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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Cancer Prev Res (Phila). Author manuscript; available in PMC 2016 July 1.
Published in final edited form as:
PMCID: PMC4491032
NIHMSID: NIHMS685370

Effect of Metformin, Rapamycin and Their Combination on Growth and Progression of Prostate Tumors in HiMyc Mice

Abstract

In this study, we compared the effect of oral administration of metformin (MET) and rapamycin (RAPA) alone or in combination on prostate cancer (PCa) development and progression in HiMyc mice. MET (250 mg/kg body weight in the drinking water), RAPA (2.24 mg/kg body weight microencapsulated in the diet) and the combination inhibited progression of prostatic intraepithelial neoplasia lesions to adenocarcinomas in the ventral prostate (VP). RAPA and the combination were more effective than MET at the doses used. Inhibition of PCa progression in HiMyc mice by RAPA was associated with a significant reduction in mTORC1 signaling that was further potentiated by the combination of MET and RAPA. In contrast, treatment with MET alone enhanced AMPK activation but had little or no effect on mTORC1 signaling pathways in the VP of HiMyc mice. Further analyses revealed a significant effect of all treatments on prostate tissue inflammation as assessed by analysis of the expression of cytokines, the presence of inflammatory cells and NFκB signaling. MET at the dose utilized appeared to reduce PCa progression primarily by reducing tissue inflammation whereas RAPA and the combination appeared to inhibit PCa progression in this mouse model via the combined effects on both mTORC1 signaling as well as on tissue inflammation. Overall, the current data support the hypothesis that blocking mTORC1 signaling and/or tissue inflammation can effectively inhibit PCa progression in a relevant mouse model of human PCa. Furthermore, combinatorial approaches that target both pathways may be highly effective for prevention of PCa progression in men.

Keywords: Metformin, Rapamycin, Prostate cancer, mTORC1, Inflammation

Introduction

Worldwide, prostate cancer (PCa) is the second most commonly diagnosed non-cutaneous cancer in men and the sixth most common cause of death (1). In the United States, it is the most frequently diagnosed non-cutaneous cancer and the second leading cause of cancer-related mortality in this population (2). An estimated 233,000 new cases will have been diagnosed in 2014 in the U.S. with an estimated 29,480 deaths (3). Although the median age for diagnosis of PCa is 66 (2), the onset of preclinical disease may occur in adults as early as 30 years of age (4). Since there can be a considerable time for the disease to progress to clinically evident cancer, there is ample opportunity for chemopreventive strategies to be applied for the successful management of PCa [reviewed in (4, 5)]. A number of agents, including sulforaphane, curcumin, green tea (or EGCG), resveratrol and various NSAIDS have shown potential chemopreventive effects in vivo in either animal models of PCa or in clinical studies (6-9). Many of these agents target inflammatory signaling pathways including STAT3 and NFκB, in addition to other cellular signaling pathways associated with PCa development and progression (10-12).

Metformin (MET) is a drug widely used for the treatment of type II diabetes (13) and its use has been shown to be associated with reduced cancer incidence and mortality (14). Several recent in vitro and animal studies also support the anticancer activity of MET in different cancer types including PCa (15-17). MET is a potent activator of AMPK, which is a serine/threonine protein kinase that acts as a central metabolic sensor involved in cellular energy homeostasis (18). AMPK activation also leads to inhibition of mammalian target of rapamycin (mTORC1) signaling through phosphorylation of tuberous sclerosis 2. It is now well documented that mTORC1 signaling plays an important role in oncogenic processes (19), and dysregulation of this signaling pathway has been reported in various cancers including PCa (20, 21).

Rapamycin (RAPA) is a macrolide used clinically as an immunosuppressant in organ transplant patients and to treat autoimmune disorders (22). It acts as a potent inhibitor of mTORC1 by binding with immunophilin FK506 binding protein (FKBP12) (23). Data in the literature also suggests that RAPA has potent cancer chemopreventive properties in a number of mouse models, including mouse models of PCa (24-26). In addition, data compiled from more than 30,000 kidney transplant recipients found that the use of mTOR inhibitors as maintenance immune-suppressive therapy produced a remarkable reduction in non-melanoma skin cancer incidence compared with cyclosporine A, a calcineurin inhibitor (27). A dramatic decrease in the incidence of skin malignancies was also observed in transplant patients who were converted to mTOR inhibitors after 3 months of treatment with cyclosporine A (28).

