Genetic ablation of FASN attenuates the invasive potential of prostate cancer driven by Pten loss

Abstract Loss of the tumor suppressor gene Pten in murine prostate recapitulates human carcinogenesis and causes stromal proliferation surrounding murine prostate intraepithelial neoplasia (mPIN), which is reactive to microinvasion. In turn, invasion has been shown to be regulated in part by de novo fatty acid synthesis in prostate cancer. We therefore investigated the effects of genetic ablation of Fasn on invasive potential in prostate‐specific Pten knockout mice. Combined genetic ablation of Fasn and Pten reduced the weight and volume of all the prostate lobes when compared to single knockouts. The stromal reaction to microinvasion and the cell proliferation that typically occurs in Pten knockout were largely abolished by Fasn knockout. To verify that Fasn knockout indeed results in decreased invasive potential, we show that genetic ablation and pharmacologic inhibition of FASN in prostate cancer cells significantly inhibit cellular motility and invasion. Finally, combined loss of PTEN with FASN overexpression was associated with lethality as assessed in 660 prostate cancer patients with 14.2 years of median follow‐up. Taken together, these findings show that de novo lipogenesis contributes to the aggressive phenotype induced by Pten loss in murine prostate and targeting Fasn may reduce the invasive potential of prostate cancer driven by Pten loss. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.


Introduction
Prostate cancer (PCa) is the second most common cancer in men worldwide and is the second leading cause of cancer death in American men (World Health Organization -Globocan, 2016). Deletions and mutations in the tumor suppressor gene phosphatase and tensin homologue (PTEN), which encodes the PTEN protein, are among the most frequent alterations found in prostate cancer, particularly in the metastatic setting [1][2][3]. Loss of PTEN expression is found in 17% of primary and 40.7% of metastatic prostate cancer and is associated with worse prognosis and shorter recurrence-free survival [3][4][5][6][7][8][9], especially in ERG-fusion negative samples [10,11]. PTEN loss of activity due to mutations or deletions results in PIP3 accumulation and activation of the PI3K/AKT pathway [12,13]. In fact, PI3K/AKT is the most frequently activated pathway in human malignant neoplasms, including prostate cancer [1,10,14].
Activation of PI3K/Akt signaling enhances invasive potential by modulating different targets in the cell, which facilitates prostate cancer progression. In human cells, PTEN overexpression decreases cell proliferation and migration due to reduced AKT phosphorylation [15]. Markers of invasion such as urokinase-type plasminogen activator (uPA) and matrix metalloproteinase (MMP)-9 are downregulated with PTEN expression or PI3K/AKT blockade [16]. In human and murine prostate cancer cells, increased expression of Cxcl12 and its receptor Cxcr4 follows PI3K/Akt activation, inducing cellular invasion and tumor growth [17]. Bone metastases are enhanced by angiogenesis induced by N-cadherin through PI3K/AKT signaling [18], and by activation of the bone morphogenetic protein (BMP) signaling cascade through the PI3K/AKT-NF-κB axis [19].
Fatty acid synthase (FASN) enzyme is an androgenregulated gene, responsible for de novo synthesis of long-chain fatty acids. Lipid synthesis is essential for new cellular membrane synthesis required during cell division, for the post-translational modification of signaling molecules, as well as for energy storage [20]. FASN facilitates oncogenesis when overexpressed in the mouse [21]. FASN is expressed at low to undetectable levels in normal tissues, except in breast during lactation, in liver, and in proliferative phase endometrium [22]. Also, non-malignant cells typically acquire palmitate from the diet [23] and FASN inhibition minimally affects normal cells, including prostate [24]. On the other hand, elevated levels of FASN were found in prostates of transgenic mice of prostate adenocarcinoma (TRAMP) and its levels increased with age, tumor progression, and metastasis [25]. FASN is overexpressed in human prostate cancer, especially in the metastatic, castrationresistant setting [26][27][28][29][30], and is associated with poor prognosis [29,31]. The lipid biosynthesis process has been associated with invasion and metastasis. Inhibition of acetyl-CoA carboxylase (ACC), another essential enzyme for the biosynthesis of fatty acids, decreases invadopodia formation and the ability of prostate cancer cells, among others, to invade [32]. In addition, FASN inhibition with siRNA in LNCaP cells reduces pseudopodia and invadopodia formation, cell adhesion, migration, and invasion [33]. Also, FASN inhibition with shRNA mediates actin cytoskeletal remodeling by decreasing the palmitoylation of Rho GTPases and the downstream activation of Cdc2, resulting in reduced prostate cancer cell migration [34]. Finally, FASN inhibition negatively regulates cytosolic phospholipase A2 (PLA2G4A) and estradiol 17-beta-dehydrogenase 12 (HSD17B12) in several prostate cancer cell lines. These enzymes, responsible for arachidonic acid and androgen production, respectively, when downregulated cause a decrease of downstream genes such as RGS2, SPAG16, VWF, and RAP2B, which are also involved in cellular motility, proliferation, and extracellular matrix interaction [33].
Somatic heterozygous deletion of Pten in mice results in prostatic intraepithelial neoplasia (PIN) by 10 months of age, with approximately 100% penetrance but not metastasis [35]. In contrast, prostate-specific homozygous deletion of Pten results in PIN, which progresses to invasive carcinoma by 12-29 weeks. Lymph node metastases occur in 45% and lung metastases in 27% of the mice [35]. Overexpression of FASN and altered metabolism in prostate cancer cells are associated with inactivation of PTEN [28,36,37]; in contrast, PTEN expression is inversely correlated with FASN expression in prostate cancer [38], while inhibition of PTEN leads to the overexpression of FASN in vitro. Here, we characterize the role of FASN inactivation in modulating the invasive propensity of PTEN knockout (KO) in a genetically engineered modified mouse (GEMM) model.

