© 2018. Published by The Company of Biologists Ltd | Journal of Cell Science (2018) 131, jcs213579. doi:10.1242/jcs.213579

RESEARCH ARTICLE PEDF regulates plasticity of a novel lipid–MTOC axis in prostate -associated fibroblasts Francesca Nardi1, Philip Fitchev1, Omar E. Franco1, Jelena Ivanisevic1, Adrian Scheibler1, Simon W. Hayward1, Charles B. Brendler1, Michael A. Welte2 and Susan E. Crawford1,*

ABSTRACT 2008; Du and Che, 2017). Unlike normal prostate fibroblasts (NFs) Prostate tumors make metabolic adaptations to ensure adequate and wound healing myofibroblasts, CAFs remain chronically active energy and amplify regulators, such as centrosomes, to owing to the influx of soluble tumor-derived factors, and they sustain their proliferative capacity. It is not known whether cancer- strongly resist reversion to a normal quiescent phenotype (Li et al., associated fibroblasts (CAFs) undergo metabolic re-programming. 2007; De Waver et al., 2008; Franco and Hayward, 2012; Kalluri, We postulated that CAFs augment lipid storage and amplify 2016). In addition to the crucial role CAFs have in remodeling the centrosomal or non-centrosomal microtubule-organizing centers microenvironment, a limited number of studies have shown that (MTOCs) through a pigment epithelium-derived factor (PEDF)- CAFs can serve as a local source of energy to help tumors in dependent lipid–MTOC signaling axis. Primary human normal sustaining growth. In breast cancer, CAFs assist in meeting the prostate fibroblasts (NFs) and CAFs were evaluated for lipid energy demands of tumor cells by secreting lactate and pyruvate as content, triacylglycerol-regulating , MTOC number and energy metabolites through the glycolytic pathway, a well-described distribution. CAFs were found to store more neutral lipids than NFs. metabolic adaptation within the tumor microenvironment (TME) Adipose triglyceride lipase (ATGL) and PEDF were strongly known as the Warburg effect (Warburg, 1956; Pavlides et al., 2009; expressed in NFs, whereas CAFs had minimal to undetectable Gonzalez et al., 2014). Cancer cells can, in turn, utilize these energy levels of PEDF or ATGL . At baseline, CAFs demonstrated metabolites in the mitochondrial tricarboxylic acid (TCA) cycle, MTOC amplification when compared to 1–2 perinuclear MTOCs thereby, promoting energy production to increase their proliferative consistently observed in NFs. Treatment with PEDF or blockade capacity. This cooperation between CAFs and cancer cells requires of lipogenesis suppressed lipid content and MTOC number. In some intrinsic metabolic sensors to detect energy deficits to summary, our data support that CAFs have acquired a tumor-like mobilize nutrients and accelerate the cell cycle for rapid growth. phenotype by re-programming lipid metabolism and amplifying Although glucose is often the first source for metabolic needs, more MTOCs. Normalization of MTOCs by restoring PEDF or by blocking recent studies revealed that tumor cells actively mobilize lipogenesis highlights a previously unrecognized plasticity in intracellular lipid stores to support their growth (Gazi et al., 2007; centrosomes, which is regulated through a new lipid–MTOC axis. Nieman et al., 2011). To date, however, no studies have focused on metabolic re-programming in CAFs or the impact of a lipid-rich This article has an associated First Person interview with the first microenvironment on the phenotype of CAFs. author of the paper. Intracellular lipid metabolism and homeostasis are regulated by selective proteins and highly dynamic organelles called lipid KEY WORDS: PEDF (SERPINF), ATGL (PNPLA2), CAF, Centrosome, droplets (LDs). LDs are sites of lipid storage, membrane synthesis MTOC, β-catenin and trafficking of cargo proteins throughout the cytoplasmic compartment (Murphy, 2001). They are formed by a core of INTRODUCTION neutral lipids containing triacylglycerol (TAG) and cholesterol esters Cancer-associated fibroblasts (CAFs), a specialized group of (CEs) that are surrounded by a phospholipid monolayer, with proteins activated fibroblasts, are important contributors in tumor–stroma either embedded in this monolayer or attached to its surface (Zehmer crosstalk by facilitating the proliferation of cancer cells and by et al., 2009; Khor et al., 2013). LDs participate in lipid flux by remodeling the extracellular matrix to support tumor invasion undergoing an active cycle of lipolysis; this metabolic process involves (Franco and Hayward, 2012; Gandellini et al., 2015; Banerjee et al., several proteins, most of which are localized on the surface of the LD. 2017). Despite their pro-tumorigenic role, most studies have This group of surface proteins includes adipose triglyceride lipase documented epigenetic changes in CAFs rather than a gain of an (ATGL; officially known as PNPLA2) (Zechner et al., 2009), oncogene or loss of a tumor suppressor, which is more common in comparative identification 58 (CGI-58; officially known as their neighboring tumor epithelial cells (Hu et al., 2005; Qiu et al., ABHD5) (Young and Zechner, 2013; Boeszoermenyi et al., 2015), members of the perilipin family (Khor et al., 2013) and pigment 1Department of Surgery, NorthShore University Research Institute, Affiliate of epithelium-derived factor (PEDF; officially known as SERPINF1) University of Chicago Pritzker School of Medicine, Evanston, IL 60201, United (Chung et al., 2008; Borg et al., 2011; Zhang et al., 2015), and they States. 2Department of Biology, University of Rochester, Rochester, NY 14627, stimulate lipolysis and the release of free fatty acids (FFAs). Factors that United States. regulate lipogenesis tend to reside in the cytoplasm but crosstalk *Author for correspondence ([email protected]) between other TAG pathway members is essential to maintain the net lipid balance in normal cells. For example, while diacylglycerol O- F.N., 0000-0003-1568-8517; A.S., 0000-0002-5472-4632; S.E.C., 0000-0003- 3890-5000 acyltransferase 1 (DGAT1) promotes TAG synthesis (Sachdev et al., 2016), the G0/G1 switch protein 2 (G0S2) acts as a potent inhibitor of

