Sin1 promotes proliferation and invasion of prostate cancer cells by modulating mTORC2-AKT and AR signaling cascades

Journal Pre-proof Sin1 promotes proliferation and invasion of prostate Cancer cells by modulating mTORC2-AKT and AR signaling cascades

Yunchuanxiang Huang, Guanying Feng, Jingshu Cai, Qian Peng, Zhenglin Yang, Chunhong Yan, Lu Yang, Ziyan Wang PII:

S0024-3205(20)30197-1

DOI:

https://doi.org/10.1016/j.lfs.2020.117449

Reference:

LFS 117449

To appear in:

Life Sciences

Received date:

4 December 2019

Revised date:

9 February 2020

Accepted date:

17 February 2020

Please cite this article as: Y. Huang, G. Feng, J. Cai, et al., Sin1 promotes proliferation and invasion of prostate Cancer cells by modulating mTORC2-AKT and AR signaling cascades, Life Sciences(2020), https://doi.org/10.1016/j.lfs.2020.117449

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© 2020 Published by Elsevier.

Journal Pre-proof Sin1 Promotes Proliferation and Invasion of Prostate Cancer Cells by Modulating mTORC2-AKT and AR Signaling Cascades Author: Yunchuanxiang Huang1,*, Guanying Feng1,*, Jingshu Cai1, Qian Peng2, Zhenglin Yang3,4, Chunhong Yan5,6,#, Lu Yang1,# and Ziyan Wang3,4,# 1 School of Medicine, University of Electronic Science and Technology of China, Chengdu, China

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2 Sichuan Cancer Hospital & Institute, Sichuan Cancer Center, School of Medicine, University of Electronic Science and Technology of China, Chengdu, China

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3 Key Laboratory for Human Disease Gene Study, Sichuan Academy of Medical Sciences and Sichuan Provincial People’s Hospital, School of Medicine, University of Electronic Science and Technology of China, Chengdu, China

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4 Research Unit for Blindness Prevention of Chinese Academy of Medical Science (2019RU026), Sichuan Academy of Medical Science & Sichuan Provincial People’s Hospital, Chengdu, Sichuan, China 5 Georgia Cancer Center, Augusta University, Augusta, GA, USA

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6 Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta University, Augusta, GA, USA. * Those two authors contributed equally to this work. # Those authors are corresponding authors. Correspondence: Dr. Ziyan Wang or Dr. Lu Yang, School of Medicine, University of Electronic Science and Technology of China, Chengdu, China E-mail: [email protected] & [email protected] Abstract: Aims: Prostate cancer (PCa) is the most common type of cancer and a major cause of death in men worldwide. Aberrant Androgen receptor (AR) and PI3K-AKT

Journal Pre-proof signaling are very frequent in PCa patients and, therefore, considered as therapeutic targets in the clinic. Sin1 is an essential component of mTORC2 complex, which determines full AKT activation and PCa development in PTEN-/- mice. Here we examined the role of Sin1 in human PCa cell lines and respective tumor samples. Main methods: Western blotting and immunohistochemistry (IHC) were performed to analyze the expression of Sin1-mTORC2-AKT related proteins in human PCa cells, as

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well as prostate tumors and normal tissue counterparts. Cell viability and

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invasion assays were also pursued in the presence or not of Sin1 in PCa cells. Immunoprecipitation assays were additionally carried out to examine the

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interaction of Sin1 with AR.

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Key findings:

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We have presently demonstrated that high levels of Sin1 expression in human PCa tissues correlate with cancer progression. Sin1-mediated cell proliferation

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and invasion of PCa cells occurs by regulating mTORC2-AKT signaling, epithelial-mesenchymal transition and matrix metalloproteinases. Moreover,

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androgens are able to induce Sin1 expression, which is further translocated to the nucleus of PCa cells. Finally, Sin1 interacts with AR to suppress its transcriptional activity. Significance:

Taken together, these data indicate that both Sin1-mediated mTORC2-AKT signaling and Sin1-AR interaction regulate PCa development. Hence, Sin1 may be considered a novel biomarker of PCa progression. Keywords: Sin1, AR, mTORC2, AKT, Prostate Cancer Introduction: Multiple risk factors, including genetic alterations, high-fat diet and aging, participate in promoting the initiation and development of prostate cancer

Journal Pre-proof (PCa). So far, PCa is still considered the most common type of malignancy and one of the major causes of death in men. In western countries, like United States and Australia, its incidence and mortality are immediately below skin cancer1,2. Contrarily, the incidence of PCa is relatively lower in eastern countries, including China. Between 1994 and 2002, the annual growth rate of PCa in China reached 13.4 (age standardized rate per 1 million population), as compared with 2.1 between 1988 to 1994. This representative difference was mostly due to changes on life style as well as the percentage of the aging

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population3,4.

Androgen receptor (AR) signaling is largely responsible by controling the

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growth of prostate gland as well as PCa5,6. By acting at the classical transcriptional level, androgen-bound AR translocates to the nucleus and

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binds AR-responsive DNA elements to further regulate gene transcription 7.