In the current study, we evaluated the ability of orally administered MET, RAPA and a combination of MET + RAPA on PCa development and progression in HiMyc mice (29). In this model, over-expression of c-Myc in the prostate is directed via the ARR2Pb probasin promoter resulting in the development of prostatic lesions that share molecular and histopathologic features with human prostate tumors (29). Prostatic epithelial expression of c-Myc in the dorsolateral prostate (DLP), ventral prostate (VP), and anterior prostate (AP) lobes results in complete penetrance of PIN as early as 2 to 4 weeks of age, which progressed to locally invasive adenocarcinomas within 6 to 12 months of age (29). MET, RAPA and the combination inhibited progression of prostatic intraepithelial neoplasia (PIN) lesions to adenocarcinomas (ACs) in the VP of HiMyc mice. RAPA and the combination were more effective than MET at the doses used. Based on mechanistic studies performed, the current results suggest that targeting mTORC1 together with inflammatory signaling pathways may be an effective strategy for prevention of PCa progression.

Materials and Methods

Reagents

RPMI-1640 and fetal bovine serum (FBS) were obtained from Life Technologies. MET and RAPA were purchased from Sigma-Aldrich and LC laboratories, respectively. Antibodies against AMPK, pAMPKThr172, mTOR, pmTORSer2448, p70S6K, pp70S6KThr389, S6 ribosomal protein (S6 Ribo), pS6 RiboSer235/236, pS6 RiboSer240/244, pNFκBp65Ser536, ULK1, pULK1Ser555, cMyc, GAPDH, cyclin D1, and PARP were purchased from Cell Signaling. Antibodies for p27, pNFκBp50Ser337, IκBα were from Santa Cruz Biotechnology and β-actin from Sigma-Aldrich. Antibodies for CD45 and CD3 (T-lymphocytes) were obtained from Abcam.

Study design

HiMyc mice (29) were obtained from the NIH MMRRC on an FVB/N genetic background and mice were bred in-house for the current experiments. All diets were purchased from Test Diets and mice were fed chow based diet containing either 14 mg/kg microencapsulated RAPA [RAPA diet equivalent to 2.24 mg/kg body weight or 14 mg/kg eudragit (control diet)] (30). Mice were placed on control diet at 4 to 5 weeks of age for a 1-week equilibration period and then randomized into the following dietary groups (n=18) for the duration of the study: a) control diet fed ad libitum; b) RAPA diet fed ad libitum; c) control diet fed ad libitum + MET (250 mg/kg BW) in drinking water; and d) RAPA diet fed ad libitum + MET in drinking water. The body weight and food consumption were determined every two weeks. Groups of mice were terminated by CO2 asphyxiation, and the genitourinary tract including urinary bladder, prostate lobes (AP, VP, DLP), seminal vesicle and urethra was taken after 18 weeks of treatment for histopathologic diagnosis and immunohistochemistry (IHC) and immunofluorescence (IF) analyses. An additional set of 10 mice from each diet group was used for protein and RNA analyses (isolated after 10 weeks of treatment). For all the studies, mice were housed in suspended polycarbonate cages on autoclaved hardwood bedding at room temperatures of 20°C-22°C, relative humidity of 60-70%, and 14/10-hour light/dark cycle.

Histologic analyses

For histologic analyses, the genitourinary tract was removed, fixed in 10% formalin, embedded in paraffin, and transversely sectioned. Sections of 4 μm were stained with hematoxylin and eosin for histopathologic diagnosis as previously described (31). Under the experimental conditions employed, HiMyc mice developed lesions primarily in the VP and DLP, with fewer lesions in the AP. Furthermore, at ~6 months of age, >90% of the mice developed invasive tumors primarily in the VP. In contrast, at the same time point, the number of invasive tumors in the DLP and AP was significantly lower. Thus, the current analyses focused on the VP that displayed a more homogeneous and consistent development of the lesions from hyperplasia to invasive ACs within the 6-month time frame of these experiments.

Cell culture

LNCaP cells were purchased from ATCC. These cells were maintained in RPMI-1640 medium with 10% FBS. The murine prostate tumor cell line, HMVP2, was derived from the VP of one year old HiMyc transgenic mice as previously described and cultured in RPMI-1640 medium containing 10% FBS (32). Cell lines were authenticated by genetic biomarkers. Mycoplasma test was performed by PCR amplification (Applied Biological Materials Inc.) and 4',6-diamidino-2-phenylindole (DAPI) staining. All cells were cultured in 95% air and 5% CO2 at 37°C.