Mice
Animal studies were performed according to ARRIVE guidelines and approved by the Dana-Farber Cancer Institute (DFCI) Institutional Animal Care and Use Committee (IACUC, Protocol number: 13-014) in accordance with the NIH Guide for the Care and Use of Laboratory Animals; to the state and federal Animal Welfare Acts; and to institutional guidelines [Beth Israel Deaconess Medical Center (BIDMC) and DFCI]. All animals were maintained in an isolated environment in barrier cages. The generation of single and double KO mice for Fasn and Pten was conducted in three phases: colony establishment (phase I), generation of the experimental breeders (phase II), and generation of experimental cohorts (phase III). Phases I and II were conducted at the Preclinical Murine Pharmacogenetics Core Facility (Department of Medicine, BIDMC) and phase III was conducted at DFCI. Details are provided in the Supplementary materials and methods.

Prostate tissue dissection and processing
At the endpoints (12 or 40 weeks), mice were weighed and euthanized by CO 2 inhalation followed by cervical dislocation. Prostates were collected, weighed, and the anterior prostate (AP), dorso-lateral prostate (DLP), and ventral prostate (VP) lobes were dissected as previously described [39], individually weighed and measured, and immediately fixed in 10% formaldehyde. Prostate tissues were paraffin-embedded for histology and immunohistochemistry.

Immunohistochemistry and Herovici's staining
Immunohistochemical staining of FASN, PTEN, p-Akt, and Ki-67 was performed on the Leica Bond Rx automated immunostainer (Leica Microsystems, Wetzlar, Germany). Details are provided in Supplementary materials and methods. Herovici's collagen staining was performed as recommended by the manufacturer (StatLab, McKinney, TX, USA) and described by Friend [40]. Briefly, following xylene and ethanol washes, slides were incubated with Herovici solution (to stain young collagen and reticulum blue, mature collagen red, and cytoplasm yellow), washed with 1% acetic acid, and incubated with Weigert's hematoxylin. Reactive stroma was calculated only in the prostate lobes of animals where Fasn KO occurred in more than 50% of the epithelial cells by measuring the area of stroma normalized by the total area of the lobe using ImageJ software (National Institutes of Health, Bethesda, MD, USA).

Wound healing assay and proliferation
Pten +/− cells (8 × 10 5 ) were seeded in 24-well plates in medium containing 500 nM IPI-9119 (US Patents 8,546,432 and 9,346,769, developed by Infinity Pharmaceuticals, Cambridge, MA, USA) or vehicle (DMSO). After 20 h, confluent cell monolayers were scraped with a sterile 200-μl pipette tip to create cell-free areas. After three washes with PBS, medium containing the drug or vehicle was replaced and cells were incubated for 24 h. Images were obtained at 0 and 24 h and cell migration was analyzed with ImageJ. To confirm a specific FASN effect on migration, we also transfected Pten +/− cells for 24 h with 50 nM Fasn siRNA (#MSS274301 and #MSS236464; ThermoFisher Scientific, Waltham, MA, USA) and control siRNA (#12935300; ThermoFisher) before seeding cells as a confluent monolayer. Following wound formation, cells were imaged at 0 and 24 h (a total of 48 h incubation with siRNA). To exclude interference of proliferation, we concomitantly counted viable cells. Cells were plated as described and after 48 h were trypsinized and counted using the Vi Cell XR analyzer (Beckman Coulter, Fullerton, CA, USA) based on the trypan blue exclusion method.