Received 29 November 2017; Accepted 10 May 2018 ATGL, and increased activity of one or both of these proteins favors Journal of Cell Science

1 RESEARCH ARTICLE Journal of Cell Science (2018) 131, jcs213579. doi:10.1242/jcs.213579 ectopic lipid accumulation in many cell types (Harris et al., 2011; within CAFs and TAG-regulating proteins, such as PEDF, result in Schweiger et al., 2012; Khor et al., 2013; Cerk et al., 2014). Many aberrant MTOC amplification and create a tumor permissive tumors, including prostate cancer, have a significantly lower level of microenvironment. In the present study, we show that the PEDF (Halin et al., 2010), while some head and neck tumors have a deficiency of PEDF in prostate CAFs results in an increase in mutation in G0S2 (Tokumaru et al., 2004), suggesting that the intracellular LDs, as well as in an MTOC amplification phenotype that pathologic imbalance in the TAG pathway is a common mechanism for consists of multiple MTOCs, consistent with ncMTOCs, dispersed dysregulated lipid metabolism. Owing to the multifunctional properties throughout the cytoplasm. A plasticity of MTOC amplification in of the TAG-related proteins, altered expression of these molecules can CAFs was discovered when restoration of PEDF normalized the negatively impact other fundamental processes, including mitosis, number of MTOCs. PEDF had a similar activity in reducing MTOCs angiogenesis and (Yamagishi and Matsui, 2014; Wang et al., in prostate cancer cells. A novel lipid–MTOC signaling axis was 2015; Zagani et al., 2015; Grace et al., 2017). observed when DGAT1 was inhibited to suppress intracellular LD LDs utilize microtubules (MTs) as tracks for directional movement density, concurrently also reducing the number of MTOCs. An (Bostrom et al., 2005; Orlicky et al., 2013; Welte, 2015) and one study unexpected interaction between MTOCs and LDs was noticed when found that cytoplasmic LDs tend to localize near the microtubule- LDs were found to carry centrosomal proteins, i.e. pericentrin and organizing center (MTOC) in HEK293 cells (Orlicky et al., 2013); γ-tubulin, suggesting that LDs in CAFs can acquire a MTOC-like however, no biological explanation was proposed regarding any phenotype. Taken together, these data suggest that lipid-laden specific function LDs might exert close to an MTOC area. The best- CAFs can modulate MTOC distribution and number through a new studied MTOC is the centrosomal MTOC (cMTOC), which is located PEDF-dependent lipid–MTOC axis. in the perinuclear area, generates a radial MT array and the mitotic spindles during mitosis (Bornens, 2002, 2012). More recent studies RESULTS have expanded the view on MTOCs when differentiated cells were Unlike NFs, human prostate CAFs are deficient in PEDF and found to often develop alternative MTOCs defined as non- ATGL centrosomal MTOCs (ncMTOCs) (Sanchez and Feldman, 2017). It is well known that PEDF exerts anti-tumor and anti-angiogenic These structures are located throughout the cytoplasm and, unlike functions in cancer (Crawford et al., 2001; Filleur et al., 2009; cMTOCs, generate a dynamic and disorganized MT array (Sanchez Becerra and Notario, 2013). PEDF can also regulate the lipid and Feldman, 2017). These findings suggest that ncMTOCs are content through ATGL in other cell types (Chung et al., 2008; Borg involved in non-mitotic processes, such as cell polarity, migration, et al., 2011) and functions as a Wnt inhibitor (Protiva et al., 2015). invasion and intracellular trafficking (Bartolini and Gundersen, At baseline (i.e. in untreated control cells), PEDF (50 kD) was 2006). All of these processes are critically important in the TME. always highly expressed in NFs, whereas in CAFs little to no We postulate that direct crosstalk exists between lipid storage detectable PEDF protein was consistently found (Fig. 1A,B). organelles and MTOCs, and that changes in neutral lipid content Next, we determined whether a pro-lipogenic (oleic acid, OA) or

