-17 receptor B (IL-17RB) and its ligand IL-17B promoted tumorigenicity in breast cancer cells and impeded acinus formation in immortalized normal mammary epithelial cells. External signal transmitted through IL-17RB activated NF-κB to up-regulate anti-apoptotic factor Bcl-2 and induce etoposide resistance. Elevated expression of IL-17RB had a stronger correlation with poor prognosis than HER2 in breast cancer patients. Interestingly, breast cancer patients with high expression of IL-17RB and HER2 had the shortest survival rate. Depletion of IL-17RB in trastuzumab-resistant breast cancer cells significantly reduced their tumorigenic activity, suggesting that IL-17RB and HER2 play an independent role in breast carcinogenesis. Furthermore, treatment with antibodies specifically against IL17RB or IL-17B effectively attenuated tumorigenicity of breast cancer cells. These results suggest that the amplified IL-17RB/IL-17B signal pathway may serve as a therapeutic target for developing treatment to manage IL-17RB associated breast cancer. The crosstalk between cancer cells and their microenvironments also contributes to cancer initiation and progression. Adipocyte is the most abundant cell type in mammary glands, and larger breast size is associated with an increased risk for breast cancer. However, how adipocytes in mammary gland affect breast cancer formation is. . III .

(5) . not completely understood. Here, we showed that primary adipocytes derived from mammary glands promote malignant growth of distinct type of tumor cells independent of its status of estrogen receptor α (ER-α). Through differential microarray analyses and RNAi knockdown screening, monocarboxylate transporter 2 (MCT2), responsible for transporting β-hydroxybutyrate, a ketone body, was identified as a critical factor that mediated the promotion of tumorigenicity associated with the adipocytes. Treating MCT2-expressing breast cancer cells with β-hydroxybutyrate, a physiological HDAC inhibitor, increased histone H3K9 acetylation, a gene activation mark, up-regulated tumor-promoting genes including cytokine IL1β as well as adipokine lipocalin 2 (LCN2), and enhanced tumorigenic activity. Consistently, elevated expressions of MCT2 as well as IL1β and LCN2, were significantly correlated with poor prognosis in breast cancer patients. These results suggest a novel mechanism of a specific crosstalk between MCT-2-expressing cancer cells and β-hydroxybutyrate secreting adipocytes in mammary gland microenvironment for promoting breast cancer malignancy.. . IV .

(6) . 誌謝. 五年的博⼠士⽣生涯沒想到轉眼間就已到了尾聲，在這期間不僅學習到許多實驗技巧與專業知識，也認識很多很棒的朋友。︒｡這幾年中，除了經歷過實驗做不出的無⼒力與灰⼼心，也享受得到結果的喜悅與感動，這將是⼀一輩⼦子最珍貴的回憶。︒｡在此⾸首先要感謝我的指導教授李⽂文華⽼老師與許⾦金⽟玉⽼老師，由衷佩服兩位⽼老師對於實驗與科學的熱忱與執著，感謝⽼老師耐⼼心指導實驗上的問題，每次討論總是可以提出精闢的⾒見解與建議，讓我獲益良多，也讓實驗有新的⽅方向。︒｡感謝潘⽟玉華⽼老師對於每次報告所提出的寶貴意⾒見，使我的研究可以更趨完善。︒｡另外也要感謝⼝口試委員林敬哲⽼老師、︑､張明富⽼老師、︑､張智芬⽼老師、︑､李明學⽼老師以及莊⽴立民⽼老師的不吝指正，得以改善論⽂文與研究缺失。︒｡此外也要感謝實驗室的好夥伴，博皓、︑､恆祥、︑､芳儀、︑､Ｗendy、︑､世嘉、︑､婕琳、︑､秉坤、︑､春美、︑､咏霖、︑､孟涵、︑､柳臻、︑､瑞芸、︑､以瑛、︑､⽂文⼼心、︑､俐⽂文、︑､韻如、︑､珮珣、︑､政遠，因為你（妳）們⼀一路上的幫忙與打氣使我能夠順利完成博⼠士論⽂文。︒｡另外也感謝我的⽗父母，你們默默地⽀支持、︑､包容與體諒，讓我在最低潮的時候有⼀一個溫暖的地⽅方可以依靠。︒｡最後要感謝的⼈人是我的太太玲淵，謝謝妳⼀一路上的⽀支持與陪伴，讓我博⼠士⽣生涯可以無後顧之憂的進⾏行。︒｡除了上班外，還要幫我照顧兩個可愛的⼩小⿁鬼頭及處理家務。︒｡這幾年來⾟辛苦妳了，如果沒有妳，我想我的博⼠士論⽂文也無法順利完成，謝謝妳。︒｡謹以此⽂文獻給我最愛的家⼈人與朋友. . . V .

(7) . Table of contents 中⽂文摘要 ........................................................................................................................... I Abstract ............................................................................................................................ III 誌謝 .................................................................................................................................. V Table of contents ............................................................................................................. VI Chapter I- Overview and rationale ................................................................................. 1 Breast cancer .............................................................................................................. 2 IL-17 family and tumorigenesis ................................................................................. 4 Microenvironment of breast cancer cells ................................................................... 6 Fibroblast and breast tumorigenesis ........................................................................... 7 Adipocytes and breast cancer ..................................................................................... 8 MCT2 (monocarboxylate transporter 2) ..................................................................... 9 Chapter II- Autocrine/paracrine mechanism of Interleukin-17B receptor promotes breast tumorigenesis through NF-κB mediated anti-apoptotic pathway .................. 12 Introduction .............................................................................................................. 13 Materials and Methods ............................................................................................. 15 Results ...................................................................................................................... 22 Discussion ................................................................................................................ 29 Figures and tables ..................................................................................................... 32 Chapter III- β- hydroxybutyrate secreted from adipocytes in mammary glands promotes malignancy of breast cancer expressing monocarboxylate transporter 2 57 Introduction .............................................................................................................. 58 . . VI .

(8) . Materials and Methods ............................................................................................. 60 Results ...................................................................................................................... 66 Discussion ................................................................................................................ 74 Figures and tables ..................................................................................................... 80 References ..................................................................................................................... 108 Curriculum vitae .......................................................................................................... 123 Appendix ....................................................................................................................... 125 . . VII .

(9) . Chapter I- Overview and rationale. . . 1 .

(10) . Breast cancer Breast cancer is one of the malignant diseases leading to cell death in women worldwide. In 2015, there will be an estimated 231,840 new cancer cases diagnosed and 40,290 cancer deaths in the United States. Although the incidence of breast caner is declined during the last decade, it remains the first and second in cancer incidence and death rate in the United States, respectively (Siegel et al., 2015). Compared to the United States, the incidence of breast cancer in Taiwan is constantly increased since 1979 to 2011. (Statistics form Health Promotion Administration, Ministry of Health and Welfare, Taiwan. Download is available at http://www.hpa.gov.tw). It ranks first and fourth in frequency for both diagnosis and cause of death in Taiwan women, respectively (Fig. I1).. Proportion of major cancer incidence, 2011 (8,140)colon 16%. 25%. breast (10,056). (7,920)liver 15%. 15%. colon (5,947). (6,938) lung 13%. 10%. lung (4,121). (6,308) oral cavity 12%. 8%. liver (3,372). (4,628) prostate. 9%. 5%. thyroid (1,954). (2,430) stomach. 5%. 4%. uterus(1,722). (2,063) esophagus. 4%. 4%. cervix (1,673). (1,590) skin. 3%. 3%. stomach (1,394). (1,389) urinary bladder. 3%. 3%. ovary (1,240). (1,123) nasopharynx. 2%. 3%. skin (1,395). (9,436 ) others 18%. 19%. 51,965 men. others (7,843) 40,717 women. Fig. I-1. Cancer incidence of 10 common cancers in Taiwan, 2011 Adapted from http://www.hpa.gov.tw. Based on the gene expression profiling and immunohistochemistry (IHC), breast cancers can be classified into at least 5 groups: luminal type A&B (both are estrogen receptor positive, ER+), HER2 (human epidermal growth factor receptor positive, HER2/neu+), basal like (also called triple negative, ER-/PR-/HER2-), and normal breast . . 2 .