Western blotting

Levels of phosphorylated and total proteins were measured by Western blot with slight modifications of previous methods (31). Briefly, LNCaP and HMVP2 cells were treated with MET (0.5 mM), RAPA (1.0 nM) or MET (0.5 mM)+RAPA (1.0 nM) for 24 h. After incubation, cells were lysed in RIPA buffer. Alternatively, individual VP lobes were excised, crushed into powder under liquid nitrogen and lysed in RIPA buffer. Proteins were separated by SDS-PAGE gel and transferred to nitrocellulose membranes. After blocking for 1h, the membranes were probed with specific primary antibodies overnight at 4°C. Following secondary antibody (GE Healthcare) incubation, membranes were visualized using a commercial chemiluminescent detection kit (Pierce Biotechnology). Except where noted, all results were confirmed in at least three independent experiments.

Quantitative real-time PCR (qPCR)

Total RNA was isolated from the individual VP lobes by using Qiagen RNeasy mini kit (Qiagen) according to the manufacturer's protocol and subjected to reverse-transcription as previously described (31). mRNA levels of genes were quantitatively determined by qPCR using the Viia7 Real Time PCR System (Applied Biosystems) with SYBR Green Master Mix (Qiagen). The relative abundance of the mRNA was normalized against glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA for quantitative evaluation.

IHC and IF analyses and toluidine blue staining

IHC analyses were performed on formalin fixed, paraffin-embedded prostate tissue sections (31). Briefly, tissue sections were deparaffinized with serial incubation and washing in xylene, 100-70% ethanol and water followed by antigen unmasking with citrate buffer. The samples were then blocked for 1h at room temperature and incubated with primary antibodies overnight at 4°C. Proteins were detected with biotinylated secondary antibodies, followed by peroxidase-conjugated avidin/biotin (Vector Laboratories) and 3,3’-diaminobenzidine (DAB) substrate (Dako) and then visualized with light microscopy. Quantification of IHC analyses for pS6Ribo was scored based on a four point scale criteria from negative (0+) to intense (3+) as described elsewhere (33). For IF staining (34), paraffin embedded prostate tissue sections were detected with fluorochrome-conjugated secondary antibodies and visualized using an Olympus BX60 fluorescence microscope. Toluidine blue staining was used to identify mast cells as described elsewhere (35). Briefly, tissue sections were deparaffinized, stained with 0.1% toluidine blue solution for 1 minute, washed with water, dehydrated with 70-100% ethanol and xylene solution. Mast cells were visualized and counted with light microscopy.

Statistical analyses

Statistical analyses were performed for differences in incidence of prostate lesions between groups using Fisher's exact test. Comparison of changes in protein, protein phosphorylation and mRNA levels was accomplished using one-way ANOVA followed by Bonferroni's multiple comparison tests. Significance was set at p<0.05.

Results

Effect of MET, RAPA and MET + RAPA on PCa progression in HiMyc mice

The effect of oral administration of MET, RAPA and MET + RAPA on PCa progression in HiMyc mice is shown in Fig. 1A. The incidence of hyperplasia and low-grade PIN (lgPIN) in the VP of all the treatment groups was similar after 18 weeks of treatment. However, treatment with MET, RAPA and MET + RAPA slightly decreased the incidence of hgPIN (Fig. 1A) although this was not statistically significant. The incidence of both in situ ACs and locally invasive ACs was significantly reduced by treatment with both RAPA and MET + RAPA. In this regard, RAPA treatment alone decreased in situ ACs by 41% (p<0.02) and completely suppressed formation of locally invasive ACs (p<0.001), compared to the control group. The combination of MET + RAPA significantly (p< 0.005) decreased in situ ACs by 51% (Fig. 1A) and also completely suppressed the formation of locally invasive ACs (p<0.001). MET given alone reduced the incidence of in situ ACs by 27% (not statistically significant) and significantly decreased the incidence of invasive ACs by 50% (p< 0.01) (Fig. 1A). The decreases in in situ ACs and ACs by the combination were not statistically significant when compared to the RAPA only group but the decrease in ACs was statistically significant (p<0.01), when compared to the MET only treated group. Representative lesions for each of the treatment groups are shown in Fig. 1B. All mice were closely monitored throughout the experimental period and did not reveal any signs of toxicity. Furthermore, there were no significant differences in body weight (Supplemental Fig. 1A) or food consumption (Supplemental Fig. 1B) across the different treatment groups during the course of the experiment. Thus, oral administration of MET, RAPA and the combination of MET + RAPA significantly decreased the incidence of malignant PCa lesions in the VP of HiMyc mice without apparent toxicity.