Fig. 1. Deficiency of PEDF in CAFs and modulation of TAG-related proteins. (A) Western blot analysis of PEDF (50 kD) and G0S2 (11 kD) levels in NF and CAF control (CTR) cells and in NFs and CAFs treated with DGAT1 inhibitor (DGAT1in.), OA or both (OA+DGAT1 in). (B) PEDF density normalized to that of GAPDH in NFs and CAFs treated as in A. (C) G0S2 density normalized b to that of GAPDH in NFs and CAFs treated as in A. (D) Western blot analysis of ATGL (56 kD) and CGI-58 (40 kD) levels in NF and CAF control (CTR) cells and in NFs and CAFs treated with OA, DGAT1 inhibitor or OA+DGAT1 inhibitor. The weaker bands (indicated by the arrow) were used for the quantification analysis in E and F. (E) ATGL density normalized to that of GAPDH in NFs and CAFs treated as in A. (F) CGI-58 density normalized to that of GAPDH in NFs and CAFs treated as in A. Western blot analysis was replicated at least three times and performed in triplicate. Values are the means±s.e.m. Student’s unpaired t-test. *P<0.05, **P<0.01. Journal of Cell Science

2 RESEARCH ARTICLE Journal of Cell Science (2018) 131, jcs213579. doi:10.1242/jcs.213579 anti-lipogenic (DGAT1 inhibitor) environment can alter the LD less molecular mass disappeared in CAFs treated with OA+DGAT1 content by modifying the levels of PEDF. When NFs were treated inhibitor, while it persisted in NFs in response to the same treatment. with OA (lipid stimulus), PEDF protein levels were markedly However, the slight increase in intensity of the band at the higher reduced (Fig. 1A,B), suggesting a mechanism that allows stromal molecular mass in CAFs could be a shift of the lower molecular cells to increase lipid storage. When CAFs were tested under any weight protein (Fig. 1D). Whether the higher molecular weight condition, PEDF was not detectable by western blot. Only low protein observed in OA-stimulated cells represents another isoform levels of the ATGL inhibitor G0S2 were detectable in CAFs of ATGL is not clear. Unlike PEDF and ATGL, levels of the ATGL (Fig. 1A,C). Western blotting was performed to also assess the activator CGI-58 remained relatively constant in untreated NFs and levels of ATGL (56 kD) and CGI-58 (40 kD) in NFs versus CAFs in CAFs (controls), with only a modest reduction within the treatment response to different treatments (Fig. 1D). ATGL and CGI-58 are groups (Fig. 1D,F). These results suggest that PEDF, ATGL and important proteins that are located on the surface of LDs and G0S2 in stromal fibroblasts are sensitive to microenvironmental regulate intracellular lipid metabolism by promoting lipolysis stimuli, and are likely to contribute to the net lipid content. (Eichmann et al., 2015; Lord et al., 2016). Similar to the pattern observed with PEDF, NFs expressed high levels of ATGL at LD density is increased in CAFs compared to that in NFs baseline and the protein was reduced by treatment with OA [122.7± To determine whether changes in TAG-modulating proteins result 1.2 vs 62.7±2.4 (control vs OA); P<0.01; Fig. 1E]. At baseline, in dysregulated lipid storage in prostate stromal cells, we quantified CAFs expressed less ATGL than NFs (64.2±7.0 vs 122.7±1.2; the number of baseline LDs in NFs and CAF, and tested whether P<0.05) and the amount was relatively unchanged after inhibition of the LD density does change in response to various stimuli. The cells DGAT1 (Fig. 1D,E). Interestingly, when NFs or CAFs were lipid were stained with Oil-Red-O, which specifically stains neutral stimulated with OA, a second, more intense, band of higher lipids, constituting the core of LDs (red staining in Fig. 2A,B). molecular mass appeared above the ATGL band, which persisted Under control conditions, both cell types revealed intracellular after DGAT1 inhibitor was added to OA. The less-intense band of LDs, although they were different in number and distribution.

Fig. 2. Higher density and diffuse localization of large LDs in CAFs versus NFs. (A,B) NFs (A) and CAFs (B) were treated with 200 µM OA, 1 µM DGAT1 inhibitor or 200 µM OA+DGAT1 inhibitor for 24 h. After fixation, LDs were visualized with Oil-Red-O (red). Scale bars: 10 µm. (C) Average of the total number of LDs per NF (black) and CAF (gray). (D) Number of LDs per cell and LD size defined by the area: small (0.1-1.00 µm2), medium (M) (1.1-2.5 µm2) and large (L) (>2.5 µm2). n=65. The analysis was replicated at least three times and performed in triplicate. Values are means±s.e.m. Student’s unpaired t-test.