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Additionally, androgen-bound AR has an alternate non-transcriptional role towards MAPKs activation8. Androgen can also trigger the interaction of AR

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with filamin A to regulate cell motility and adhesion9. Based on the mechanistic role of AR in PCa, current standard treatment of localized or advanced

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prostate PCa initially involves androgen deprivation therapy (ADT) 10,11. However, due to the feedback activation of PI3K-AKT signaling upon androgen deprivation, a more aggressive condition (i.e. castration-resistant prostate cancer,CRPC) might eventually evolve12,13. The PI3K/AKT pathway is a major signaling pathway, which is frequently altered during cancer progression14. Currently, a number of cancer treatments are based on PI3K-AKT inhibition to further achieve the expected therapeutic effects15,16. The full activation of AKT requires phosphorylation at two distinct protein sites17,18. PDK1 phosphorylates AKT at position T308 in the kinase domain, while mTORC2 phosphorylates AKT at S473 in the C-terminal hydrophobic motif19. The mTOR kinase, which is also a target of rapamycin,

Journal Pre-proof assembles into two distinct complexes, called mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2)20,21. In addition to mTOR, mTORC1 contains Raptor, PRAS40 and mLST8/GβL and regulates cell growth by modulating the activity of S6 kinase and 4E-BP protein. On the other hand, mTORC2 has three special components (RICTOR, Sin1 and PROTOR), binds to different substrates and plays an indispensable role in cell growth, proliferation and metabolism22,23. mTORC1 and mTORC2 seem to play distinct roles in PCa progression. For instance, mTORC2 activity is not necessary for normal

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prostate epithelial cells, but is critical for the development of prostate cancer

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caused by PTEN deletion24. On the other hand, targeting mTORC1 by Rapamycin to inhibit PI3K-AKT signaling in PCa cells typically increases AR

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transcriptional activity25. These observations suggest that mTORC1 and

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mTORC2 have different regulatory features in AR signaling.

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Sin1 is one of the key components of mTORC2. In fact, recent studies have revealed that Sin1 is crucial for the integrity and activity of mTORC2 26.

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Ubiquitination of GβL can disrupt the interaction between Sin1 and mTORC2, which will lead to mTORC1 organization27. In contrast, de-ubiquitination of GβL

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promote mTORC2 organization. These findings indicate that there is a dynamic equilibrium between mTORC1 and mTORC2, which ultimately determines AKT activation. Therefore, Sin1 is on the interface of mTORC1, mTORC2 and AKT.

Sin1 is highly expressed in breast, colon and liver cancers28,29,30, and it is involved in regulating cell invasion and EMT 29. Sin1 overexpression can decrease the anti-cancer effect of Nitidine Chloride31. Recent studies have revealed that Sin1 is also expressed in PCa cells, where it promotes tumor growth by regulating the assembly and activity of mTORC2 32,33. These data point out that Sin1 may participate in the progression of multiple types of

Journal Pre-proof cancer by affecting mTORC2-AKT signaling. Consequently, the detailed characterization of Sin1’s role in prostate cancer is warranted. In the present study, we examined the role of Sin1 in human PCa cell lines and related tumor samples. We reveal that Sin1 regulates cell proliferation and invasion by activating mTORC2-AKT pathway. Additionally, Sin1 can translocate to nucleus and interact with AR to inhibit the transcription of AR responsive genes, therefore forming a negative feedback loop between AKT

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and AR pathways. Finally, Sin1 is highly expressed in human PCa tissues,

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suggesting its potential use as a novel PCa biomarker.

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Results:

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High Sin1 expression is a putative biomarker for prostate cancer

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Current studies have indicated that Sin1 is involved in the progression of multiple cancers28,29,30. In this context, we first analyzed Sin1 expression data

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retrieved from the Oncomine database. We found that high expression of Sin1 was closely related to PCa progression. Compared to normal tissues, Sin1

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was highly expressed in tumor tissues (Fig. S1A). Interestingly, Sin1 expression gradually elevated from T0 to T3 (Fig. S1B), reiterating the correlation with cancer progression. To further explore the profile of Sin1 levels in PCa tissues, IHC staining was performed in 3 normal, 67 paired-normal and 137 human prostate cancer tissues (Table 1). As indicated, Sin1 protein levels were increased in prostate tumors when compared with respective normal tissues (Fig. 1C). In particular, the intensity of Sin1 staining was significantly higher in 43 out of 56 prostate tumor samples (76.8%) (Fig. 1A). Notably, Sin1 levels enhanced according to the prostate pathological grading (Gleason score). Prostate tumor samples of Gleason 7-8 showed much higher Sin1 staining as compared to samples of Gleason 6 (Fig. 1D), indicating that Sin1 may promote the early progression of prostate tumors. However, due to lack of

Journal Pre-proof tumor samples at Gleason stage 9-10, we could not confirm whether Sin1 participates during the whole process of PCa development. Prostate gland is mainly made up of basal and luminal epithelial cells, which both contribute to PCa progression34. To examine where Sin1 is expressed in human prostate, we carefully evaluated the location of Sin1 in stained prostate sections. We observed that Sin1 was expressed in basal (red arrow) and luminal cells (black arrow), with a strong staining both in the cytoplasm (red

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arrow head) and nucleus (black arrow head) (Fig. S2). In accordance with the

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Sin1 function in the cytoplasmic AKT activation, the majority of Sin1 positive sections showed cytoplasmic staining, but 35 out of 137 tumor samples