Figure 1
Effect of MET, RAPA and MET + RAPA on incidence of prostate lesions in HiMyc mice. Panel A, HiMyc mice were administered MET (250 mg/kg body weight in drinking water), RAPA (14 mg/kg microencapsulated in the diet) or their combination for 18 weeks starting ...

Effect of MET, RAPA and MET + RAPA on AMPK and mTORC1 signaling in VP of HiMyc mice

As shown in Fig. 2A, treatment with MET led to increased phosphorylation of AMPKThr172 and this was greater in mice that received MET + RAPA. As expected, RAPA alone had little or no effect on AMPK activation. In contrast, MET alone had little or no effect on mTORC1 signaling (as assessed by phosphorylation of both p70S6K and S6 Ribo) (Fig. 2B). However, treatment with RAPA alone produced a significant reduction of phospho-p70S6KThr389 and phospho-S6 RiboSer240/244. Interestingly, the combination of MET + RAPA significantly decreased phospho-mTORSer2448 as well as levels of phospho-p70S6K and phospho-S6RiboSer240/244 (Fig. 2A and 2B). The effect on phospho-S6Ribo was further confirmed by IHC staining of VP sections (Fig. 2C and 2D). These data indicate that the ability of RAPA and MET+ RAPA to inhibit PCa progression in HiMyc mice correlated with significant inhibition of mTORC1 signaling whereas the inhibitory effect of MET on PCa progression did not.

Figure 2
Effect of MET, RAPA and MET + RAPA on AMPK and mTORC1 signaling. Protein lysates were prepared from the VP of HiMyc mice (pooled sample of n=4-5 mice/group) treated with MET, RAPA or MET+RAPA for 10 weeks and Western blot analyses were performed for the ...

Effect of MET, RAPA and MET + RAPA on AMPK and mTORC1 signaling in cultured mouse and human PCa cells

Similar to the results seen in vivo after administration of the compounds to HiMyc mice, treatment of HMVP2 cells with MET (0.5 mM) alone led to significant activation of AMPK but did not produce significant effects on mTORC1 phosphorylation and mTORC1 downstream signaling (again measured by phosphorylation of p70S6K and S6 Ribo)(Fig. 3A and 3B). However, both RAPA (1.0 nM) and the combination of MET (0.5 mM) + RAPA (1.0 nM) significantly increased the phosphorylation of AMPK and decreased the phosphorylation of mTORSer2448, p70S6KThr389, S6 RiboSer240/244 (again see Fig. 3A and 3B). As shown in Supplemental Fig. 2A and 2B, the combination of MET + RAPA also significantly increased the levels of phospho-ULK1Ser555 and p27. However, no increases were observed in apoptosis marker, cleaved PARP with MET, RAPA or the combination in these cells. Thus, these results examining the effects of the agents on AMPK and mTORC1 signaling in cultured HMVP2 cells are very similar to the results seen in the VP of HiMyc mice following oral administration of the compounds.

Figure 3
Effect of MET, RAPA and MET + RAPA on AMPK and mTORC1 signaling in mouse (HMVP2) and human (LNCaP) PCa cells. Cells were treated with MET (0.5 mM), RAPA (1.0 nM) or MET (0.5 mM) + RAPA (1.0 nM) for 24h and Western blot analyses were performed for pAMPK ...

The effect of MET, RAPA and MET + RAPA on AMPK and mTORC1 signaling in cultured human PCa cells (i.e., LNCaP) was also evaluated. Treatment of LNCaP cells with both MET and RAPA alone led to AMPK activation, which was further potentiated by treatment with the combination (Fig. 3C and 3D). Similar to HMVP2 cells, both RAPA and the combination reduced mTORC1 signaling (measured by phosphorylation of p70S6K and S6 Ribo) in LNCaP cells (again see Fig. 3C and 3D) whereas MET had little or no effect on mTORC1 downstream signaling. The combination but not the individual compounds also decreased cyclin D1 and c-Myc levels (Supplemental Fig. 3A and 3B) and similar to the mouse PCa cells, there was no increase in the apoptosis marker, cleaved PARP. Collectively, these data using cultured PCa cells from both HiMyc mice and humans showed similar results to those observed from the in vivo experiments and further substantiate a lack of effect of MET on mTORC1 signaling and in some cases greater effects with the combination compared to RAPA alone at the concentrations used.