*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. Journal of Cell Science

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CAFs showed a significantly higher mean LD density compared to lipolytic proteins (Fig. 1A,D) could be one mechanism responsible that of NFs (188.5±17.8 vs 66.8±6.8; P<0.0001) (Fig. 2C). To for the size reduction in LDs and the decrease in LD density simulate a lipogenic microenvironment of ectopic lipid observed. accumulation, such as observed in obesity, NFs and CAFs were subjected to a 24 h treatment with 200 µM OA. This treatment Treatment with DGAT1 inhibitor reduces proliferation of regimen increased the mean number of LDs >2-fold in NFs (141.4 CAFs ±8.0 vs 66.8±6.8; P<0.0001), whereas an almost 1.5-fold increase CAFs are known to proliferate at a higher rate than NFs (Kalluri, was evident in CAFs (280.8±30.1 vs 188.5±17.8; P<0.05) 2016). Given that our data showed elevated levels of stored lipids in (Fig. 2C). LD density in NFs never reached the baseline LD level CAFs versus NFs, it was unclear whether modulation of observed in CAFs. To assess whether the lipid-rich phenotype in intracellular lipid would alter cellular proliferation. To assess CAFs or NFs can be suppressed, DGAT1 inhibitor was used to whether reduction of LD density impacts on proliferation of NFs block lipogenesis. This treatment drastically decreased the mean and CAFs, cells were treated for 24 h with DGAT1 inhibitor (that LD density in both cell types, whether added alone (NFs: 2.6±0.4 inhibits lipogenesis). Cell proliferation was then analyzed based on vs 66.8±6.8, P<0.0001; CAFs: 3.3±0.5 vs 188.5±17.8, P<0.0001) the percentage of cells that stained positivity of the proliferating cell or together with OA (NFs: 60.1±11.3 vs 141.4±8.0, P<0.0001; nuclear antigen (PCNA); cells treated with DGAT1 inhibitor were CAFs: 88.4±12.7 vs 280.8±30.1, P<0.0001) (Fig. 2C). To compared to untreated control cells (Fig. 3A,B). Our results showed determine whether or not suppression of lipid resulted in a that treatment with DGAT1 inhibitor reduced in both change in the activation of CAFs, we confirmed that CAFs treated NFs (1.2±0.1 vs 2.1±0.3; P<0.05) and CAFs (1.5±0.2 vs 4.3±0.5; with OA still express alpha smooth muscle actin (α-SMA) and P<0.001) (Fig. 3B). Moreover, in support of other studies, untreated vimentin (data not shown). CAFs demonstrated increased proliferation rate compared to NFs (4.3±0.5 vs 2.1±0.3; P<0.001). Growth of NFs and CAFs was also Compared with NFs, CAFs exhibit heterogeneity in the analyzed by using the MTT Cell Proliferation Assay (Fig. 3C) and, cytoplasmic distribution and size of LDs again, our data showed that treatment with DGAT1 inhibitor, LD localization and size have been shown to influence LD significantly decreased proliferation of both NFs (OD: 0.20±0.00 vs intracellular trafficking and signaling, and the patterns are cell 0.23±0.00; P<0.05) and CAFs (OD: 0.39±0.00 vs 0.50±0.00; type specific (Nielsen et al., 2017). The control group of NFs P<0.0001) when compared to untreated controls (Fig. 3C). These revealed a consistent phenotype, with LDs concentrated in the results suggest that the higher proliferative baseline capacity of perinuclear region of the cell showing a ring-like configuration. This CAFs is due to ready access to stored lipids and/or loss of growth perinuclear distribution did not change, even when LD density inhibition by PEDF. Moreover, elevation of PEDF in NFs could be increased after the treatment with OA. However, when NFs were one mechanism to suppress growth when lipogenesis is blocked by treated with DGAT1 inhibitor, the size of the perinuclear LDs was DGAT1 inhibitor (Fig. 1A). reduced (Fig. 2A). At baseline, LDs in