Western blotting
To confirm FASN knockdown with siRNA, we perform a western blot [20] in the same conditions and at the same time points as the functional assays (proliferation, wound healing, and invasion). Forty-eight and 72 h after siRNA incubation, Pten +/− cells were collected and lysed in RIPA buffer with the addition of phosphatase and protease inhibitor cocktail (Sigma-Aldrich and Roche, Basel, Switzerland). Protein quantification was performed using the Bradford protein assay (Bio-Rad, Hercules, CA, USA) and equal amounts of protein were resolved on precast Tris-glycine polyacrylamide gels (Invitrogen). After transfer to nitrocellulose blotting membrane (Sigma), samples were incubated with primary antibodies to FASN (#3180; CST, Danvers, MA, USA) and vinculin (V9131; Sigma-Aldrich).

Study population
We included 660 men diagnosed with prostate cancer who were participants in the Physicians' Health Study (PHS) [42] or Health Professionals Follow-Up Study (HPFS) [43]. The men were diagnosed between 1983 and 2004 and had archival prostate (prostatectomy and TURP) tumor materials for immunohistochemical evaluation. The PHS included two randomized trials investigating aspirin and vitamin supplements in the prevention of cardiovascular disease and cancer among a total of 29,071 male physicians, including 22,071 men aged 40-84 years starting in 1982 and an additional 7,000 men aged ≥50 years starting in 1999, with all receiving annual follow-up questionnaires. The HPFS is an ongoing cohort of 51,529 male health professionals who have been followed with biennial questionnaires since 1986. Incident prostate cancers (ICD-9: 185) were selfreported and confirmed through medical record and pathology report review. Further details have been provided previously [44].

Clinical and follow-up data
We abstracted data on tumor stage, prostate-specific antigen (PSA) level at diagnosis, and treatments from medical records and pathology reports. Standardized histopathologic review by study pathologists of H&E slides of each case provided uniform Gleason grading [45]

Statistical methods for human data
To evaluate if the association of tumor FASN with clinical outcomes is modified by PTEN status, we dichotomized protein expression of FASN based on the median expression as described by Nguyen et al [46] and cross-classified dichotomized FASN and PTEN tumor status. For PTEN, we used the scoring and validation methods described previously [11]. Details are provided in Supplementary materials and methods.

Results
Fasn genetic ablation reduces prostate tumor progression mediated by Pten  Figure S3B,C) mice when compared with Pten single knockout mice. Similar results were observed for AP at 12 weeks ( Figure 1D,E) but not at 40 weeks (supplementary material, Figure S3D,E). Only animals where Fasn KO occurred in more than 50% of the epithelial cells (supplementary material, Figure S4) were included in the analysis.