Effect of MET, RAPA and MET + RAPA on inflammatory signaling and inflammation in the VP of HiMyc mice

Since previous studies have suggested that both MET and RAPA may affect inflammation signaling pathways (36-39), the effect of MET, RAPA and the combination on the expression of various chemokines, cytokines, growth factors and angiogenesis factors associated with inflammation was examined in RNA samples isolated from the VP of HiMyc mice on the different treatment regimens. As shown in Fig. 4A, treatment with MET or RAPA alone significantly decreased the mRNA levels of IL1α and IL1β (p<0.05). MET alone also significantly decreased mRNA levels of IL23, TNFα and CXCL12 (p<0.05). The combination of MET + RAPA appeared to further decrease the expression of these inflammatory mediators compared to RAPA or MET alone although statistical significance was achieved only for CCL5 (compared to either MET or RAPA) and for IL-23 and CXCL12 (compared to RAPA). MET treatment alone also significantly (p<0.05) decreased the expression of both VEGFB and IGF-1 (Fig. 4B and 4C) whereas treatment with RAPA had little or no effect on mRNA levels for these genes. Again, the combination treatment produced greater inhibition of IGF-1, IGF-1R and VEGFB compared to RAPA and produced stronger inhibition of HIF1α and VEGFA compared to both agents.

Figure 4
Effect of MET, RAPA and MET + RAPA on inflammation and angiogenesis markers. RNA was isolated from the VP of HiMyc mice treated for 10 weeks. qPCR analyses of gene expression is shown for CCL5, IL-1α, IL-1β, IL-6, IL-23, TNF-α, ...

As shown in Fig.5, IF staining of prostate tissues from untreated HiMyc mice on the control diet showed significant infiltration of inflammatory cells (measured by CD45+ cells) whereas HiMyc mice treated with MET, RAPA and MET+RAPA had very few CD45+ cells (Fig. 5, panels A and D). Further analyses confirmed that mice treated with MET, RAPA and MET+RAPA had significant decreases in the number of both T-lymphocytes (CD3+ cells; Fig 5, panels B and E) and mast cells (O-toluidine blue stained cells; Fig. 5, panels C and F) in the VP compared to the control group. Thus, both MET and MET + RAPA significantly reduced expression of many of the genes examined whereas RAPA treatment alone only significantly reduced expression of IL-1α/β. However, all treatments reduced tissue inflammation as assessed by the presence of several types of inflammatory cells.

Figure 5
Effect of MET, RAPA and MET + RAPA on inflammatory cells in VP of HiMyc mice. Representative stained sections of the VP of HiMyc mice treated 18 weeks with either a control diet or diets containing MET, RAPA or RAPA + MET. Panels A, sections stained with ...

Effect of MET, RAPA and MET + RAPA on NFκB signaling in VP of HiMyc mice

As shown in Fig. 6A (upper panels), MET, RAPA and MET + RAPA strongly inhibited the phosphorylation of the p50 subunit of NFκB as measured by IF staining of tissues from the VP of treated mice. Since IκBα acts as an inhibitor of NFκB signaling (40), staining for IκBα was also performed in tissues from the VP of treated mice. As shown in the lower panels of Fig. 6A, the level of IκBα was higher in the VP of HiMyc mice treated with MET, RAPA and MET + RAPA compared to the mice in the control group. The status of pNFκBp65Ser536 was also evaluated by Western blot (Fig. 6B). The level of pNFκBp65Ser536 in VP of HiMyc mice was decreased by all three treatments, with the greatest decreases seen with MET and MET + RAPA treatment compared to the RAPA only treated groups. Collectively, these data indicate that treatment with MET, RAPA and MET + RAPA significantly decreased NFκB signaling in the VP of HiMyc mice.

Figure 6
Effect of MET, RAPA or MET+RAPA on NFκB signaling. Panel A, IF staining of representative sections of VP from HiMyc mice treated 18 weeks with MET, RAPA or MET+RAPA for pNFκBp50 (Top panels) and IκBα (bottom panels) as ...