CAFs had a significantly different distribution pattern from NFs. LDs were diffusely CAFs show amplification of MTOCs at baseline and plasticity distributed throughout the cytoplasm from the perinuclear region in response to a lipid stimulus to the leading edge of the cells, and this pattern persisted after the Amplification of the centrosome – or, in general, abnormalities of treatment with OA. When CAFs were treated with DGAT1 the MTOC – has been observed in many tumors and can disrupt inhibitor, the few remaining LDs were mainly located in the astral microtubule organization within cells, promote aneuploidy perinuclear region although some were also found in the peripheral and/or directly promote tumorigenesis, possibly independent of zone (Fig. 2B). genomic instability (Zyss and Gergely, 2009). Since CAFs, unlike Considering the importance of LD size for cargo proteins and cancer cells, are cells that are genetically relatively stable within the signaling, LD size differences were evaluated. Specifically, LDs TME, we sought to determine whether CAFs had acquired any were divided in three groups based on their surface area: small LDs MTOC aberrations similar to those of tumor cells and to test whether (S-LDs, 0.1–1.0 µm2), medium LDs (M-LDs, 1.1–2.5 µm2) and a microenvironmental stimulus, such as a lipid-inducing challenge large LDs (S-LDs, >2.5 µm2). Under baseline conditions, both NFs is capable of modulating MTOC number. To assess MTOC density and CAFs contained mainly small and some medium-sized LDs. in CAFs compared to NFs, we performed immunofluorescence Compared to NFs, the mean number of S-LDs and M-LDs in CAFs staining of the pericentriolar matrix protein pericentrin, commonly was higher (S-LDs: 138.6±15.3 vs 62.0±7.1, P<0.0001; M-LDs: used to denote the presence of both types of MTOC, i.e. cMTOC 48.6±11.9 vs 4.2±0.8, P<0.0001) (Fig. 2D). In control CAFs and and ncMTOC (Dictenberg et al., 1998). As expected, all NFs NFs no L-LDs were observed; but, unsurprisingly, treatment with revealed one or two pericentrin-stained foci located very close to the OA increased the number of M-LDs and L-LDs in both NFs and nucleus (Fig. 4A). The MTOC number in NFs remained the same CAFs compared to control cells (NF M-LDs: 71.5±5.6 vs 4.2±0.8, despite any exogenous treatment regimen. In contrast, the MTOC P<0.000; NF L-LDs: 13.8±1.3 vs 0.5±0.2, P<0.0001; CAF M-LDs: density and distribution in CAFs were strikingly different. At 133.9±20.5 vs 48.6±11.9, P<0.01; and CAF L-LDs: 24.1±6.1 vs baseline, CAFs demonstrated a significant increase in pericentrin 1.3±0.3; P<0.01) (Fig. 2D). However, the mean number of S- and foci (Fig. 4B,C) compared to NFs. These MTOCs localized not only M-LDs in CAFs was still higher than in NFs (S-LDs: 106.9±18.0 vs to the perinuclear region but were also spread to the cytoplasmic 57.7±4.8, P<0.01; M-LDs: 133.9±20.5 vs 71.5±5.6, P<0.001). edge of the cell (Fig. 4B,C,E). We discovered that the MTOC Finally, to determine whether the DGAT1 inhibitor impacts on LD number in CAFs varied significantly and appeared to act as a size, both NFs and CAFs were treated with either DGAT1 inhibitor metabolic sensor because alteration of lipid content in response alone, or DGAT1 inhibitor with or without OA. Treatment with to a microenviromental stimulus changed MTOC density. DGAT1 inhibitor decreased the size of LDs significantly, especially Administration of OA markedly increased MTOC density those of S-LDs in both NFs and CAFs (Fig. 2D). A concurrent (156.7±16.2 vs 69.1±10.6; P<0.0001) and treatment with DGAT1 increase in lipolysis due, in part, to the marked expression of pro- inhibitor simultaneously reduced the density of LDs and ncMTOCs Journal of Cell Science