Prostate-specific Fasn KO reduces stromal reaction surrounding intraepithelial neoplasia and proliferation in Pten KO context
To investigate the reason for the decreased size and weight of prostate lobes in F/P-dKO compared with P-KO mice, histopathological analysis was performed in all prostate lobes and mouse genotypes that had been generated. As control, we confirmed that the prostates from the F-KO group of 12-week-old ( Figure 2A) and 40-week-old mice (supplementary material, Figure S5A) were histologically normal and, as expected, positive for PTEN but negative for p-Akt staining. Also, we observed that whereas Fasn KO showed a chimeric pattern of inactivation ( Figure 2A and supplementary material, Figure S5A), all epithelial cells in Pten KO mice exhibiting PIN were FASNpositive (Figure 2A). Histopathological analysis of experimental groups revealed that the areas of PIN and stroma of anterior and ventral prostate lobes in P-KO mice were 41.05% more extensive compared with F/P-dKO mice (p < 0.0001).
We next studied the impact of FASN inactivation on the stromal reaction. We observed a dramatic difference in the quantity of reactive stroma when the ventral lobes of the P-KO ( Figure 2B) and F/P-dKO groups ( Figure 2C) were compared. The stromal areas in the VP and AP of 12-week-old ( Figure 2D,E) and the VP of 40-week-old F/P-dKO ( Figure 2F,G and supplementary material, Figure S5C) mice were reduced in comparison to P-KO mice (supplementary material, Figure S5B). Importantly, Ki-67 positive staining of epithelial cells in the stroma was reduced by 27.7% and 26.1% in the AP ( Figure 3A) and VP ( Figure 3B), respectively, in the F/P-dKO group when compared with P-KO. On the other hand, proliferation in the acini was reduced by 6.7% and 7.2% in the AP and VP, respectively, in the F/P-dKO group in comparison with P-KO.
Several areas of microinvasion were identified in the P-KO prostate ( Figure 4D,E), while basal layer integrity was preserved in F-KO ( Figure 4A,B) and F/P-dKO mice ( Figure 4G,H). The Herovici stain ( Figure 4C, F,I) demonstrated that the stroma of prostates in the F-KO group ( Figure 4C) was not reactive, devoid of de novo synthesized collagen (blue fibers), while the intense stromal reaction in the invasive areas of the P-KO group ( Figure 4C) is formed by new collagen fibers permeating old and mature collagen fibers (pink). On the other hand, we observed a sparse stroma in the F/P-dKO group, formed by new collagen fibers between tumor islands that are surrounded by old and mature collagen fibers ( Figure 4F), reflective of the loss or deceleration of the invasive phenotype.

FASN depletion reduces PCa cell invasiveness
To confirm the negative effect of Fasn KO on the ability of prostate cancer cells to migrate and invade, we inhibited de novo fatty acid synthesis by genetic and pharmacologic means in Pten +/− prostate cancer cells. The selective FASN inhibitor IPI-9119 [24] reduced both cell migration ( Figure 5A, left panel, and 5B) and invasion ( Figure 5A, right panel, and 5C) in Pten +/− prostate cancer cells, but not their viability ( Figure 5D). Similar results were obtained with FASN knockdown by siRNA ( Figure 5E,F), which was confirmed by western blot ( Figure 5G).

FASN is required for prostate cancer progression 295
Combined Pten loss and FASN overexpression is associated with lethal prostate cancer  Figure 6 show the results of cross-classified FASN/PTEN with lethal prostate cancer. In the multivariable models, tumors with PTEN loss and high FASN expression were associated with a higher risk for lethal progression (HR 2.0; 95% CI 1.1-3.9; p interaction = 0.03).
Several studies have reported that pharmacological or genetic FASN inhibition results in cell cycle arrest, apoptosis, and reduction of prostate cancer progression in xenograft models [24,47,51,52]. Previous studies in

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DC Bastos, CF Ribeiro et al prostate cancer were mostly conducted with FASN inhibitors such as C75, orlistat, and triclosan [53][54][55], whose off-target effects as well as instability and poor solubility/oral availability have limited their use in the clinical setting [56]. However, we recently demonstrated that IPI-9119, a specific and selective inhibitor of FASN, impairs the growth of mCRPC xenografts and human organoids while inducing substantial metabolic reprogramming [24]. In addition, FASN inhibition with TVB-3166 reduced both tubulin palmitoylation and expression, and the combination TVB-3166/paclitaxel decreased prostate (22Rv1) xenograft growth [51]. TVB-2640 (ASC-40, Alcletis and 3V-Bioscience), which shares molecular similarities with TVB-3166, is being used in a phase I clinical trial for patients with solid tumors (NCT02223247) as well as in phase II trials for colon cancer (NCT02980029), KRAS non-small cell lung carcinomas (NCT03808558), astrocytomas (NCT03032484), and ErbB2-positive breast cancer (NCT03179904).
Here, we sought to gain insights into the mechanism responsible for halting progression of castrationresistant prostate tumors with a deficient Pten background. First, we observed that prostate-specific Fasn deletion is associated with a significant reduction of the stromal area surrounding expanding PIN lesions induced by Pten KO. Stromal reaction is likely associated with microinvasion and is typical in P-KO tumors as they advance. Prostate lobes were in fact hardened and enlarged in P-KO mice compared with F/P-KO mice. Inhibition of lipid metabolism has been associated with decreased cellular migration and invasion in several cancer cell lines [57][58][59][60][61][62][63]. In addition, the stroma may play a significant role in tumor progression [64] and contributes to gland enlargement and tumor weight. Thus, lobe weighing is useful to indirectly assess tumor FASN is required for prostate cancer progression 297 progression. Acini in P-KO mice are spaced far apart and occupy proportionally less space compared with F/P-KO. Also, proliferation of epithelial cells dispersed in the stroma was reduced in F/P-dKO compared with P-KO. Although the reduction of epithelial cell proliferation in acini was also significant in the F/P-dKO group, this reduction was less pronounced compared with the Ki-67 staining reduction in the stroma. These results suggest that prostate-specific FASN inactivation affects cancer progression in the early phases of carcinogenesis (12-week-old mice) and that these effects are maintained during tumor progression