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(25.5%) exhibited a strong nuclear Sin1 staining (Figs. 1B and 1E), suggesting

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that Sin1 may have putative nuclear effects. Altogether, these results reveal

Sin1

enhances

cell

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that Sin1 plays an important role in PCa progression. proliferation

and

invasion

by

regulating

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mTORC2-AKT pathway and epithelial-to-mesenchymal transition (EMT) Both Sin1 mRNA and protein are highly detected in several human tumor

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tissues and multiple cancer cell lines28-30, suggesting a potential role of Sin1 in tumor development and progression. Some studies have provided strong evidence for the role of Sin1 in tumor proliferation and invasion 29. We have also found that Sin1 expression was correlated with the aggressiveness of PCa (Figs. S1D-S1G). Therefore, we examined the regulatory effect of Sin1 in the viability and invasive ability of PCa cell lines (LNCaP, PC3 and C4-2), using MTT and invasion assays. We have found that Sin1 overexpression increases cell viability and invasion capability of LNCaP and PC3 cells, which typically express low levels of endogenous Sin1. Consistently, knocking down Sin1 expression by siRNAs blocked cell proliferation and invasion (Figs. 2A-2C and 3C). Since C4-2 cells have higher Sin1 levels (Fig. S3A), increased levels of exogenous Sin1 did not affect cell proliferation but enhanced cell invasion,

Journal Pre-proof while Sin1 knockdown dramatically inhibited cell proliferation and invasion of C4-2 cells (Figs. 3A-3C). Sin1 is an important component of mTORC2 and regulates the assembly of mTORC2 complex, which further activates AKT to promote cell survival 35. To determine whether Sin1 induces the proliferation of PCa cells via mTOCR2-AKT signaling, we examined the mTORC2 assembly and AKT activation in C4-2 and LNCaP cells, respectively. Western blot analysis

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showed a dramatic upregulation of mTOR, phosphorylation of Sin1 and Rictor,

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accompanied by an elevation of phosphorylated AKT and a slight increase of Rictor in Sin1-overexpressing LNCaP cells (Fig. 2D). In contrast, mTORC2

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and AKT signaling decreased in Sin1 knockdown C4-2 cells (Fig. 3D).

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Epithelial-mesenchymal transition (EMT) plays an important role in the

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conversion of primary PCa into an invasive malignancy36,37. Typically, epithelial cells undergoing EMT lose specific markers, such as E-cadherin, and instead

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acquire the expression of mesenchymal markers, including Vimentin and Snail. Sin1 is known to promote EMT in liver cancer29. Thus, we first investigated the

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mRNA levels of EMT-related markers in Sin1-overexpressing PC3 cells. The result indicated that Sin1 overexpression decreases the mRNA levels of E-cadherin and, instead, increases Vimentin and Snail mRNAs (Figure 2E). To further determine the mechanism by which Sin1 regulates EMT, we treated PC3 cells with inhibitors of AKT (MK-2206) or mTOR (Rapamycin) after transient overexpression of Sin1 in vitro. The protein levels of Vimentin increased upon Sin1 overexpression but, interestingly, could be inhibited by MK-2206 and Rapamycin treatment (Figs. 2G and 2H). Matrix metalloproteinases (MMPs) also participate in promoting prostate cancer invasion and metastasis38,39. After overexpressing Sin1 in LNCaP cells, we carried out qRT-PCR assays to detect the expression of MMP family members. No change on MMP7 and MMP13 expression was observed but,

Journal Pre-proof contrarily, we found a significantly increased expression of other MMPs, including MMP1, MMP3 and MMP10 (Fig. 2F). Collectively, these results demonstrate that Sin1 is crucial for cell proliferation and invasion by regulating EMT and mTORC2-AKT signaling. Sin1 is transcriptionally regulated by androgens in androgen-dependent PCa cells Prostate epithelial proliferation is strongly associated with the hormonal activity

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of androgens34 . To test whether Sin1 expression is induced by androgens, we

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treated LNCaP and C4-2 cells with a synthetic androgen (R1881) for further

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immunoblotting analyses. As expected, R1881 was able to induce the expression of the AR target gene NKX3.1. At the same time, we also found

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elevated Sin1 levels in LNCaP cells as previously reported in CWR22R3

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cells33. Inaugurally, we have also shown an increased phosphorylation of Sin1 (Fig. 4A) and a rapidly enhanced Sin1 mRNA level (Fig. 4C), suggesting that

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androgens can induce Sin1 at the transcriptional level. To explore this notion, we carried out dual-luciferase activity assays using the Sin1 promoter.