Discussion

The current study was designed to evaluate the effect of oral administration of MET, RAPA and their combination on PCa development and progression in an established mouse model of PCa. All treatments inhibited PCa progression in HiMyc mice but had little or no effects on the development of lgPIN and hgPIN lesions at ~6 months of age (i.e., following 18 weeks of treatment). The effect of MET on PCa progression at the doses used in this study was observed primarily on the incidence of locally invasive ACs whereas both RAPA and MET + RAPA produced statistically significant decreases in in situ ACs as well as complete inhibition of locally invasive ACs in the VP of HiMyc mice. Both MET and RAPA have been shown to decrease PCa cell growth in culture and to inhibit growth of PCa cells in xenograft models either alone or in combination with other agents (41-45). In addition, both MET and RAPA have been shown to inhibit development and/or progression of tumors in a number of mouse primary tumor models (16, 46, 47). The current results clearly demonstrate the efficacy of both of these compounds and the potential for increased efficacy with the combination for inhibiting PCa progression in this mouse model. Although the combination of MET + RAPA was not more effective than RAPA alone in terms of effects on tumor incidence, the combination was more effective in some cases at reducing relevant signaling pathways and gene expression changes. Therefore, further studies using different dosing combinations could reveal potential additive or synergistic effects of this combination. Collectively, the current data show that long-term oral dosing of MET, RAPA and MET+RAPA significantly reduces PCa progression in HiMyc mice.

Recently, the effect of short-term treatment with MET (200 mg/kg body weight in drinking water for 4 weeks) was evaluated in HiMyc mice starting at 5 weeks of age (48). This short-term treatment was reported to significantly inhibit formation of both PIN as well as PCa lesions through a mechanism involving downregulation of c-myc. While treatment related effects on the development of PIN lesions were not observed in the current study, under the experimental conditions employed, MET given alone significantly inhibited progression of PIN lesions to PCa. In other studies using HiMyc mice, a short-term treatment with RAD001 (given orally by p.o. tube daily for 14 days) did not revert mPIN lesions in these mice even though mTORC1 was inhibited by this treatment regimen (49). These data suggested that development of mPIN lesions in HiMyc mice did not depend on mTORC1 signaling. The current data with more long-term treatment using RAPA are consistent with this earlier study but also demonstrate a dependence of tumor progression in HiMyc mice, at least in part, on mTORC1 signaling. Finally, short-term treatment with RAPA (given i.p. daily for 14 days) in 12-14 month old PTEN+/− mice (C57BL/6 genetic background) was shown to reduce mTORC1 signaling and to inhibit proliferation of prostate lesions in these mice. A reduction in incidence of all detectable lesions was seen but these reductions in incidence were not statistically significant due to the low numbers of animals used in that study (50). The current data clearly demonstrate that RAPA given in the diet in a microencapsulated (and more bioavailable) form for an extended period of time inhibited the progression of PCa in HiMyc mice.

A number of experiments were performed to examine potential mechanisms for the effects of MET, RAPA and the combination of MET + RAPA on PCa progression. As shown in Fig. 2, treatment with RAPA significantly inhibited mTORC1 downstream signaling to p70S6K and S6 Ribo in the VP of HiMyc mice. Importantly, the addition of MET with RAPA further potentiated the inhibition of mTORC1 signaling. Further studies using both mouse and human PCa cells in culture confirmed these effects on mTORC1 signaling (Fig. 3). Notably, MET given alone had little or no effects on mTORC1 signaling in VP of HiMyc mice although AMPK activation was observed with this compound (Figs. 2 and and3).3). Thus, although MET is known to inhibit mTORC1 through both AMPK-dependent and -independent mechanisms (51), its ability to inhibit PCa progression could not be attributed to inhibition of mTORC1 signaling. These observations were also confirmed in cultured mouse and human PCa cells where MET, at relatively high concentrations, failed to significantly inhibit mTORC1 downstream signaling.