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DC Bastos, CF Ribeiro et al (40-week-old mice), especially in the VP. In general, effects on weight, volume, and stromal reaction were more pronounced in the VP than in the AP, especially at 40 weeks. We attribute this to the following reasons: the commonly observed accumulation of liquids in 40-week-old mouse AP, which affects the weight, size,  FASN is required for prostate cancer progression 299 and distance between the acini in the AP, but does not occur in the VP; that the stroma of normal VP is thinner and more delicate than the prominent connective tissue in the stroma of normal AP [65] makes even subtle changes more easily identified in the VP. Consistent with these findings, we have previously shown that knockout of both PI3K mediators (p110 alpha and beta) is required to reverse PIN in the VP, while the loss of p110 beta alone is needed to reverse it in the AP [66]. Thus, the VP is biologically the lobe that requires full PI3K signaling in tumorigenesis and is therefore the more appropriate lobe to assess the differences that we observed. Additionally, it is important to highlight that prostates of F-KO mice were histologically normal, positive for PTEN, and negative for p-Akt staining. Our group has been working with Fasn KO mice for many years [67] and we have found no histological differences between prostate, or other organs, in WT and Fasn KO mice of the same genetic background at all ages (unpublished data). In addition, it is widely reported that pharmacologic inhibition of FASN minimally affects normal prostate cells [24]. Also, we observed that whereas F-KO showed a chimeric pattern of inactivation in epithelial cells, PIN lesions arising in P-KO are all FASN-positive.
Inhibition of acetyl-CoA carboxylase (ACC) decreases invadopodia formation and the ability of prostate cancer cells to invade [32]. In addition, FASN inactivation with siRNA in LNCaP cells reduces pseudopodia and invadopodia formation, cell adhesion, migration and invasion, and FASN inhibition negatively regulates PLA2G4A and HSD17B12 in several prostate cancer cell lines [33]. Here, we demonstrated that FASN inhibits tumor progression by reducing the migration and invasion of Pten heterozygous prostate cancer cells. Further, in vitro Fasn inactivation by shRNA affects the adhesion, migration, and invasion of prostate cancer cell lines via actin cytoskeletal remodeling, achieved by decreasing the palmitoylation of the GTPase RhoU and consequent inactivation of Cdc42 [34].
FASN overexpression was previously associated with PTEN inactivation [28]. Bandyopadhyay et al [38] demonstrated that inhibition of the tumor suppressor gene Pten was inversely correlated with FASN expression in prostate cancer in the clinical setting. In that study, 43 patients with follow-up of 5 years were examined. Inhibition of PTEN led to the overexpression of FASN in vitro [27]. Here, we validated and extended these data in a cohort of 660 prostate cancer patients with median follow-up of 14.2 years, sufficient to adequately assess outcome [68]. Furthermore, we correlated PTEN and   . Interestingly, FASN did not differ by PTEN status, but tumors with PTEN loss and high FASN expression had the most advanced pathologic and clinical TNM stage. Importantly, in the multivariate models, tumors with PTEN loss and high FASN levels were associated with a higher risk for lethal progression. Considering that the new FASN inhibitor TVB-2640 is the first-in-class in clinical trials, the data provide a rationale for utilizing FASN inhibitors in the treatment of prostate cancer, especially those with Pten loss.
In conclusion, we demonstrate that Fasn genetic ablation reduces invasive potential in the well-established prostate-specific Pten knockout mouse model, which arguably reflects one of the most frequent genetic alterations in human prostate cancer [1]. The reduction in stromal reaction induced by invading Pten-null cells suggests that de novo fatty acid synthesis is required for the invasive and migratory potential of Pten-loss prostate cancer cells. Thus, targeting lipid metabolism represents an attractive therapeutic strategy in advanced prostate cancer, especially in tumors harboring Pten loss.

SUPPLEMENTARY MATERIAL ONLINE
Supplementary materials and methods