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Accordingly, the activity of the Sin1 promoter was significantly elevated upon R1881 stimulation in LNCaP cells (Figure 4D). To confirm the direct regulation of Sin1 expression by AR signaling, AR antagonist MDV3100 was further utilized. After adding MDV3100 to R1881-stimulated LNCaP cells, the androgen-induced Sin1 protein and mRNA expression were equally inhibited (Figs. 4E, 4F and 4G). In contrast to the androgen-dependent LNCaP cells, Sin1

was

constitutively

expressed

and

phosphorylated

in

the

castration-resistant C4-2 cells (Fig. 4B). Thus, our data indicate that AR signaling plays a vital role in the regulation of Sin1 expression, as well as its phosphorylation, in androgen-dependent PCa cells. Androgen increases the stability and nuclear accumulation of Sin1 protein in androgen-dependent but not in castration-resistant PCa cells

Journal Pre-proof Previous studies revealed that dihydrotestosterone increases Sin1 stabilization and nuclear accumulation, both in castration-resistant CWR22R3 cells and androgen-dependent LNCaP cells, affecting the assembly and activity of the mTORC2 complex33. In this regard, here we obtained some contrasting data. For this, LNCaP and C4-2 cells were treated with cycloheximide (CHX) to block protein synthesis, after 2-hour stimulation with R1881. The levels of Sin1 protein were then examined for 48 hours after treatment. As indicated, Sin1 levels decreased in DMSO-treated LNCaP cells, while R1881-stimulated cells

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sustained Sin1 protein levels (Figs. 5A and 5B). Contrarily, Sin1 protein was

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continuous degraded in castration-resistant C4-2 cells, independently of

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DMSO or R1881 treatment (Figs. 5C and 5D).

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To clarify whether Sin1 nuclear translocation causes an increase on protein stability, we examined the sub-cellular distribution of Sin1 after R1881

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stimulation in both LNCaP and C4-2 cells. We observed an increase on Sin1 levels (both total and phosphorylated Sin1) in LNCaP cells, followed by

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increased yields of Rictor and mTOR (mTORC2 components) as well as phosphorylation of Ser-473 AKT, both in the cytosolic and nuclear fractions

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(Figs. 6A and 6B). Conversely, R1881 did not affect the distribution of Sin1 in C4-2 cells. In fact, independently of any androgen treatment, high levels of Sin1 were detected in the cytoplasm of C4-2 cells, potentially recruiting more Rictor and mTOR proteins towards the assembly of mTORC2 and AKT activation (Figs. 6C and 6D). Altogether, our data demonstrates that Sin1 is protected from androgen-dependent degradation only in androgen-dependent PCa cells (Figs. 5 and 6). Sin1 interacts with androgen receptor to regulate its transcriptional activity Considering that Sin1 is able to translocate into the nucleus upon androgen stimulation, we hypothesized that Sin1 could interact with AR. To explore the

Journal Pre-proof probability of a Sin1-AR interaction, co-immunoprecipitation assays were carried out using HEK293T cells co-transfected with AR-His and Sin1-DDK constructs. Co-immunoprecipitation of ectopic Sin1 and AR proteins could be detected using either anti-His or anti-DDK antibodies (Figs. 7A and 7B). Furthermore, we confirmed the endogenous interaction between AR and Sin1 in LNCaP (Fig. 7C) and C4-2 cells (Fig. 7D). Since Sin1 expression can be induced and translocated into nucleus upon exposure to androgens, we speculated whether the interaction between AR and Sin1 would be also

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enhanced. Upon R1881 treatment, the Sin1-AR interaction in LNCaP cells was

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dramatically increased (Fig. 7C). The median interaction between Sin1 and AR in C4-2 cells was also detected (Fig. 7D), which indicated that this interaction

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might occur out of the nucleus. To further determine the putative role of

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Sin1-AR interaction, we carried out AR-mediated luciferase reporter assays to

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verify whether Sin1 could affect AR transcriptional activity. We revealed that Sin1 overexpression, combined with R1881 stimulation, suppressed AR

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transactivation (Fig. 7E). At the same time, overexpression of Sin1 in LNCaP cells downregulated the transcription levels of AR-target genes, including PSA,

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NKX3.1 and TMRPSS2 (Fig. 7F). Taken together, this data suggest that Sin1 interacts with AR to further inhibit its transcriptional activity. Discussion

Despite the great potential of targeting of androgen/AR axis to control the growth and progression of primary PCa, the emergence CRPC is still a major issue in the therapeutic area5. A number of preclinical and clinical studies are currently aiming to enhance the therapeutic effect(s) of ADT as well as to investigate the molecular mechanisms of PCa progression and hormone resistance10 . Considering the abnormally activated PI3K-AKT signaling, induced by PTEN loss in the majority of PC patients, the suppression of cancer cell growth by PI3K or AKT inhibitors suggests an alternate revenue for CRPC

Journal Pre-proof treatment. Still, PI3K-AKT inhibition recalls AR activation, where PTEN deficiency appears to have a major role12. As previously mentioned, a complete AKT activation requires its phosphorylation at S473 by mTORC2. Hence, mTORC2 is critical for the development of PCa caused by PTEN loss24. The mTORC2 complex should also be involved in castration resistance due loss of PTEN function. Sin1 has been well described to regulate the integrity and activity of mTORC2

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via phosphorylation of two threonine residues (T86 and T398) at the N-terminus and PH domain, respectively26,40. Recent studies have revealed

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that ubiquitination and de-ubiquitination of GβL can regulate the interaction

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between Sin1 and mTORC2 and, ultimately, affects AKT activation 27. Thus, a

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dynamic equilibrium between mTORC1 and mTORC2 appears to be modulated by Sin1. Here we show that Sin1, a key component of mTORC2, is

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regulated transcriptionally by androgen-dependent AR signaling in PCa. Changes on Sin1 levels, after DHT stimulation in vitro, have been previously

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demonstrated33 . Nevertheless, we presently demonstrate, for the first time, that both Sin1 protein and mRNA levels increase upon stimulation with R1881.