The lack of effect of MET on mTORC1 downstream signaling through p70S6K and S6 Ribo led to consideration of additional mechanisms for its inhibitory effects on PCa progression. Previous studies from our laboratory demonstrated a strong local anti-inflammatory effect of low dose RAPA when given topically to mice during the process of skin tumor promotion (37). Furthermore, other studies have shown anti-inflammatory effects of RAPA (36), and more recently anti-inflammatory effects of MET (38, 39). Therefore, markers of inflammation were evaluated in the VP of HiMyc mice treated with MET, RAPA and MET + RAPA. As shown in Fig. 4, treatment with MET, RAPA and the combination significantly decreased expression of IL1α and IL1β. MET and the combination also significantly inhibited expression of IL-23, TNF-α, CXCL12, VEGFB and IGF-1 whereas the combination also significantly reduced expression of HIF-1α, VEGFA and IGF-1R. The effect of MET given alone as well as MET + RAPA on CXCL12 is quite interesting. In this regard, CXCL12 is known to play a role in PCa progression (52). Notably, all three treatments also significantly decreased the infiltration of inflammatory cells (CD45+, cells, T-lymphocytes and mast cells) into the VP of HiMyc mice compared to the control animals (Fig.5). These results indicate that all treatments inhibited tissue inflammation and this effect may have contributed to their ability to inhibit PCa progression in HiMyc mice, especially in the group treated with MET.

Recent evidence suggests that MET has inhibitory effects on NFκB signaling (53). In addition, some of the genes whose expression was analyzed in this study are known to be regulated by NFκB signaling (40). Therefore, the status of NFκB signaling was also evaluated. The NFκB transcription factor family in vertebrates consist of p65 (RelA), RelB, cRel, p50/105 and p52/100 with all having highly conserved dimerization and DNA binding domains (40). The p50 subunit, which lacks a transactivation domain, is essential for NFκB DNA binding, and the phosphorylation of p50 at Ser337 is required for efficient DNA binding of NFκB (54). IF analysis of prostate tissues from treated HiMyc mice showed that phospho-p50Ser337 was dramatically decreased by all of the treatments compared to tissue from the control mice (Fig. 6A). The most important and common transactivating p65/p50 heterodimers of NFκB remain bound with inhibitor protein IκBα in the cytoplasm in an inactive form (40). A wide variety of stimuli including cytokines (TNF-α, IL-1 etc.), bacterial and viral products cause phosphorylation and proteosomal degradation of IκBα through activation of the upstream inhibitor of IκBα, leaving the free, active form of NFκB for rapid nuclear translocation (40). As also shown in Fig. 6A, IκBα levels were higher in the VP of HiMyc mice treated with MET, RAPA and MET+RAPA indicating that the active form of NFκB was comparatively lower in the VP of these groups compared to the control group. The phosphorylation of pNFκBp65 at Ser536 was also reduced in the VP of HiMyc mice by all treatments (Fig. 6B). Since NFκB regulates the expression of various genes including proinflammatory cytokines, chemokines, growth factors, angiogenesis and adhesion molecules, inhibition of NFκB signaling (as indicated by decreased phosphorylation of both p50 and p65 as well as reduced IκBα degradation) by MET, RAPA and MET+RAPA could explain the decreased expression of many of the genes examined in Fig. 4. Reduced NFκB signaling could also explain, at least in part, the reduced infiltration of inflammatory cells observed in the VP of treated mice.

In conclusion, the current results demonstrate that long-term oral dosing of HiMyc mice with MET, RAPA and MET+RAPA decreased PCa progression without apparent toxicity. Mechanistic studies revealed that RAPA and the combination of MET+RAPA significantly inhibited mTORC1 signaling whereas MET did not. However, all treatments produced significant reductions in inflammatory gene expression, infiltration of inflammatory cells and reduced NFκB signaling in the VP of treated HiMyc mice. MET and the combination of MET + RAPA also reduced expression of several angiogenesis genes and IGF-1/IGF-1R. These data suggest that RAPA and MET + RAPA inhibited PCa progression via effects on both mTORC1 and on tissue inflammation and inflammation signaling whereas the effects of MET were associated primarily with the latter. Although we were unable to show that the combination of MET + RAPA was more effective than either agent alone in preventing PCa progression, this was most likely due to the dose of RAPA used which completely suppressed formation of invasive ACs. However, analyses of mTORC1 and inflammation signaling suggested the possibility that careful dose selection could reveal possible additive or synergistic effects of this combination in future studies. Collectively, the current results suggest that targeting mTORC1 and/or inflammation signaling may be an effective strategy for reducing PCa progression and PCa specific mortality.

Supplementary Material

Acknowledgments

Grant Support: This study was supported by NIH grant P50 CA140388 and Start-up funds from the University of Texas at Austin. Dr. Achinto Saha was supported by Cancer Prevention Research Institute of Texas postdoctoral trainee award under grant RP101501 from the State of Texas.

Footnotes

The authors disclose no potential conflicts of interest.

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