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Fig. 3. DGAT1 inhibitor treatment reduces cell proliferation in NFs and CAFs. (A) NFs and CAFs were treated with DGAT1 inhibitor (DGAT1in.) for 24 h, and after fixation, the cells were stained with PCNA antibody. Scale bars: 10 μm. (B) Percentage of cells positive for PCNA staining/total cells was evaluated. n=200 cells. (C) NFs and CAFs were treated with DGAT1 inhibitor and the proliferation rate was analyzed by the MTT Proliferation Assay. n=50 cells. The cell proliferation analysis was replicated at least three times and performed in triplicate. Values are means±s.e.m. Student’s unpaired t-test. *P<0.05, ***P<0.001, ****P<0.0001.

(40.5±7.0 vs 69.1±10.6; P<0.05) (Fig. 4C,D). This MTOC number, both agents were used in one experimental group. No sensitivity to intracellular lipid content suggests a new signaling significant advantage was observed beyond the ‘normalization’ of pathway between MTOCs and LDs. In addition to the number of MTOC number by treatment with PEDF alone (Fig. 5A,C). MTOCs, the composition of the MT network in CAFs was different Moreover, the few [1–2 MTOCs remaining post treatment tended from the pattern observed in NFs. The MT network in CAFs to be slightly larger and were located in the perinuclear region of the appeared to be less organized, especially in perinuclear and cell, the typical site of cMTOCs (Fig. 5B)]. Assessment of the peripheral regions, compared with the more organized MTs in pericentrin ‘dot’ area in control CAFs revealed a variation in size NFs (Fig. 4A,B). with a mean area of 13.9±1.0 µm2 (Fig. S1). When CAFs were treated with PEDF the 1–2 pericentrin dots left appeared more Restoration of PEDF in CAFs selectively reduces the density uniform in size and overall larger with a mean area of 23.1±3.6 µm2. of MTOCs, MTs and LDs These larger pericentrin dots were often irregular and multi-lobular To assess whether restoration of PEDF normalized MTOC number in shape, suggesting an aggregation of several MTOCs. These data or the MT network in CAFs, cells were treated for 48 h with 10 nM suggest that PEDF treatment not only normalizes the number of PEDEF, PEDF+OA or PEDEF+DGAT1 inhibitor. The MTOC MTOCs but also promotes clustering or aggregation of several density and MT morphology were analyzed (Fig. 5A,B) and MTOCs. Treatment with PEDF also triggered an unexpected but revealed that treatment of CAFs with exogenous PEDF, compared to consistent structural change in the MT network. The PEDF- untreated CAFs, significantly reduced the number of pericentrin- deficient CAF phenotype of dense and disorganized MTs was stained MTOCs by 98.1% (1.3±0.1 vs 69.1±10.6; P<0.0001) remodeled to a less dense and more radial MT configuration in the (Fig. 5C). When lipid-stimulated CAFs were treated with PEDF, the perinuclear region when PEDF was restored (area occupied by MTs: MTOC number normalized to close to one MTOC (1.4±0.2 vs 43.0%±0.6 vs 35.8%±0.8; P<0.001) (Fig. 5A,D). The peripheral 156.7±16.2; P<0.0001) (Fig. 5A,C). To determine if the addition of MT component in CAFs that remained post-treatment was less

DGAT1 inhibitor synergized with PEDF in modulating the MTOC dense but retained higher complexity. Journal of Cell Science

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Fig. 4. Amplification and plasticity of MTOCs in CAFs. NFs (A) and CAFs (B) were stained for α-tubulin (red) and pericentrin (green) to visualize the MT network and the MTOCs, respectively. The nucleus was stained blue by DAPI. Scale bars: 10 µm. (C) CAFs were treated with OA or DGAT1 inhibitor and compared to untreated controls (CAF CTR). The cells were stained with mouse anti-pericentrin antibody and DAPI to identify the MTOCs (green) and nucleus (blue), respectively. Scale bars: 10 µm. (D) The total number of pericentrin-stained MTOCs was counted per single cell. (E) The number of pericentrin-stained MTOCs was stratified based on their localization: perinuclear (black) and peripheral (gray) regions. n=50. All the experiments were replicated at least 3 times and performed in triplicate. Values are means±s.e.m. Student’s unpaired t-test. *P<0.05, **P<0.001, ****P<0.0001.

To assess if restoration of PEDF in CAFs reduced neutral Prostate cancer cells demonstrate MTOC amplification lipid content, CAFs were treated with 10 nM PEDF for 48 h, which is normalized after PEDF treatment fixed and LDs stained with Oil-Red-O. Adding exogenous Centrosome number and MTOC abnormalities have long been PEDF promoted a 73.3% decrease in the number of LDs per implicated in cancer; they can cause chromosomal instability and cell compared to CAF baseline (50.4±5.3 vs 188.5±17.8; loss of tissue architecture, two of the most common phenotypes P<0.0001) (Fig. 5G). Interestingly, when PEDF treatment was observed during tumorigenesis (Nigg, 2006). To assess MTOC combined with addition of DGAT1 inhibitor, CAFs showed density in prostate cancer cells, immunofluorescence staining was increased lipid catabolism with a significant decrease in the performed in LNCaP and PC3 cells to detect the presence of the number of LDs per cell compared to treatment with PEDF alone pericentriolar matrix protein pericentrin. Both LNCaP and PC3 cells (2.1±0.5 vs 50.4±5.3; P<0.0001) or DGAT1 inhibitor alone demonstrated an increase in pericentrin-positive foci (within the (2.1±0.5 vs 4.2±0.7; P<0.05) (data not shown). These data range of 3–10 per cell), with many of the ncMTOCs diffusely suggest that PEDF can synergize with the DGAT1 inhibitor to located throughout the cytoplasm of the cell (Fig. 5E,F). Since decrease LD density in CAFs. PEDF treatment of CAFs markedly addition of PEDF to CAFs resulted in a significant change in MTOC reduced the number of MTOCs to 1–2 per cell, whereas this amplification by normalizing their numbers back to ∼1 perinuclear same treatment was only able to suppress LD density to a mean centrosome, we investigated whether PEDF had the same effect in of 50 LDs/cell. cancer cells. The analysis revealed that, compared to untreated Journal of Cell Science