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In addition, consistent with previous reports33 , we could exhibit that Sin1 is able to translocate into nucleus of androgen-dependent LNCaP cells. Increased Sin1 recruits mTOR and Rictor (both in cytoplasm and nucleus) to form mTORC2 and then activate downstream AKT signaling to boost cell proliferation. Besides the structural role as a mTORC2 component, Sin1 can also activate AKT by interacting with other proteins. Indeed, Sin1 can interact with DNA-PKCs to mediate X-ray-induced AKT activation and osteoclast differentiation, and to promote UV-induced survival of skin cells41,42. In the present study, we detected, for the first time, a direct interaction between Sin1 and AR. As a crucial partner of the mTORC2-AKT pathway, Sin1 also takes

Journal Pre-proof part in suppressing AR transcriptional activity, forming a negative feedback loop between AKT and AR signaling cascades. In general, Sin1 acts via mTORC2-AKT to promote cell growth in cancer cells and, based on current evidences, it has considered as an oncogene. Besides, Sin1 has also been reported to act as ATF2 or p38 co-factor to regulate gene transcription in several cell types, which could possibly lead to other unknown functions 42,43. Furthermore, we detected increased levels of PDCD4 mRNA while inhibiting

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Sin1 with the androgen receptor antagonist MDV3100 (Figure S4). PDCD4

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has been reported to regulate Sin1 translation in colon cancer44, and AR signaling can repress PDCD4 expression by miR-21 to promote PCa growth

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and castration resistance45. Based on our results, AR signaling appears to also

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regulate Sin1 expression in PCa via the miR21-PDCD4 axis.

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In contrast to previous reports33 , we verified that castration resistant C4-2 cells express a consistently higher level of total and phosphorylated Sin1. Protein

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degradation and nuclear accumulation of Sin1 in C4-2 cells are not altered upon androgen stimulation. These contrasting results may be due to the use of

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distinct prostate cell lines. Respectively, CWR22R3 and C4-2 cells are originated from androgen-dependent CWR22 and LNCaP which underwent androgen-depleted

condition

and

then

acquired

androgen-refractory

characteristics46,47. Still, CWR22R3 cells present higher phosphorylation of ERK and detectable PTEN expression, but no PI3K-AKT activation and PTEN loss as compared to LNCaP. Since mTORC2 has also been involved in PTEN-deficiency PCa24, we speculated whether the function of Sin1 could also depend on PTEN loss. We have also detected high Sin1 levels in DU145 cells (containing PTEN heterozygosis), while C4-2 cells presented the highest Sin1 levels (Fig. S3A), indicating that Sin1 may play a decisive role in promoting cell survival even after ADT. We also detected some medium Sin1-AR interaction in C4-2 cells, even without androgen stimulation. These findings suggest that

Journal Pre-proof Sin1 can bind to cytoplasmic AR. Protein interaction with extranuclear AR is also shown in binding to filamin A in NIH3T3 and HT1080 cells to enhance cell motility48. Since C4-2 cells are not responsive to exogenous androgens, we assumed that Sin1 could bind to AR variants located in cytoplasm. Since the full-length AR shares some common sequence with AR variants 49, the specific binding domain of AR with Sin1 needs further evaluation. The prostate tissue samples used in our study were derived from Chinese

50,51

could impact the function of Sin1 in PCa. Analysis of Sin1

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heterogeneity

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population. Hence, racial differences related to tumor incidence and

expression in PCa patients by RNA-Seq indicated a differential expression

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pattern in Caucasian and African-americans. Caucasians presented a

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relatively higher Sin1 level, while African-americans showed lower levels (Fig.

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S3B), indicating some distinct functions of Sin1 in different races. The exact location of Sin1 in human prostate cells has not been previously

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examined. Here we found that, in human prostate tumors, Sin1 can locate both in the cytoplasm and nucleus. Consistent with our results, Sin1 is able to

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translocate into nucleus to acquire its biological function. Based on the Sin1 staining in human prostate sections, Sin1 expression may occur in both basal and luminal cells, suggesting that Sin1 may regulate the function of different cell types during prostate carcinogenesis. Conclusion: Our studies demonstrate that Sin1 is closely related to PCa progression. Sin1 promotes cell proliferation and invasion by regulating mTORC2-AKT signaling and EMT. Sin1 is a novel partner of AR, and the interaction of Sin1 with AR has a crucial role in the transcriptional regulation of AR-responsive target genes. Sin1 augments mTORC2-AKT activity in PCa cells by two different mechanisms: (1) maintenance of high levels of Sin1 expression by

Journal Pre-proof AR-mediated transcription, and (2) inhibition of Sin1 degradation by androgen-mediated nuclear translocation and AR interaction (Fig. 8). Altogether, our data indicate that Sin1 is important for survival of PCa cells and may be considered a potential biomarker or therapeutic target for the treatment of human PCa. Materials and Methods

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Immunohistochemistry

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Tissue array chips of prostate cancer were purchased from Shanghai Outdo Biotech Co. LTD (Pudong, SH, CHN). Anti-Sin1 (NB110-40424, 1:200) from

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Novus Biologicals was used for immunohistochemical staining (IHC).