(11) . like (Fig. I-2a)(Vargo-Gogola and Rosen, 2007). Each subtype of breast cancer shows different risk factors of incidence, treatment response, and even disease progression risk. Among these different types of breast cancer, overexpression of nuclear receptor ER and membrane bound receptor HER2/neu protein accounts for approximate 85% of breast cancer. Compared to luminal ER+ breast cancer, the basal type and HER2/neu+ breast. REVIEWS. cancers exhibit high a. Luminal subtype A. Luminal subtype B. b. ERBB2+. Basal Normal subtype breast-like. 48. 72. 1.0. Probablity. 0.8 0.6 0.4 0.2 0.0. 0. 24. 96. Overall survival (months). Is the expression of CD44 +CD24 –ESA + markers broadly indicative of tumour-initiating subpopulations malignancy and suggests poor that within cell lines? Accumulating evidence their expression is heterogeneous within cell lines and breast cancers19,29–31. Furthermore, their relationship to prognosis (Fig.functional I-2b). clinical outcome is unclear30–32. More studies (using limiting dilution transplantation of cells isolated by FACS (fluorescent activated cell sorting)) that utilize Fortunately, the the different subtypes of breast cancer are required to conclude definitively whether each subtype contains subpopulations of tumour-initiating cells and whether development of they display identical or distinct cell surface markers. Improved methods and markers are needed to identify and characterize tumour-initiating cells within cell hormone and targeting lines and breast cancers. One promising approach may be to use the stem cell marker aldefluor33 in FACS analysis coupled with immunohistochemistry (IHC) using therapy (Tamoxifen an anti-aldehyde dehydrogenase 1 (ALDH1) antibody (M. Wicha, personal communication). Notwithstanding these limitations, several studies indicate that certain and Trastuzumab, also cell lines can be used to investigate the cellular and molecular distinctions between the tumour-initiating and non-tumour-initiating subpopulations19,26,28,34.. called Herceptin) for. Figure 2 | The identification of breast cancer subtypes by molecular profiling. a | The concept that breast cancer is not a single disease is demonstrated by the 2D versus 3D culture conditions. The molecular profilNature Reviews | Cancer Fig. I-2. The identification of breast by that molecular profiling. increasing number of gene-expression profilingcaner studies,subtypes which suggest there are at + above indicate that + cell lines have ing studies described five subtypes invasive ductal carcinoma approximately (a)least Breast cancerofis heterogeneous that(IDC) canthat be constitute classified into at least 5 many of the genetic and genomic alterations found in 10,131 80% of all breast cancers . A dendogram shows clustering of 115 breast tumours into primary breast cancers. However, most of these studies subtypes according expression profiling. (b)didThe prognostic outcomes the five subtypes of IDC.gene Grey branches indicate tumours that not correlate with any were performed using cell lines cultured on plastic12,13,35. . Invasive lobular carcinomas, which also display distinct gene-expression forsubtype each132subtype of invasive breast cancer. A principal limitation of in vitro cell culture studies is profiles, constitute an additional 10–15% of breast cancers (not shown)133. Ten that the culture conditions used to propagate these cells additional rare types of breast cancer havefrom also been although 7: collectively Adapted Natdescribed, Rev Cancer., 659-672, 2007 create an environment that differs markedly from the these account for less than 10% of newly diagnosed cases each year (see the Mayo Clinic web page on breast cancer). b | The prognostic outcomes for each subtype of IDC breast microenvironment. This caveat must be considare shown as overall survival. The ERBB2+ and basal subtypes demonstrate the worst ered when discussing the fidelity with which cell lines prognoses, whereas the luminal subtype A shows the most favourable outcome. model breast cancer (FIG. 3). Recently, it has been demonstrated that the prognostic outcomes of the subtypes were Recently, to determine whether gene expression, like 134 not different when a pathologically complete response to therapy was achieved . It morphology, is more faithfully recapitulated in breast canhas been suggested that the distinct prognostic outcomes between the subtypes may cer cell lines when grown in 3D reconstituted basement reflect the differential responses of the bulk of the tumour and tumour-initiating cell membrane (rBM) cultures, the molecular profiles of 25 populations to chemotherapy and targeted therapies135. Reproduced with permission breast cancer cell lines cultured in 2D versus 3D conditions from REF. 132 ¢ (2007) National Academy of Sciences, USA.. ER and HER2 breast cancer. treatment. significantly. improve. the clinical outcomes, respectively. However, development of drug resistance, limited response rate, and cancer recurrence remain to be resolved. Thus, identification of novel. were compared molecular profiles of pathways involved in breast cancer tumorigenesis is urgently needed. Nottosurprisingly, offer new targets individual cell lines were more similar to themselves than 36. MCF-7 sub-line, MCF-S, forms mammospheres, exhibits. the CD44 CD24 expression profile and is enriched by for developing the corresponding treatments. 1000-fold in tumour-initiating capacity compared with +. Limiting dilution transplantation An experimental method for estimating the number of cells that have stem or progenitor or tumour-initiating behaviour within a population of cells.. Aldefluor. . An aldehyde dehydrogenase (ALDH) substrate that allows the identification and isolation of stem or progenitor cells based on the observation that these cells have high ALDH activity.. –. the parental cells19. Other laboratories have examined subpopulations of MCF-7 cells that demonstrate reduced sensitivity to radiation and enhanced self-renewal capabilities in mammosphere assays26–28. However, the tumour-initiating capability of these subpopulations was not reported. Recently, Kuperwasser and colleagues tested the tumour-initiating potential of CD44+CD24– populations within breast cancer cell lines directly by performing limiting dilution transplantation experiments. These markers did not correlate with tumour-initiating capability, but instead were indicative of a basal subtype (C. Kuperwasser and C. Fillmore, personal communication). However, an ESA+ fraction within the CD44+CD24– subpopulation showed enrichment for tumour-initiating capability and increased resistance to chemotherapeutic agents.. 662 | SEPTEMBER 2007 | VOLUME 7. to other cell lines grown in the same culture conditions, indicating that the 3D culture environment does not promote global changes in gene-expression patterns. However, a group of signal transduction genes were identified that significantly correlated with cells grown in the 3D environment. Altered expression of these genes coupled with post-transcriptional gene regulation probably accounts for the morphological and behavioural differences of cells grown in 3D compared with 2D cultures37. Bissell, Brugge and colleagues have pioneered the 3D culture methods of breast epithelial and tumour cell lines, and readers are directed to a number of excellent reviews on modelling the microenvironment in 3D cultures38–41. Three-dimensional culture models have been used to investigate the critical signalling pathways that regulate tumour biology. For example, Weaver and colleagues42 have demonstrated how one feature 3 of breast cancer biology, stromal rigidity and its effects on morphogenesis, can be modelled in 3D cultures, www.nature.com/reviews/cancer.