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Fig. 5. Restoration of PEDF normalizes MTOC number and MT density. CAFs were treated with 10 nM PEDF±200 µM OA or 1 µM DGAT1 inhibitor for 48 h. (A) CAFs were stained with rabbit anti-α-tubulin, mouse anti-pericentrin antibodies and DAPI to show the MT network (red), the MTOCs (green) and the nucleus (blue), respectively. Scale bars: 10 µm. (B) CAFs were stained with mouse anti-pericentrin antibody (green) and DAPI (blue) to visualize the MTOCs and the nucleus, respectively. Scale bars: 5 µm. (C) The number of pericentrin foci was counted in CAFs. n=50. (D) MT density was determined by the area occupied by microtubules (in %) per cell occupied by microtubules. n=50. (E) LNCaP and PC3 cells were stained with mouse anti-pericentrin antibody and DAPI to visualize the MTOCs (green) and the nucleus (blue), respectively. Scale bars: 10 µm. (F) The number of pericentrin-positive foci in LNCaP and PC3 per single cell. n=50. (G) The change in MTOCs and LD densities were analyzed in CAFs treated with PEDF versus the control (CTR). Neutral lipids in LDs were stained with Oil-Red-O (red) in CAFs treated with PEDF. Scale bar: 10 µm. n=50. All the experiments were replicated at least 3 times and performed in triplicate. Values are means±s.e.m. Student’s unpaired t-test. ***P<0.001, ****P<0.0001. control cells, treatment with exogenous PEDF significantly reduced most likely to represent ncMTOCs and/or pericentrin-positive the number of pericentrin-positive MTOCs in both LNCaP cells LDs. When CAFs were treated with exogenous PEDF, the number (1.4±0.1 vs 5.2±0.5; P<0.0001) and PC3 cells (1.3±0.1 vs 4.7±0.6; of foci positive for centrin and pericentrin reduced to 1–2 P<0.0001) (Fig. 5E,F). (CAFs+PEDF versus CAFs CTR: 1.3±0.1 vs 4.0±0.2; P<0.0001), demonstrating that PEDF is also able to normalize the cMTOCs in CAFs demonstrate amplification of both cMTOCs and CAFs (Fig. 6A,C). To assess MT nucleation, re-growth assays ncMTOCs were performed and analyzed in control and PEDF-treated cells To determine whether the amplified MTOC population in CAFs (Fig. 6D). At baseline, after 30 s of regrowth, NFs showed a single exhibited typical features of centrosomes, immunofluorescence MT array that appeared organized, whereas CAFs exhibited staining for pericentrin and centrin was performed (Fig. 6A). NF several f MT nucleation foci and their MT arrays appeared less control cells consistently had a single pericentrin-positive/centrin- symmetrical (Fig. 6D magnification). Treatment with PEDF positive focus. In contrast, CAF, LNCaP and PC3 control cells reduced the number of MTOCs in CAFs to 1 and, in this group, demonstrated three or more structures with dual staining for the MTs appeared shorter (Fig. 6D), which concurs with our other centrin and pericentrin supporting that these are consistent with finding demonstrating reduced MT density in PEDF-treated cells cMTOCs (CAFs versus NFs: 4.0±0.2 vs 1.2±0.1, P<0.0001; (Fig. 5A,D). Since the analysis of MTOC amplification in CAFs LNCaP versus NFs: 3.4±0.2 vs 1.2±0.1, P<0.0001; PC3 vs NFs: revealed more pericentrin-stained ncMTOCs than expected and 4.1±0.2 vs 1.2±0.1, P<0.0001) (Fig. 6A,B). Some other structures the distribution patterns involved regions away from the nucleus, in these cells stained only positive for pericentrin and most were we investigated whether or not LDs interact more directly with Journal of Cell Science

7 RESEARCH ARTICLE Journal of Cell Science (2018) 131, jcs213579. doi:10.1242/jcs.213579

Fig. 6. CAFs demonstrate amplification of both cMTOCs and ncMTOCs. (A) NFs, CAFs, LNCaP and PC3 cells were stained with mouse anti-pericentrin, rabbit anti-centrin 1 antibodies and DAPI to visualize the cMTOCs (green) and/or ncMTOCs (red) and the nucleus (blue), respectively. Scale bars: 5 μm. (B) The number of centrosomes was counted in NFs, CAFs, LNCaP and PC3 cells. n=25. (C) The number of centrosomes was counted in CAFs treated with PEDF and compared to CAFs baseline. n=25. (D) MT regrowth assay was performed in NFs and CAFs and the cells were fixed after 30 s and stained for pericentrin and α-tubulin. Scale bars: 10 μm. (E) CAFs were stained with mouse anti-γ-tubulin antibody, BODIPY and DAPI to visualize γ-tubulin (red), lipid droplets (green) and nuclei (blue), respectively. Scale bars: 5 μm. Arrowheads indicate colocalization; boxed areas in merged images indicate the magnified areas in bottom right corner of each image. n=50. All the experiments were replicated at least 3 times and performed in triplicate.