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Procedures were carried out as described before52. After antigen retrieval, prostate sections were subjected to IHC staining according to the

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manufacturers’ protocols, using SP-link Detection and DAB Kits (ZSGB-Bio, BJ, CHN). The level of staining intensity was graded between 0 and 3, which

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corresponded to no staining (0), weak (1), median (2) and strong staining (3). The staining extent ranged from 0 to 4, which was respectively used for

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grading coverage percentage of immunoreactive tumor cells (0%, 1-25%, 26-50%, 51-75%, 76-100%). The overall staining score was obtained after multiplying the staining intensity and extent score grading from 0 to 12. Grades from 0 to 3 were considered negative staining, while Grades 4 to 12 were positive. HE and IHC stained slides were independently assessed by two experienced pathologists, and determined as above. Cell Culture and Transfection Prostate cell lines were originated from ATCC. PC3, LNCaP and C4-2 cells were maintained in RPMI 1640 medium containing 10% fetal bovine serum (FBS). DU145 and RWPE-1 cells were cultured in DMEM medium supplemented with 10% FBS. The MAPKAP1/Sin1 and control (scrambled)

Journal Pre-proof siRNAs were purchased from Santa Cruz (Dallas, TX, USA). The pCMV6-Entry-Sin1-DDK-Myc (PCMV-Sin1) plasmid was from Origene (Beijing, China). The pcDNA3.1-AR-His plasmid and pGMAR-Lu firefly luciferase reporter vector containing human Sin1-promoter were constructed by Youbio (Hunan, China). The pGMAR-Lu firefly luciferase plasmid with AR responsive elements (ARE) as well as the pGMAR-TK renilla luciferase plasmid were retrieved from Genomeditech (Shanghai, China). Cells were set up at proper

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density one day before transfection with Lipofectamine 3000 (Thermo Fisher).

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Hormone Stimulation Assay

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Cells were cultured in charcoal stripped medium (phenol red-free RPMI medium (Gibco) supplemented with 10% charcoal stripped FBS (Gibco) and 1%

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nonessential amino acid (NEAA). After removing the endogenous hormone,

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cells were cultured overnight culture and then treated with 10nM Estradiol (Selleck) or 1nM R1881(Sigma-Aldrich). Rapamycin and MK-2206 (Selleck)

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were eventually used to inhibit AKT.

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Western Blotting Assays

Western blotting was set as described previously53. Cell fractionation was performed using NE-PER Nuclear and Cytoplasmic Extraction kit (Solarbio, BJ, CHN). To produce whole cell lysates, cell pellets were resuspended in RIPA buffer containing protease inhibitor cocktail and phosphatase inhibitor cocktail (Roche). Thereafter, protein samples were resolved by sodium dodecyl sulfate–polyacrylamide electrophoresis for further immunoblotting analyses. Anti-Sin1 (1:1000, 12860), anti-p-Sin1 (1:1000, 14716), anti-AKT (1:1000, 2920), anti-p308-AKT (1:1000, 13038), anti-p473-AKT (1:1000, 4060), anti-S6 (1:1000, 2217), anti-p-S6 (1:1000, 5364), anti-p-Rictor (1:1000, 3806), mTOR (1:1000, 2983), anti-Vimentin (1:1000, 5741) and anti-PR (1:1000, 3172) were purchased from CST (Danvers, MA, USA). anti-Rictor (1:5000, ab70374) and

Journal Pre-proof Androgen receptor (1:5000, ab74272) were from Abcam (Cambridge, MA, USA). Anti-His (66005-1-Ig) and anti-DDK (66008-3-Ig) were from Proteintech (Rosemount, IL, USA). Immunoprecipitation Cells were rinsed twice with ice-cold PBS and lysed with CO-IP buffer containing protease inhibitors. The soluble fractions were collected after

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centrifugation. For immunoprecipitation, anti-DDK or anti-AR antibody was added to the lysates and incubated overnight with rotation at 4°C. Next,

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Protein G-Sepharose was added and incubation was extended by one

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additional hour. Immunoprecipitates were washed extensively with lysis buffer, and immunoprecipitated proteins were recovered by adding 1x loading buffer

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Cell Viability Assay

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and boiling for 5 mins, followed by resolution on 10% SDS-PAGE gels.

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MTT (Methylthiazolyldiphenyl-tetrazolium bromide) was purchased from Solarbio (Beijing, China) . PBS was used to dissolve MTT and to ensure the

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concentration of 5 mg/ml. MTT was added to each sample and incubated in 37℃ incubator for 4hrs. DMSO was used to dissolve the sedimented solutions. Absorbance at 490 nm was measured to estimate cell viability. Cell Invasion Assay

FBS-free medium was used to dissolve the Matrigel (BD), which was carefully pipetted into the bottom of a transwell chamber. After 4-hr incubation in 37℃ incubator, the supernatant from the chamber was carefully discarded. Normal medium was then added into the bottom of the well. FBS-free medium was prepared for the cell suspension. A total of 1x106 cells were added into a single chamber. After 24-hr incubation in 37℃ incubator, cells were stained with crystal violet. Stained chambers were photographed and the number of

Journal Pre-proof invasive cells were counted. Real-time Quantitative PCR Total RNA was extracted using TrIzol (Solarbio, BJ, CHN). The cDNA Synthesis was performed with Goldenstar™ RT6 Mix (Tsingke, BJ, CHN). The qRT-PCR reactions were prepared with SYBR Green, and procedures followed according to manufacturer’s protocol (Tsingke,BJ, CHN). The primer

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sequences are shown in Table S1.