(12) . IL-17 family and tumorigenesis IL-17 family is a large cytokine family that consists of six IL-17 cytokines and five IL-17R receptors (Fig. I-3). IL-17A and IL-17F, mainly secreted from T helper 17 Immunity. (TH17) cells, are the most well-characterized members in the IL-17 family (Iwakura et Review al., IL-17A. IL-17A+F. IL-17F. IL-17E (IL-25). IL-17B. IL-17C. IL-17D. Act1. Act1. TRAF6 ?. Act1. TRAF3. TRAF6. either. form. homodimer with IL-. with IL-17F to bind. TAK1. FnIII. ERK MAPK. IL-17A. 17A or heterodimer. Act1. TRAF6. TAK1. IL-17RD. IL-17RB. Unknown. IL-17RA. IL-17RE. IL-17RC. IL-17RB. can IL-17RA. 2011).. Unknown. MAPK. to the receptor IL-. SEFIR. 17RA/IL-17RA, AP-1. NF-κB. C/EBP. AP-1. NF-κB. Fig. I-3. IL-17 and the IL-17receptor families. Six IL-17 family cytokines (IL-17A to IL-17F) and five IL-17R family receptors (IL-17RA to IL-17RE) have been identified. After binding of an IL-17A or IL-17F homodimer or heterodimer to IL-17R (the heterodimer of IL-17RA and IL-17RC), Act1 associates with IL-17RA and/or IL-17RC through its SEFIR domains. Subsequently, the complex associates with TRAF6, leading to the activation of NF-κB, MAPK-AP-1, and C/EBP. Similar to signaling via IL-17R, IL-17E (IL-25) binding to IL-25R (heterodimer of IL-17RA and IL-17RB) results in activation of NF-κB, MAPK-AP-1, and C/EBP via recruitment of Act1 TRAF6. not IL-17RA, an intracellular TRAF6-binding CD4+ and contains Th17 cell development stilland remains elusiveIL-17RB, (reviewed but in Korn CD8+ T cells, these cells constitutively express et al., 2009). IL-23R, IL-1R, and RORgt. Likewise, respectively; NKT cells produce IL-17A motif, which activates NF-κB. IL-17B and IL-17C bind to IL-17RB and IL-17RE, The Th17 cellthe lineage is heterogeneous population. In addition in the presence of IL-1 and in combination with TCR stimhowever, downstream signaling pathway is unknown. The ligand(s) forIL-23 IL-17RD is also unknown. to IL-17A and IL-17F double-positive cells, populations that are ulation. These two T cell populations (gd-17 and NKT-17) can FnIII, fibronectin III-like domain; SEFIR, similar expression to FGF, IL-17R, and Toll-IL-1R family only IL-17A or IL-17F positive have been identified. The mecha- rapidly produce IL-17A and IL-17F in response to proinflammadomain. nisms that regulate IL-17A and IL-17F production also differ; tory cytokine stimulation and may therefore provide an essential Adapted from Immunity., 34: 149-162, 2011 IL-17F is expressed earlier than IL-17A during Th17 cell develop- initial source of these two cytokines. Figure 1. IL-17 and the IL-17 Receptor Families. Six IL-17 family cytokines (IL-17A to IL-17F) and five IL-17R family molecules (IL-17RA to IL-17RE) have been identified. After binding of an IL-17A or IL-17F homodimer or heterodimer to IL-17R (the heterodimer of IL-17RA and IL-17RC), Act1 associates with IL-17RA and/or IL-17RC through its SEFIR domains. Subsequently, the complex associates with TRAF6, leading to the activation of NF-kB, MAPK-AP-1, and C/EBP. Downstream of IL-17R, TRAF3 also associates with Act1 to inhibit Act1-TRAF6-mediated activation of transcription factors. Act1-independent ERK activation also contributes to IL-17R signaling via an unknown molecule (?). Similar to signaling via IL-17R, IL-17E (IL-25) binding to IL-25R (heterodimer of IL-17RA and IL-17RB) results in activation of NF-kB, MAPK-AP-1, and C/EBP via recruitment of Act1 and TRAF6. IL-17RB, but not IL-17RA, contains an intracellular TRAF6-binding motif, which activates NF-kB, but not AP-1, through TRAF6 binding. IL-17B and IL-17C bind to IL-17RB and IL-17RE, respectively; however, the downstream signaling pathway is unknown. The ligand(s) for IL-17RD is also unknown. FnIII, fibronectin III-like domain; SEFIR, similar expression to FGF, IL-17R, and Toll-IL-1R family domain.. ment (Lee et al., 2009). Although underlying molecular mechanisms have not been described, it is likely that several mediators, such as transcription factor or T cell receptor (TCR) signaling, distinctly regulate the production of the cytokines. Indeed, defiH (Yang ciency of RORa selectively reduced IL-17A production et al., 2008b), and IL-17A expression was more sensitive to the strength of TCR signaling (Gomez-Rodriguez et al., 2009). In addition to Th17 cells, a wide variety of T cells also produce IL-17A and IL-17F. These cytokines are produced by cytotoxic CD8+ T cells (Tc17) under conditions that are similar to those required by Th17 cells, but different from those required by IFN-g producing CD8+ T cells (Tc1). Similarly, distinct populations of gdT (gd-17) cells and NKT (NKT-17) cells produce IL-17A and IL-17F (reviewed in Cua and Tato, 2010). However, IL-23 and IL-1 can directly induce gd-17 cell development in the absence of IL-6 and TCR ligation because, unlike naive. or. C/EBP. More recently, innate lymphoid populations of neutrophils, monocytes, natural killer cells, and lymphoid tissue inducer (LTi)-like cells have been shown capable of rapidly producing IL-17A and IL-17F (Cua and Tato, 2010). In addition, IL-17A is produced by intestinal Paneth cells (Takahashi et al., 2008), whereas IL-17F mRNA, but not IL-17A mRNA, is expressed in colonic epithelial cells (Ishigame et al., 2009), suggesting that IL-17A and IL-17F from nonlymphoid cells may also regulate immune responses. Substantial efforts are underway to clarify the mechanisms that control IL-17A and IL-17F production in these cell types, and the relative contributions of the resulting cytokines in immune responses.. IL-17RA/IL-17RC, respectively. (Chang. and Dong, 2007). The main function of IL17A/IL-17F signaling. pathway is involved in T 17 cells regulation and production of several pro-inflammatory cytokines. Dysregulation of IL-17A related pathway is associated with many autoimmune diseases (Kirkham et al., 2014). IL-17E (also known as IL-25) is produced Signaling Mechanism of IL-17A and IL-17F Both Il17ra!/! and Il17rc!/! mice fail to respond to both IL-17A and IL-17F, indicating that both IL-17RA and IL-17RC are. by mucosal epithelial cells and many immune cells. It has been reported that IL-17E Immunity 34, February 25, 2011 ª2011 Elsevier Inc. 151. regulates type-2 immune response and stimulates TH2-type cytokines production (Dong, 2008). However, the function of IL-17B, IL-17C, and IL-17D remain elusive.. . . 4 .

(13) . It has been reported that chronic inflammation plays an important role in cancer development including angiogenesis, growth promotion, invasion, and metastasis of tumor cells (Lu et al., 2006). The infiltrating of IL-17A producing TH17 cells in tumor microenvironment contributes to generating a pro-inflammatory niche and promotes tumor progression in inflammation-associated gastric and pancreatic cancer, respectively. ofactivates factorsdownstream includedsignaling antiangiogenic (Iida et al., 2011; McAllister et al., 2014). IL-17A, which. pro teins (ATIII and VBP), proinflammatory of STAT3 to up-regulate several pro-survival genes, cytokines also promotes invasionand of breast (IL-1F7 IL-25), and growth and differentiation proteins (FGF11 and cancer (Wang et al., 2009; Zhu et al., 2008). Compared to the promoting role of IL-17A BMP10). IL-25 exhibited the most poten in tumorigenesis, Furuta et al. found an opposite role of IL-17E in breast cancer cytotoxic activity toward breast cancer cells whereas the other factors exhibited cytostat tumorigenesis (Furuta et ic activity. Here, we focused on the mech Secretion of ILanismal.,of2011). action of IL-25 and its potentia as a therapeutic agent in breast cancer. 17E from non-malignant IL-25 is a proinflammatory cytokine epithelial that ismammary expressed highlycells in certain organs such induces as testis,caspase-mediated prostate, and spleen, and i expressed in low amounts in other organ apoptosis IL-17E including normalin breast (11, 24). It is the most receptor distant(also member of the IL-17 family o known as ILproteins, sharing only 16 to 30% sequence 25R, with comprised of ILhomology the other family member R renders cells sensitive to apoptotic after treat-(IL-17RA/ Fig. I-4. Cytotoxic activity of IL-25signaling (IL-17E) is specific to IL-25R (25). 17RA/ It plays a roleIL-17RB in proinflammatory IL-17RB heterodimer) expressing breast cancer cells. Non-malignant MECs otein expressed in mutation wild-type do not express IL-25Ranalyses. and are resistantWt, to apoptosis induced byfullIL-25. responses of lymphatic, kidney, and lung with a deletion in TRAF6 binding domain (amino acids cells heterodimer) expressing by inducing production of T helpe Adapted D376–387). from Sci Transl Med., 78ra31, 2011 n in the DD-like region (amino acids (B)3: Westcytokines (11, 19, 24, 26) 2 (TH2)–type breast IL-17RB are also the receptors for IL-17A and IL-17B, nts (Wt, DTRAF6, or cancer DDD)cells. afterIL-17RA IL-25Rand were ectopically ndogenous IL-25R amounts in parental cells (Ctrl). b-Actin The function of IL-25 in other tissues re respectively (Fig. I-4). Contradictory to the roles of IL-17E and its receptor in apoptosis, n of IL-25R is essential for apoptotic signaling mediated by mains to be elucidated. Weinshow here that IL-25 is temporally ct cleavage of caspase and that PARP in MCF10Aofcells that is observed it is worth3noting overexpression IL-17RB murine leukemia cells, L-25 (500 ng/ml, ~25 nM) for varying time periods. b-Actin up-regulated in developing normal mam suggesting oncogenic rolecleaved of this receptor 2000). IL-17B, another cognate mary glands and induces caspase-mediated uncleaved protein; whiteanarrowheads, protein.(Tian (D) et al., specific to breast cancer cells that express IL-25R. Non- apoptosis of breast cancer cells without af ligand of IL-17RB, may also play a role in promoting tumorigenesis (Fig. I-4). Thus, we resistant to apoptosis induced by IL-25. Breast cancer cells fecting nonmalignant MECs either in cul duced apoptosis. hypothesize that the interaction of another cognate ligand with IL-17RB may ture IL-17B or in mice. The reason behind the resistance of nonmalignant cells to IL-25 5 lly distinct apoptotic reg- is the differential expression of the receptor, IL-25R, high in breast can nd a putative DD-like mo- cer cells but low or absent in nonmalignant MECs (Fig. 5, A and B) ating the intricacy of the IL-25R overexpression contributes to tumorigenic potential, as shown by.

(14) . exert a different function rather than IL-17E induced apoptosis in breast carcinogenesis. To test this possibility, my first study focuses on elucidating the role of IL-17RB/IL-17B signaling in breast cancer tumorigenesis.. Microenvironment of breast cancer cells Growth of cancer cells is subjected to multiple signals regulation including autocrine/paracrine signals from cancer cells as well as various cell types in the local environment (also known as microenvironment). Accumulating evidence suggests that Place et al. Breast Cancer Research 2011, 13:227 Page 2 of 11 the microenvironment of tumor cell plays a vital role in cancer initiation and progression http://breast-cancer-research.com/content/13/6/227. of many cancers (Liotta. A Fibroblasts Macrophages T cells B cells Neutrophils Extracellular matrix Blood vessels Myoepithelial cells Luminal epithelial cells Basement membrane Secreted factors MMPs Cross-talk. Normal. B. and Kohn, 2001). The microenvironment breast fibroblast,. in. comprises adipocytes,. endothelial as well as. Normal. DCIS Enhanced angiogenesis. immune cells (Fig. I-. Activated and heterogeneous fibroblasts. 5A). During the breast. Aberrant autocrine/ paracrine regulatory loops. cancer progression, the. Altered myoepithelial cells. numbers of fibroblasts. Increased leukocyte infiltration. Figure 1. Alterations of the microenvironment from normal duct to in situ transition. (A) Schematic (transverse) view of a normal breast duct composed of a layer of luminal epithelial cells encircled by myoepithelial cells (green) and surrounded by a continuous basement membrane. Stroma containing fibroblasts, immune cells, and vasculature surrounded by the extracellular matrix maintains the normal tissue structure. (B) Longitudinal view of the normal duct and in situ carcinoma. In ductal carcinoma in situ (DCIS), epigenetically and phenotypically altered myoepithelial cells (shown as brown cells) are surrounded by a still largely continuous basement membrane. Altered myoepithelial cells in DCIS are unable to aid polarization and organize the structure of the normal duct. At the same time in the stroma, the numbers of fibroblasts and infiltrated leukocytes are increased and angiogenesis is enhanced. Cancer-associated fibroblasts (shown as yellow-green fibroblasts) and infiltrated leukocytes elevate secretion of growth factors, cytokines, chemokines, and matrix metalloproteinases (MMPs) to promote tumor progression. Potential crosstalk between cell-cell and cell-matrix interactions are aberrantly regulated by both autocrine and paracrine networks of proteolytic enzymes, cytokines, and chemokines (red arrows; not all possible interactions are indicated). Interactions between stromal and cancer cells may interact with each other via paracrine signaling rather than direct cell-cell contact.. Fig. I-5. Alterations of the microenvironment from normal duct to in situ transition. (A) Normal breast duct consists of a luminal epithelial layer encircled by myoepithelial cells and surrounded by basement membrane. (B) Longitudinal view of the normal duct and in situ carcinoma. In ductal carcinoma in situ (DCIS), the numbers of fibroblasts and infiltrated immune cells are increased and the angiogenesis is also enhanced. Adapted from Breast Cancer Res., 13: 227, 2011. serial analysis of gene expression (SAGE). In addition, genetic changes were detected by cDNA array comprehensive genomic hybridization and single nucleotide polymorphism arrays. The results of this study. and infiltrated immune cells are increased and the angiogenesis is also enhanced. (Fig.. I-5B).. demonstrated altered gene expression patterns in each cell type analyzed during breast cancer progression. Myoepithelial cells from normal breast tissue and DCIS had the highest number of diﬀerentially expressed genes.. The phenotypic change in microenvironment can be observed even in the early stage of breast cancer, ductal carcinoma in situ (DCIS). Allinen et al. isolated and examined the. . . 6 .

(15) . global gene expression profiles of major cells types including fibroblasts, infiltrated immune cells, endothelial and luminal/myoepithelial cells from normal breast tissue, DCIS, and IDC (invasive ductal carcinoma) lesions (Allinen et al., 2004). The results demonstrated that gene expression profiles are altered in each cell types during breast cancer progression. A significant fraction of these altered genes encode secreted proteins and cell surface receptors, suggesting that the activation of aberrant autocrine/paracrine loops occurs. Deciphering of crosstalk between breast cancer and stromal cells in the microenvironment may lead to develop novel therapeutic strategies and targets.. Fibroblast and breast tumorigenesis In most of cancer microenvironment studies, cancer-associated fibroblasts (CAFs) attract a lot of attention in the last decade. In the tumor stroma, connective tissue fibroblasts adjacent to cancer cells can be activated (or educated) as CAFs, which can be defined by expression of alpha smooth muscle actin (αSMA) (Paunescu et al., 2011). Although the detailed mechanisms for the activation of CAFs remain uncertain, TGF-β and CXCL12/SDF-1 are regarded as the main factors that contribute to CAFs activation (Kojima et al., 2010). Compared to inactivated normal fibroblasts, activated CAFs play important roles in several tumor-promoting functions including sustaining proliferative signaling, inducing angiogenesis, activating invasion and metastasis (Hanahan and Coussens, 2012). In breast cancer, CAFs promote breast cancer progression and metastasis via several growth factors and chemokines secretion. For example, HGF and CXCL12/SDF-1 secreted from CAFs promote breast cancer cell growth through their receptors c-Met and CXCR4, respectively (Orimo et al., 2005; Rong et al., 1992; Tyan et al., 2011). The secretion of CXCL12/SDF-1 from CAFs also promotes angiogenesis via recruiting endothelial progenitor cells in breast cancer (Orimo et al., 2005). In sum, these. . . 7 .

(16) . studies suggest that CAFs play an active role in breast tumorigenesis.. Adipocytes and breast cancer Compared to the role of CAFs in breast cancer tumorigenesis, little attention has been given to the adipocytes despite the fact that adipocytes are the most abundant stromal partners in human mammary gland. Originally, adipocytes is regarded as energy storage cells, have clearly emerged as endocrine cells in the last decade. Adipocytes can secrete many factors such as hormones, growth factors, chemokines and proinflammatory molecules, also known as adipokines (Rajala and Scherer, 2003). Thus, tumor-surrounding adipocytes become a good candidate to evaluate heterotypic interaction between stroma and tumor cells, especially in breast cancer. Moreover, it has been reported that obesity and the larger breast size are associated with increased risk of breast cancer, suggesting that local adiposity in breast may have a significant impact on breast cancer progression (Berclaz et al., 2004; Calle et al., 2003; Kusano et al., 2006). Understanding the interaction between breast cancer cells and adipocytes in microenvironment may not only provide new targets for novel therapeutic strategy development but also confer new insights into the connection between obesity and breast cancer risk. The influence of adipocytes on breast cancer cells has been investigated. Adipokines secreted from adipocytes may be important for this interplay (Vona-Davis and Rose, 2007). For instance, leptin, collagen VI and interleukin-6 secreted from adipocytes, promote breast cancer growth and invasion (Dirat et al., 2011; Iyengar et al., 2005; Surmacz, 2007). The expression of metalloproteinase (MMP)-11/stromelysin-3 in adipocytes is induced by breast cancer cells at the invasive front, indicative of a role in extracellular matrix (ECM) remodeling during breast cancer development (Andarawewa . . 8 .

(17) . et al., 2005). Furthermore, estrogen secreted from adipose tissue also contributes to the breast cancer tumorigenesis. Adipose tissue in breast becomes an important site for estrogen production in post-menopausal women due to the aromatase expression. Increased estrogen production is thought to contribute to ER-positive breast cancer progression (Lorincz and Sukumar, 2006). Although these findings implicate that adipocytes may play a promoting role in breast cancer progression, a systematic study to explore the crosstalk between human primary adipocytes and breast cancer cells have not been performed. Through systemic differential microarray analyses and RNAi knockdown screening, I found that MCT2 played an important role in the crosstalk between breast cancer cells and adipocytes.. MCT2 (monocarboxylate transporter 2) Monocarboxylate transporters (MCTs) family comprises 14 members, of which MCT1-4 catalyze the proton-linked transport of monocarboxylates such as lactate, pyruvate and β-hydroxybutyrate across the plasma membrane (Halestrap, 2013). All MCTs are predicted to have 12 transmembrane domains with intracellular N- and Ctermini. MCT1 is widely expressed in most tissues, while MCT4 is primarily expressed in highly glycolytic cells, such as white skeletal muscle fibers. MCT2 and MCT3 show a more limited expression pattern; MCT2 is primarily expressed in liver, kidney, and neurons, while MCT3 expression is restricted to retinal pigment epithelium and choroid plexus epithelia (Halestrap and Price, 1999). In general, MCT2 shows higher affinity to. . . 9 .