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Dual-Luciferase Activity Assays

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The pGMR-Lu plasmid expressing firefly luciferase reporter and the pGMR-TK renilla vector were co-transfected into LNCaP cells maintained in charcoal

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stripped medium (CSM). Twenty-four hours later, 1nM R1881 was added and

and

lysed

for

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cells were incubated for 24 hrs in 37oC incubator. Next, cells were collected dual-luciferase

activity

assay

using

TransDetect

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Double-Luciferase Reporter Assay Kit (Transgen Bio Tech, SH, CHN).

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Experimental procedures followed according to the kit instructions. Data and Statistical Analysis Student’s t-test was used to analyze the difference of Sin1 expression between normal and cancer tissues. One-way ANOVA test was used to evaluate the difference of Sin1 expression between normal and cancer tissue within different Gleason scores. Paired t-test was used for western blotting, cell viability, cell invasion, and dual-luciferase activity assays. P<0.05 was considered as cut-off for statistical significance. Conflicts of Interest: The authors declare no conflict of interest.

Journal Pre-proof Acknowledgements: This work was supported by National Natural Science Foundation of China (grand number: 31601137 and 81790642), the Central University Basic Scientific Research Business Expenses Special Funds (grand number: ZYGX2017KYQD169), National Key R&D Program of China (grand number: 2016YFC0905200) and Chinese Academy of Medical Sciences (grand number:

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2019-I2M-5-032).

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Author Contributions:

CXHY and GYF carried out the experiments and performed statistical analyses

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with the help of JSC and QP. CY, LY and ZLY edited the manuscript. CY, ZYW

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and LY conceived the study. ZYW analyzed the data and wrote the manuscript.

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Journal Pre-proof Figure 1. Sin1 levels are upregulated in human prostate cancer (PCa). (A) Representative IHC analysis indicating Sin1 protein levels in human prostate tumors and their paired normal tissues. (B) Representative pictures of cytosolic and nuclear location related to Sin1 expression. (C) Sin1 staining score was evaluated according to the staining intensity multiplied by the extent score. This parameter was used for comparison between prostate cancer samples and normal tissues. The p-value was calculated by Student’s t-test.

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(D) Sin1 expression was compared between normal and tumor tissues within

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different Gleason scores. The p-value was calculated by one-way AVONA test. (E) Summarized quantification of Sin1 staining location in human prostate

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tumors.

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Figure 2. Sin1 overexpression increases cell viability and invasion of

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PCa cells via mTORC2-AKT pathway

(A-C) PC3, LNCaP and C4-2 cells were transfected with PCMV-Empty or

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PCMV-Sin1 for further cell invasion and viability analyses. (D) PC3 cells were transfected with PCMV-Empty or PCMV-Sin1 to examine the content of

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Sin1-mTORC2-AKT related proteins. (E, F) LNCaP cells were transfected with PCMV-Empty or PCMV-Sin1, followed by RNA purification and qRT-PCR analysis. The mRNA levels of E-cadherin, Vimentin, Snail and some matrix metalloproteinases (MMPs) were quantified. Data were normalized according to GAPDH mRNA levels. (G, H) PC3 cells were transfected with PCMV-Empty or PCMV-Sin1, followed by 30-min treatment with 100nM Rapamycin or 10μM MK-2206. Western blotting was performed to detect respective Vimentin proteins. Data include representative immune blots and a quantification summary. Each treatment was performed in triplicates. Protein and mRNA expression data were normalized according to β-actin levels (mean±S.D., n=3).

Journal Pre-proof Figure 3. Inhibiting Sin1 decreases AKT signaling and negatively affects cell invasion and viability. (A, B) C4-2 cells were transfected with Sin1 or scrambled (control) siRNAs for further cell invasion assays. Representative pictures are shown. The number of invasion cells were presently calculated. (C) PC3, LNCaP and C4-2 cells were transfected with Sin1 or scrambled siRNAs to evaluate the impact of Sin1 gene silencing on the viability of PCa cells. (D) C4-2 cells were transfected or

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Sin1-mTORC2-AKT related proteins. Each treatment was performed in

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triplicates, and protein expression data were normalized according to β-actin

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levels (mean±S.D., n=3).

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Figure 4. Sin1 is androgen-inducible in androgen-dependent prostate

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cancer cells.

(A-C) LNCaP and C4-2 cells, cultured in charcoal stripped medium (CSM),

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were treated with 1nM R1881 for following western blotting analysis and qRT-PCR assays. (D) CSM-cultured LNCaP cells were transfected with 1μg

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luciferase reporter plasmids (driven by Sin1-promoter), followed by treatment with/without 1nM R1881. Twenty-four hours later, cells were processed to detect luciferase activity. (E-G) CSM-cultured LNCaP cells were treated with/without 1nM R1881 and androgen receptor antagonist MDV3100, followed by Sin1 and p-Sin1 immunoblotting analysis and qRT-PCR assay. Data presented include representative immune blots from three independent experiments. Protein expression data were normalized according to β-actin levels. The relative mRNA level was normalized according to GAPDH expression. All data are shown as the mean±S.D. (n=3). The p-value was calculated by Student’s t-test. Figure 5. Androgen increases Sin1 protein stability in LNCaP cells

Journal Pre-proof (A, B) CSM-cultured LNCaP cells were treated with/without 1nM R1881 for 2hrs, followed by co-treatment with 50μg/ml CHX for additional 22hrs. The quantification of Sin1 levels was shown as mean ± S.D.(n=3). (C, D) CSM-cultured C4-2 cells were treated with/without 1nM R1881 for 2hrs, followed by co-treatment with CHX for additional 22hrs. The quantification of Sin1 was normalized according to GAPDH levels (mean±S.D., n=3).