(18) . MCT STRUCTURE AND FUNCTION. 3. Table 1. Table I-1. Km values of different MCT isoforms for a ranges of monocarboxylates K Values of different MCT isoforms for a range of monocarboxylates m. Substrate. MCT1 tumor cells. MCT1 oocytes. MCT2 oocytes. MCT3 yeast. MCT4 oocytes. Formate Bicarbonate Oxamate Glyoxylate L-Lactate D-Lactate Pyruvate S-Chloropropionate R-Chloropropionate D,L-a-Hydroxybutyrate L-b-Hydroxybutyrate D-b-Hydroxybutyrate c-Hydroxybutyrate Acetoacetate a-Ketobutyrate a-Ketoisocaproate a-Ketoisovalerate b-Phenylpyruvate. [100 – 49 63 4.5 27.5 0.7 0.7 0.7 2.6 11.4 10.1 7.7 5.5 0.2 – – –. – – – – 3.5 [60 1.0 – – – – – – – – 0.7 1.3 –. – – – – 0.74 – 0.08 – – – 1.2a 1.2a – 0.8 – 0.1 0.3 –. – – – – 6 – – – – – – – – – – – – –. [500b [500b [500b [500b 28 519 153 46 51 56 824 130 [500b 216 57 95 113 [500b. Data are for Km values (mM) of endogenous MCT1 in tumor cells or for MCT1, MCT2, and MCT4 expressed in Xenopus oocytes as indicated and are taken from ref. 14 where further details may be found. The L-lactate Km for MCT3 was measured following expression in Yeast and taken from ref. 16. a D,L-Racemic mix used in these studies. b Uptake at 50 mM is very low to measure.. Adapted from INBMB Life., 64: 1-9, 2012 mouse tumor cell line by monitoring changes in intracellular pH using the fluorescent pH indicator 20 -70 -bis-(carboxyethyl)-5-6carboxy-fluorescein (BCECF) (13). Subsequently, MCT1 was expressed in Xenopus laevis oocytes that exhibit no significant endogenous MCT activity and its activity again determined either radioactively or by monitoring transport-mediated changes in intracellular pH with BCECF or pH microelectrodes (14, 15). All these techniques confirmed that MCT1 demonstrates Michaelis Menten kinetics with a broad specificity for shortchain monocarboxylates including those substituted on the 2 and 3 positions with small groups such as halides, hydroxyl, and carbonyl groups as illustrated in Table 1. In addition, the transport of unsubstituted short-chain fatty acids, such as acetate, propionate, and butyrate, is strongly facilitated by MCT1, but these substrates can also enter cells rapidly by free diffusion of the undissociated acid [see (1, 10)]. Natural occurring substrates for MCT1 include L-lactate, pyruvate, b-hydroxybutyHALESTRAP rate, and acetoacetate (1, 2) and Km values for these substrates are within the range found physiologically (1, 8). More hydrophobic ketoacids derived by transamination of amino acids may also be transported by MCT1; these include phenylpyruvate (from phenylalanine), a-ketoisocaproate (from leucine), a-ketoisovalerate (from valine), and a-keto-b-methylvalerate (from isoleucine). However, the hydrophic side chain of these substrates impairs the release of the bound substrate following transport resulting in very slow rates of net transport of these substrates. by MCT1; indeed, they act as potent competitive inhibitors of the transport of other monocarboxylates (13). Interestingly, the transport of lactate is relatively stereoselective, with D-lactate being a poor substrate compared to L-lactate, whereas such stereoselectivity is not demonstrated for 2-chloropropionate or b-hydroxybutyrate. By far, the predominant role of MCT1 is to facilitate unidirectional proton-linked transport of L-lactate across the plasma membrane. This may represent either influx or efflux of lactic acid depending of the prevailing intracellular and extracellular substrate concentrations and the pH gradient across the plasma membrane. Net rates of transport of any monocarboxylate will be determined by the difference between influx and efflux, and at thermodynamic equilibrium, the concentration ratio of monocarboxylate inside the cell to outside the cell is equal to the ratio of [H1]out to [H1]in. However, MCT1 can also exchange one monocarboxylate for another without net movement of protons [see (1–3)].. most monocarboxylates than MCT1 and MCT4 (MCT3 is less well characterized)(Table I-1)(Halestrap, 2012).. As shown in Fig. I-7, the major physiological roles of MCT1-4 are responsible. for rapid transport of lactic acid and ketone bodies (β-hydroxybutyrate and acetoacetate) across cell membrane. Both. Inhibitors of MCT1. In addition to inhibition by competing monocarboxylates such as those described above (Substrate Specificity section), numerous nonphysiological competitive inhibitors of MCT1 have been described. These include CHC analogs and stilbene disulfonates such as 4,40 -di-isothiocyanostilbene-2,20 -disulfonate (DIDS) and 4,40 -dibenzamidostilbene2,20 -disulfonate (DBDS) [see (1, 10)]. These agents have been used in some published experiments as specific MCT1 inhibitors. influx. and. monocarboxylates. efflux. of. can. be. facilitated by MCT1, depending on the concentration of protons and monocarboxylates across. Figure 2. The role of MCTs in metabolism. It should be noted cell membrane. However, Fig. The MCTs in full metabolism. that no I-7. single cellrole will of carry out the spectrum of pathways shown. Depending on the tissue and the species, MCT1 or Lactic acid and ketone bodies are influxed by MCT1 or MCT2 are and used effluxed to take up by lactic acid and bodies for oxiMCT2 MCT1 or ketone MCT4. MCT2 and MCT4 are only MCT family members. dation (e.g., heart, red muscle, and neurons) or lactic acid for and kidney). In most tissues that rely on CT and SLC16 numbers gluconeogenesis (liver Adapted from INBMB Life., 64: 1-9, 2012 involved in unidirectional been functionally char- glycolysis for their energy metabolism under normoxic condig proton-coupled lactate tions (e.g., white skeletal muscle fibers), lactic acid efflux uti10 catalyze the sodium- lizes MCT4, but MCT3 fulfils this function in the retinal piginflux and efflux, respectively. The transport of lactic acid and ketone bodies into or out yroid hormone and aro- ment epithelium. All cells export lactic acid under hypoxic conties of the other eight ditions and use whichever MCT isoform is expressed (normally details are presented in the text and refs. 1–4. e have failed to demon- ofMCT1). cells Further is important for intracellular pH maintenance and supply of respiratory fuel or range of monocarboxy- A more detailed consideration of tissue specific roles of MCTs oocytes. The figure is a is giving in the accompanying review where the role of differ2). The bar indicates the ent MCT isoforms in shuttling lactate between different cell 10 h 0.1 corresponding to a types within a tissue is discussed (8).. dues.. well reviewed elsewhere. . CHARACTERIZATION OF THE DIFFERENT MCT FAMILY MEMBERS.

(19) . gluconeogenesis substrates (Halestrap and Wilson, 2012). Although the major function of MCTs is associated with the uptake or efflux of monocarboxylates, studies on the roles of MCTs in cancer are emerging recently. For example, elevated expression of MCT1 is reported in several cancers, such as breast and colorectal cancer (Pinheiro et al., 2010; Pinheiro et al., 2008). MCT2 and MCT4 are also highly expressed in prostate cancer and renal cell carcinoma, respectively (Gerlinger et al., 2012; Pertega-Gomes et al., 2013). Furthermore, the expression of MCT1 increases the lactate to fuel cancer growth (Sonveaux et al., 2008). However, the role of MCT2 in tumorigenesis is less explored. In this study, we confer a new role to MCT2 in the communication between breast cancer cells and adipocytes.. . 11 .

(20) . Chapter II- Autocrine/paracrine mechanism of Interleukin-17B receptor promotes breast tumorigenesis through NF-κB mediated anti-apoptotic pathway. . 12 .

(21) . Introduction Aberrant expression of membrane receptor is a common feature observed in a variety of cancers. For example, the overexpression of epidermal growth factor receptor1 (EGFR) in non-small cell lung cancer (Salomon et al., 1995) and epidermal growth factor receptor-2 (HER2/neu) in breast cancer (Slamon et al., 1987) are wellcharacterized, respectively. Increased activation of membrane receptors promotes proliferation, survival and invasion ability of cancer cells via interacting with specific ligands or auto-activation. Importantly, the constitutive activation of signaling pathways also offers potential opportunities for pharmacological intervention. In breast cancer, trastuzumab (also known as (a.k.a.) Herceptin) targeting therapy has been used to treat HER2/neu+ tumor and significantly improves clinical outcome (Piccart-Gebhart et al., 2005). However, critical issues including developing drug resistance, limited response rate and cancer recurrence remain to be resolved. Thus, identification of novel cell surface receptors involved in breast tumorigenesis is urgently needed to offer new potential therapeutic targets. During the last decade, accumulating evidence has suggested a strong association between chronic inflammation and cancer development among different types of cancer (Coussens and Werb, 2002). Cancer cells may take advantage of cytokine or cytokine receptor overexpression to benefit their own growth or invasive ability via autocrine or paracrine loop. In breast cancer, several pro-inflammatory cytokines, such as IL-1 (Singer et al., 2003), IL-6 (Sansone et al., 2007) and TGF-β (Taylor et al., 2013), have been reported to promote proliferation or invasion. IL-17A (a.k.a. IL-17) mainly secreted from TH17 cells activates downstream signaling of STAT3 to up-regulate several prosurvival genes, also promotes invasion of breast cancer (Wang et al., 2009; Zhu et al., 2008). The activating of IL-17A/IL-17RA axis is also required for the initiation and . 13 .

(22) . progression of inflammation associated pancreatic intraepithelial neoplasia (PanIN) (McAllister et al., 2014). Compared to the role of IL-17A/IL-17RA in tumorigenesis, overexpression of IL-17RB in murine leukemia cells also implicates an oncogenic role of this receptor (Tian et al., 2000). However, the precise contribution of IL-17RB signaling in tumorigenesis remains to be substantiated. The interaction among IL-17 ligands and receptors are intertwined. Previously, we found that IL-25 (a.k.a. IL-17E) secreted from non-malignant mammary epithelial cells induces breast cancer apoptosis (Furuta et al., 2011). The apoptotic activity of IL-25 is mediated by differential expression of its receptor, IL-25R, which is composed of IL17RB and IL-17RA heterodimer (Rickel et al., 2008). IL-17RA and IL-17RB are also the receptors for IL-17A and IL-17B, respectively (Shi et al., 2000; Yao et al., 1995). Thus, the ligands-receptors interaction may exert differential roles in a temporal and spatial manner. It is worth noting that high expression of IL17RB was found to correlate with poor prognosis in breast cancer patients (Furuta et al., 2011). However, the precise role of IL-17RB/IL-17B signal contributes to breast carcinogenesis remains unclear. In this study, we affirmed that the amplified IL-17RB/IL-17B signal was critical for breast tumorigenesis by correlating its expression with poor prognosis based on two well-characterized independent cohorts of breast cancer patients. Gain or loss of function study of IL-17RB/IL-17B signal in non-malignant mammary epithelial cells and cancer cells further supported this notion. Amplified IL-17RB/IL-17B signal activated Bcl-2 expression to exert anti-apoptotic effect through NF-κB pathway. Importantly, treatment with IL-17RB/IL-17B specific antibodies significantly reduced tumorigenicity of breast cancer cells. These data indicate that the amplified IL-17RB/IL-17B signaling contributes to breast tumorigenesis and offers a potential therapeutic target for breast cancer. . 14 .

(23) . Materials and Methods Cell lines Human breast cancer cell lines MCF7, MDA-MB-157, MDA-MB-231, MDAMB-361, MDA-MB-468, SKBR3 and SKBR3-hr were cultured in Dulbecco’s modified Eagle’s. medium. supplemented. with. 10%. fetal. bovine. serum. and. antibiotics/antimycotics. Non-malignant mammary epithelial cell lines H184B5F5/M10 (M10) and MCF 10A cells were cultured in Minimal Essential Medium supplemented with 10% fetal bovine serum and Dulbecco’s modified Eagle’s medium/F12 supplemented with 5% horse serum, 20 ng/ml epidermal growth factor, 0.5 μg/ml hydrocortisone, 100 ng/ml cholera toxin, 10 μg/ml insulin and antibiotics/antimycotics in a humidified 37℃ incubator supplemented with 5% CO2. H184B5F5/M10 cell line was purchased from Bioresource Collection and Research Center (BCRC) in Taiwan, and others were purchased from ATCC.. Clinical specimens All human samples were obtained from National Taiwan University Hospital (NTUH). The samples were encoded to protect patient confidentiality and used under protocols approved by the Institutional Review Board of Human Subjects Research Ethics Committee of Academia Sinica (AS-IRB02-98042) and NTUH, Taipei, Taiwan (#200902001R). Clinical information was obtained from pathology reports. Patients with at least 5 years follow-up were included in this study.. Soft agar colony formation assay In one well of a 12-well plate, 2500 cells were seeded in culture medium containing 0.35% agar on top of a layer of culture medium containing 0.5% agar (M10 . 15 .

(24) . cells also used MCF 10A culture medium in soft colony formation assay). Cells were maintained in a humidified 37℃ incubator for 16 days and colonies were fixed with ethanol containing 0.05% crystal violet for quantification. For addition of rIL-17B protein or IL-17B/IL-17RB neutralization assays, anti-human IL-17B (R&D Systems), antihuman IL-17RB antibodies or rIL-17B was added to the soft agar culture every 2 days.. Xenograft assay in NOD/SCID/γnull mice Animal care and experiments were approved by the Institutional Animal Care and Utilization Committee of Academia Sinica (IACUC#080085). 2 X 106 MDA-MB361 breast cancer cells mixed with equal volume of Matrigel (BD bioscience) were injected into NOD/SCID/γnull fat pads (Shultz et al., 2005). Tumor volumes were evaluated every 4 days after initial detection. Student’s t-test was used to test the significant differences between shLacZ, shIL-17RB, and shIL-17B cells derived tumor growth. In vivo administration of IL-17RB antibody was initiated when tumors reached 50-100 mm3, and the mice were divided into a same group with comparable tumor size. For each tumor, 10 μg of IL-17RB antibody in 20 μl sterile PBS was administrated by intratumoral injection. Non-linear regression (curve fit) was used to evaluate the statistical significance of tumor growth between control and treated mice in each group.. IL-17RB antibody Recombinant IL-17RB extracellular domain that carried only a single N-linked GlcNAc at each glycosylation sites was generated by ectopic overexpression in a suspension cell culture of N-acetylglucosaminyltransferase I-deficient (GnTI-) strain HEK293 cells (Reeves et al., 2002). The resulting N-glycans, GlcNAc2Man5, was then treated with endoglycosidase Endo H to remove residual glycans. Polyclonal antibody . 16 .

(25) . generated through this immunogen was used throughout the entire work.. Immunoblotting Immunoblot analysis was performed after 8% or 12% SDS-PAGE, with overnight incubation of 1:2000 dilution of mouse polyclonal anti-IL17RB, anti-Bcl2 (OP60T, Merck), anti-Caspase3 (IMG144A, IMGENEX), or 1:1000 dilution of anti-IL17B (MAB1248, R&D Systems), anti-TRAF6 (Sc-8409, Santa Cruz), anti-HER2 (GTX61656, Genetex) antibodies followed by a 1:10000 dilution of horseradish peroxidase-conjugated anti-mouse or anti-rabbit secondary antibody (GeneTex). Signals were detected using Immobilon Western Chemiluminescent HRP Substrate (Millipore). Protein concentration was determined by the Bradford assay (Bio-Rad) before loading and verified by α-tubulin level using a 1:10000 dilution of anti-α-tubulin antibody (GTX72360, GeneTex). The intensity of western blot bands was quantified using Image J software (NIH).. Immunohistochemistry Formalin-fixed paraffin embedded primary tumor tissue sections were used. Antigen retrieval was performed using EDTA buffer (Trilogy) heated for 10 min in a microwave. Endogenous peroxidase activity was eliminated by 3％ H2O2. The slides were blocked in PBS containing 10％ FBS and then incubated with purified mouse antiIL17RB polyclonal antibody (1:100) or anti-HER2 rabbit antibody (1:100) overnight at 4 ℃. HRP conjugated rabbit/mouse polymer (Dako REAL EnVision) and liquid diaminobenzidine tetrahydrochloride plus substrate (DAB chromogen) were used for visualization. All slides were counterstained with hematoxylin, and the images were taken using an Aperio Digital Pathology System. Samples were identified as IL-17RB positive if more than 5% of the tumor cells were positive for membrane staining. . 17 .