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Figure 6. Androgen increases Sin1 nuclear accumulation. CSM-cultured LNCaP (A, B) and C4-2 (C, D) cells were treated with/without

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1nM R1881 for 8hrs, and then fractionated for cytoplasmic and nuclear protein

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extraction. Components of mTORC2 were examined by immunoblotting with the indicated antibodies. GAPDH and Lamin B1 were blotted as cytosolic and

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nuclear markers, respectively. Protein expression was normalized according to

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GAPDH or Lamin B1 levels (mean±S.D., n=3). Figure 7. Sin1 interacts with androgen receptor to regulate its

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transcriptional activity.

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(A, B) HEK 293T cells were co-transfected with Sin1-DDK and AR-His expression vectors. Cell were further lysed, and protein extracts were separately by IP using anti-His or anti-DDK. Western blotting was performed using anti-DDK or anti-His primary antibodies. Data are representative immunoblots from three independent experiments. (C) CSM-cultured LNCaP cells were treated with/without 1nM R1881. Cell were further lysed, and protein extracts were separately by IP using anti-Sin1 or rabbit IgG. Western blotting was performed using primary anti-AR antibody. Data are representative immunoblots from three independent experiments. (D) C4-2 cell extracts were IP using anti-Sin1 or rabbit IgG. Western blotting was performed using primary anti-AR antibody. (E) CSM-cultured LNCaP cells were transfected with PCMV-Sin1 or PCMV-Empty and luciferase reporter plasmid containing AR

Journal Pre-proof responsive elements. After 24hrs, cells were treated with/without 1nM R1881 for another 24hrs. Cell lysates were used to detect respective luciferase activity. (F) LNCaP cells were transfected with PCMV-Empty or PCMV-Sin1, and then subjected to qRT-PCR to measure the mRNA levels of androgen receptor-target genes SIN1, AR, PSA, NKX3.1 and TMRPSS2. Figure 8. Model of Androgen-mediated Sin1 in the regulation of cell

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viability and invasion in PCa Sin1 regulates mTORC2-AKT and AR signaling to promote invasion and

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viability of PCa cells. (A) Androgen stimulates Sin1 transcription. (B) Androgen

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elevates Sin1 protein stability by nuclear translocation. (C) Sin1 interacts with

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AR to modulate the transcription of AR-responsive genes. Figure S1. High Sin1 expression correlates with progression of human

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(A-G) Sin1 expression data, measured by RNA-seq, were retrieved from Oncomine database and used for comparison between normal and PCa

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tissues, according to different clinical grade of tumor (T stage and Gleason score) and different malignant states. Figure S2. Sin1 is expressed in basal and luminal cells of human prostate tumor tissues

Location of Sin1 staining in human prostate tumors. Basal cells (red arrow), luminal cells (black arrow), cytoplasm (red arrow head) and nucleus (black arrow head) are indicated. Figure S3. Sin1 is expressed in PCa cells (A) Western blotting of Sin1 and AKT signaling proteins in PCa cell lines. (B) Sin1 expression data, measured by RNA-seq, were retrieved from TCGA and

Journal Pre-proof used for comparison between prostate tumor samples, from different populations, and normal tissues. Figure S4. Sin1 expression is regulated by androgen receptor (AR) signaling. (A-D) CSM-cultured LNCaP cells were treated with/without 1nM R1881 and androgen receptor antagonist MDV3100 for further qRT-PCR analysis. The mRNA levels of AR, NKX3.1 PSA and PDCD4 were accessed. Data were normalized according to GAPDH expression, and shown as mean

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±S.D.(n=3). The p-value was calculated by Student’s t-test.

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Table 1. Clinicopathological characteristics in relation to Sin1 expression status in human normal and tumor tissues. Normal tissue Tumor tissue Total: 70 samples Total: 137 samples IHC IHC IHC IHC positive negative positive negative Number 35 35 132 5 <60 1 0 9 0 Age1 ≥60 33 33 123 5 <7 / / 34 3 2 Gleason score =7 / / 61 1 ≥8 / / 35 1 Extraprostatic / / 6 / extension Perineural / / 21 / invasion

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1：3 normal samples are lack of age information. 2：5 tumor samples are lack of Gleason information.

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Highlights 1. High expression of Sin1 is a novel biomarker for human prostate cancer. 2. Sin1 promotes cell viability and invasion by modulating mTORC2-AKT signaling and epithelial-to-mesenchymal transformation. 3. Sin1 expression is regulated by AR signaling transcriptionally in

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androgen-dependent prostate cancer cells.

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4. Sin1 interacts with AR to regulate its transcriptional activity.

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