(26) . Three-dimensional morphogenesis Assay In a well of an eight-well chamber slides (Labtek, Nunc), approximately 5000 M10 or MCF 10A cells were seeded in growth medium supplemented with 2% Matrigel on top of a layer of Growth Factor Reduced Matrigel (BD Biosciences) as described in (Debnath et al., 2003). The 3D morphogenesis was monitored by fluorescence microscopy confocal sectioning at day 16 after seeding.. Co-immunoprecipitation Assay The whole cell protein extract was prepared using lysis buffer (10mM Tris-HCl, 150mM NaCl, 2mM MgCl2 and 1％ Triton X-100) at 4℃ and pre-cleaned with protein A/G beads (Santa Crus) for 60 min at 4℃. IL-17RB and TRAF6 were immunoprecipitated with 1 μg antibodies against IL-17RB and TRAF6 (8028, Cell Signaling), respectively, at 4℃ overnight. Normal mouse/rabbit IgG was used as a control. The immunoprecipitated protein complex were separated by SDS-PAGE, and followed by western blot analysis. In rIL-17B treatment experiment, the cells were serum starved for 6 h before treated with rIL-17B for 5 minutes.. NF-κB reporter assay Cells of 80% confluence were transfected using Lipofectamine 2000 (Invitrogen). For NF-κB reporter assay, 0.5 μg NF-κB luciferase reporter plasmid and 50 ng of the pGL4-74 Renilla luciferase plasmid (as a transfection efficiency control) were cotransfected into cells per well (24-well plate). Cell extracts were prepared at 24 h after transfection, and the luciferase activity was measured using the Dual-Glo Luciferase Assay System (Promega) following the manufacturer’s instruction. . 18 .

(27) . RNA isolation and reverse transcription Total RNA from cell culture or clinical specimens were isolated using Trizol reagent (Invitrogen) and reverse-transcribed with High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems) for gene expression analysis according to instructions from the manufacturers.. Real-time PCR assay Quantitative real-time RT–PCR was performed using Fast SYBR-Green master mix for gene expression according to the manufacturer’s instruction and analyzed on a StepOnePlus Real-Time PCR system (Applied Biosystems). The primer sequences were shown in Table S6, and β-actin mRNA was used as an internal control for mRNA expression. Relative expression levels were calculated according to the relative ΔCt method, and the specificity of each primer pairs were determined by dissociation curve analysis.. Recombinant human IL-17B protein expression and purification The plasmid that includes the full-length IL-17B with a C-terminal six-histidine tag was transfected into human embryonic kidney 293 EBNA cell by using polyethyleneimine (Durocher et al., 2002). Transiently transfected cell was cultured in Freestyle 293 expression medium (Invitrogen) at 37℃ for 96 h. The supernatant containing secreted IL-17B was purified by nickel-affinity chromatography following the manufacture’s procedure. IL-17B was further purified with Superdex 200 size-exclusion chromatography (GE Healthcare) equilibrated in 50 mM HEPES, pH 7.5, and 150 mM NaCl. . 19 .

(28) . Stable cell lines for IL-17RB The cDNA clones of human IL-17RB-FL in pCMV6-XL5 and IL-17RB1 in pCMV-SPORT6 vector were obtained from OriGene and mammalian gene collection then cloned into pQCXIH retroviral vector (Clontech). The expression construct of IL17RB2 was generated from IL-17RB-FL and cloned into pQCXIP retroviral vector (Clontech). GP2-293 retrovirus packaging cells were cotransfected with IL-17RB variants containing retroviral plasmids and pVSVG plasmids using Lipofectamine 2000 (Invitrogen) following the manufacturer’s instruction. The retrovirus supernatant was harvested in the conditioned medium after 24 h post transfection. The M10 and MCF 10A cells were infected with retrovirus with addition of 8 μg/ml polybrene and then selected with 200 μg/ml Hygromycin B (Sigma) or 1 μg/ml puromycin (Sigma) to establish stable cell lines.. IL-17RB/IL-17B knockdown experiment For the knockdown of endogenous IL-17RB/IL-17B, the lentiviral vector carrying IL-17RB/IL-17B specific shRNA was obtained from National RNAi Core Facility (Academia. Sinica,. Taiwan).. The. target. CCATTAAGGTTCTTGTGGTTT-3’. for. human. sequence IL-17RB. were. 5’-. and. 5’-. TCTTACCATTTCCATCTTCCT-3’ for human IL-17B. shRNA against β-galactosidase was used as a negative control (shLacZ). For lentivirus production, HEK-293T cells were cotransfected with shRNA containing lentiviral vector, envelope plasmid pMD.G and packaging plasmid pCMVΔR8.91. Virus containing supernatant was harvested at 24 and 48 h post transfection. MDA-MB-361and MCF7 cells were infected with lentivirus and then selected with 1 and 2 μg/ml puromycin, respectively. . 20 .

(29) . Statistical methods The association between IL-17RB gene expression and survival in breast cancer patients was evaluated using univariate Cox proportional-hazards regression analysis. Multivariate Cox regression analysis was used to adjust the association between survival and IL-17RB gene expression level for varying clinical parameters including age, tumor size, lymph-node status, tumor grade and estrogen receptor expression. Hazard ratio was evaluated using the method of Grambsch and Therneau. No violation of the proportional assumption was detected. The optimal cut-off value of IL-17RB gene expression level for 5-year survival was determined using ROC (receiver operating characteristic) analysis. Kaplan-Meier method was plotted and log-rank test was used to evaluate the statistical significance between patients with high and low IL-17RB expression in survival.. . 21 .

(30) . Results High expression of IL-17RB promotes breast tumorigenesis We first examined the expression of IL-17RB in a panel of non-malignant mammary epithelial cells (H184B5F5/M10 and MCF 10A) and breast cancer cell lines (MCF7, MDA-MB-157, MDA-MB-231, MDA-MB-361 and MDA-MB-468) by Western blot and RT-PCR. Elevated expression of IL-17RB protein and mRNA were predominantly observed in many breast cancer cell lines (Fig. II-1A and 1B). Depletion of IL-17RB by its corresponding shRNA in two cell lines, MDA-MB-361 and MCF7, highly expressing IL-17RB (Fig. II-1C), resulted in a significant decrease in soft-agar colony formation (Fig. II-1D). IL-17RB depletion also significantly retarded tumor growth in a xenograft model using NOD/SCID/γnull mice (Fig. II-1E). Palpable tumors derived from the control (shLacZ) and IL-17RB depleted cells (sh17RB) were both observed in the first week. However, from Day 20 to 36, tumors from control cells grew faster and larger than those from IL-17RB depleted cells (Fig. II-1E). The wet weights of the tumors derived from IL-17RB depleted cells were only 40% of those from the control cells (Fig. II-1F and 1G), indicating that high expression of IL-17RB promotes tumor growth.. Membrane bound IL-17RB is critical for promoting breast tumorigenesis The gene of human IL-17RB encodes two alternative spliced isoforms. Isoform 1 contains a transmembrane domain (refers to IL-17RB1 hereafter), and isoform 2 (IL17RB2) is a secreted form without the transmembrane domain (Tian et al., 2000). The IL-17RB full-length (IL-17RB-FL) cDNA mainly transcribed IL-17RB1 and a very small amount of IL-17RB2 due to harboring an intron inside (Tian et al., 2000). To pinpoint which isoform is critical for breast tumorigenesis, the non-malignant mammary. . 22 .

(31) . epithelial cell line, M10, was transduced with retrovirus carrying IL-17RB1, IL-17RB2 or IL-17RB-FL, respectively (Fig. II-2A). These cells were seeded in the threedimensional Matrigel culture for testing their acinar forming activity. The control and IL-17RB2 overexpressing M10 cells formed acinar structure with normal hollow lumens, but M10 cells expressing IL-17RB1 or IL-17RB-FL failed to develop a proper lumenlike structure (Fig. II-2B and 2C). In addition, only cells expressing the membrane bound IL-17RB promoted colony formation (Fig. II-2D). These results indicated that overexpression of the membrane bound IL-17RB1 contributes to the transformation of normal cells to cancerous phenotypes.. IL-17RB/IL-17B signaling activates NF-κB pathway and exerts anti-apoptosis via up-regulation of Bcl-2 To elucidate how IL-17RB promotes tumorigenesis in breast cancer, we performed differential expression profiling using IL-17RB overexpressing M10 cells and IL-17RB depleted MDA-MB-361 cells. We found 72 up-regulated and 70 downregulated genes with differential expression ratio greater than 1.5 fold (Table II-1 and 2). Use of the bioinformatics database, DAVID (http://david.abcc.ncifcrf.gov/) and KEGG, we found that apoptosis and focal adhesion pathway were most likely regulated by IL17RB (Fig. II-3). The pro-apoptotic genes TNFSF10 (Wiley et al., 1995) and TRADD (Baker and Reddy, 1998) were up regulated in IL-17RB depleted cells. Conversely, antiapoptotic gene Bcl2 (Catz and Johnson, 2001) was up regulated in IL-17RB overexpressing cells (Fig. II-4A). These results were further confirmed by real-time quantitative PCR (Q-PCR) and western blot analyses (Fig. II-4B and 4C). Since IL17RB signaling activates NF-κB in human renal cell line (Lee et al., 2001) and NF-κB up-regulates Bcl-2 (Catz and Johnson, 2001), in breast cancer cells, it is likely that . 23 .

(32) . overexpression of IL-17RB may block apoptosis via NF-κB-mediated Bcl-2 upregulation in breast cancer cells. To test this possibility, we performed NF-κB reporter assay and found that the NF-κB promoter activity was up-regulated in IL-17RB1 and IL17RB-FL overexpressing cells, but downregulated in IL-17RB depleted MDA-MB-361 cells (Fig. II-4D). Furthermore, Bcl-2, but not other NF-κB downstream pro-survival genes including XIAP (Tang et al., 2001) and Survivin (Ambrosini et al., 1997), were up-regulated in IL-17RB1 and IL-17RB-FL overexpressing cells (Fig. II-4B, 4C and 5). When treating with the cytotoxic agent, etoposide (VP-16, topoisomerase II inhibitor), activation of apoptotic marker cleavage caspase-3 was reduced in IL-17RB overexpressing cells compared with the control (Fig. II-6A). In contrast, caspase-3 activation was enhanced in IL-17RB depleted cells (Fig. II-6B). These results suggested that overexpression of IL-17RB inhibited apoptosis via NF-κB-mediated Bcl-2 upregulation.. IL-17B enhances tumorigenic activity through IL-17RB IL-17B, the ligand of IL-17RB, was expressed in both normal and tumor cells by RT-PCR (Fig. II-7A); however, the level of the secreted ligand was barely detectable by ELISA. To test whether ectopic addition of IL-17B enhances tumorigenic activity of breast cancer cells, we generated recombinant IL-17B (rIL-17B) protein from mammalian cell expressing system (Fig. II-7B). Supplement with rIL-17B increased the colony formation of MDA-MB-361 cells, which express high endogenous IL-17RB, in a dose-dependent manner (Fig. II-7C). Similar results were also observed in M10 cells expressing IL-17RB-FL, but not the control (Fig. II-7D). In contrast, depletion of the endogenous IL-17B in MDA-MB-361 cells (Fig. II-8A) not only inhibited the colony formation (Fig. II-8B) but also decreased the NF-κB reporter activity (Fig. II-8C) and . 24 .

(33) . Bcl2 expression (Fig. II-8D). Consistently, the tumor size and weight were both reduced in IL-17B knockdown cells compared to shLacZ control in the xenograft model (Fig. II8E and 8F). These findings suggested that IL-17B contributes to breast tumorigenesis specifically via IL-17RB.. IL-17B signaling activates NF-κB by enhancing TRAF6 recruitment to IL-17RB Based on bioinformatics and amino acid sequences analysis in IL-17RA and IL17RB, we determined two types of putative functional domains which may involve in IL-17RB signaling: (1) the extracellular ligand binding domains (LBD) (Thr28-Leu36, Thr89-Ser96, and Ser259-His264, Fig. II-9), and (2) the intracellular TRAF6 binding domain (from Pro339-Glu341), which is critical for IL-17RB signaling transduction (Maezawa et al., 2006). To affirm that IL-17B signal transduces through IL-17RB, two mutants, ΔLBD, deleted with ligand-binding domain of Thr89-Ser96 and the other, ΔTRAF6, deleted TRAF6 binding domain, were generated (Fig. II-10A). The expressions of these two mutants were comparable in M10 cells (Fig. II-10B). Compared with the wild-type receptor, expression of these two mutants abolished IL-17RB signaling leading to the reduction of colony formation and NF-κB promoter activity (Fig. II-10C and 10D). Similarly, unlike the wild-type receptor, acinus formation of M10 cells expressing these mutants appeared to be unaffected (Fig. II-10E and 10F). Consistently, addition of rIL-17B to those cells failed to enhance their colony formation (Fig. II-10G). To trace the downstream effectors of this signaling, we tested whether IL-17B promotes the recruitment of TRAF6 to IL-17RB. TRAF6 is the factor directly binding to the TRAF6 binding domain in IL-17RB receptor upon ligand addition. This recruitment is also critical for the NF-κB signaling transduction (Maezawa et al., 2006; Ye et al., 2002). Upon rIL-17B treatment, the association between IL-17RB and TRAF6 was . 25 .

(34) . increased in a dose-dependent manner (Fig. II-11A). In contrast, both ΔTRAF6 and ΔLBD mutants failed to recruit TRAF6 (Fig. II-11B). These results suggested that IL17B bound to the extracellular domain of IL-17RB and transduced the signal through its intracellular domain by recruiting TRAF6 to activate NF-κB activity.. Antibodies targeting to IL-17RB/IL-17B inhibit tumorigenicity of breast cancer cells expressing IL-17RB To further assess the importance of the IL-17RB/IL-17B signaling, we used antibodies specific to IL-17RB and IL-17B to examine their biological consequences. Addition of IL-17B antibodies to the M10 cells expressing IL-17RB or MDA-MB-361 cells inhibited their colony forming activity (Fig. II-12A and 12B). Similarly, addition of IL-17RB antibody inhibited colony formation of MDA-MB-361 cells (Fig. II-12C). Furthermore, treatment with IL-17RB antibodies retarded tumor growth of MDA-MB361 cells in the xenograft model (Fig. II-12D). These results suggested that disruption of IL-17RB/IL-17B signaling inhibits breast tumorigenicity and use of the specific antibodies may provide a potential therapeutic strategy to treat IL-17RB positive breast cancer (Fig. II-13).. Elevated IL-17RB expression has a stronger correlation with poor prognosis than HER2 positive breast cancer. In a cohort with limited number of patients (69 patients), it was shown that the elevated expression of IL17RB is correlated with poor prognosis (Furuta et al., 2011). To affirm this previous observation, an independent larger cohort of 179 breast cancer patients (Table II-3) was further examined by immunohistochemistry (IHC). Consistently, elevated IL-17RB expression was correlated with poor prognosis (Fig. II . 26 .

(35) . 14A and 14B, p = 0.02). The correlation between IL-17RB expression and poor prognosis was statistically significant even adjusted with several clinical parameters including age, tumor size, lymph node status, and ER expression (Fig. II-14C). In addition, we also performed Q-PCR to measure IL-17RB isoform 1 transcripts amount in another independent cohort of 104 clinical breast cancer specimens (Table II-3) and used (-ΔCt=-7.55) as a cut-off value based on a ROC (Receiver operating characteristic) curve analysis to define “high or low” IL-17RB1 expression. Kaplan-Meier (KM) analysis showed that patients with high IL-17RB1 expression had a shorter survival compared to patients with low IL-17RB1 expression (Fig. II-14D, p = 0.03). The association of IL17RB1 expression and poor prognosis was statistically significant after adjusted with age, tumor size, lymph node status, grade as well as ER expression (Table II-4). These results suggest that high expression of IL-17RB1 may serve as a poor prognosis marker for breast cancer patients. Intriguingly, we found that IL-17RB expression was associated with HER2 amplification in breast cancer specimens (Table II-5). The coexistence of IL-17RB and HER2 overexpression was further affirmed by IHC in the serial paraffin embedded breast cancer tissue sections (Fig. II-15A). Patients with both high IL-17RB expression and HER2 amplification had the shortest survival rate (Fig. II-15B). Interestingly, when we compared the IL-17RB or HER2 positive group with the double negative group of patients, elevated expression of IL-17RB showed a stronger correlation to poor prognosis than HER2 amplification (Fig. II-15C). Both of these correlations were strengthened (Fig. II-15D) when the triple negative patients, who have the worst prognosis(VargoGogola and Rosen, 2007), were excluded from the cohort. These findings suggest that IL-17RB may serve as an alternative target for patients that have both HER2 amplification as well as IL-17RB expression. . 27 .