MTOCs and, possibly, carry MTOC matrix proteins, such as PEDF acts as a Wnt inhibitor and decreases the expression of pericentrin or γ-tubulin, on their surface. Immunofluorescence β-catenin in CAFs staining was performed to assess a possible colocalization PEDF is known to be an inhibitor of the Wnt/β-catenin pathway, between γ-tubulin and LDs in untreated CAFs (Fig. 6E), which regulates several processes including angiogenesis, showing that a subset of LDs appears to be positive for inflammation and fibrosis (Park et al., 2011; Protiva et al., 2015). centrosomal proteins. It is possible that pericentrin and/or γ- In prostate cancer, β-catenin is known to be overexpressed within tubulin positivity in a subset of LDs (30–40%) represents cargo the nuclear compartment, where it can chronically activate the proteins on the LD surface. Although this would be the first transduction of several implicated in tumor growth (Kypta observation of a centrosomal protein associated with LDs, these and Waxman, 2012). More recently, β-catenin has been recognized organelles are known to actively shuttle a variety of proteins to have additional activities related to centrosomes. β-catenin within the cytoplasm of several cell types (Murphy, 2001). was found to colocalize with pericentrin during interphase and Journal of Cell Science

8 RESEARCH ARTICLE Journal of Cell Science (2018) 131, jcs213579. doi:10.1242/jcs.213579 mitosis, and to be involved in centrosome segregation and β-catenin – the functional modulator of centrosome morphology mitotic spindle orientation (Bahmanyar et al., 2010; Mbom et al., and distribution – were markedly decreased in both the cytoplasmic 2013). To assess whether PEDF can change MTOCs was through and nuclear compartments of CAFs, which was confirmed by inhibition of the Wnt pathway, β-catenin levels were analyzed by quantification of total β-catenin and β-catenin phosphorylated at western blotting and immunofluorescence. Fig. 7A shows the levels Y142 (phospho-β-catenin) in western blots (CAF control cells vs of β-catenin (94 kD) in NF and CAF control cells. Compared to CAFs treated with PEDF) (Fig. 7C). These data suggest a new those in CAFs, NFs had lower levels of total-β-catenin. To mechanism for MTOC amplification in CAFs that is related to the investigate whether addition of PEDF inhibits active β-catenin, loss of the PEDF Wnt inhibitory action, thus increasing β-catenin immunofluorescence staining was performed, showing intracellular (Mbom et al., 2013; Protiva et al., 2015). localization of active β-catenin in CAFs (Fig. 7B). In control cells, β-catenin was located in both the cytoplasm and nucleus. The In NFs, RNAi of PEDF induces duplication of centrosomes presence of nuclear β-catenin was confirmed using z-stack analysis Restoration of PEDF in CAFs reduces the number of LDs and and by performing vertical and horizontal cuts through the normalizes MTOC density, thus, more resembling the phenotype of nucleus. After treatment with exogenous PEDF, levels of NFs. To assess the impact of PEDF reduction in NFs on MTOC

Fig. 7. PEDF acts as a Wnt inhibitor and decreases the expression of β-catenin in CAFs. CAFs were treated with 10 nM PEDF for 48 h. (A) Total-β-catenin (92 kD) levels were evaluated in NFs and CAFs by western blotting and normalized against those of GAPDH. (B) CAFs were stained with rabbit anti-β-catenin and DAPI to, respectively, visualize the intracellular localization of β-catenin (red) in the cytoplasm and in the nucleus (blue). To analyze whether the protein was localized inside the nucleus, z-stack analysis was performed. Scale bars: 10 µm. (C) Western blot analysis of levels of total-β-catenin and of β-catenin phosphorylated at Y142 normalized to those of GAPDH in untreated CAFs (CTR) and in CAFs treated with PEDF. (D) Western blot analysis of NFs transfected with siRNA targeting PEDF or with control siRNA. (E) NFs transfected with siRNA targeting PEDF or with control siRNA were stained with mouse anti-pericentrin, rabbit anti-centrin 1 antibodies and DAPI to, respectively, visualize the cMTOCs (green), ncMTOCs (red) and the nucleus (blue). Scale bars: 10 µm. (F) The number of centrosomes was evaluated in NFs silenced for PEDF and in its negative control. n=50. All the experiments were replicated at least 3 times and performed in triplicate. Values are means±s.e.m. Student’s unpaired t-test. ****P<0.0001. Journal of Cell Science

9 RESEARCH ARTICLE Journal of Cell Science (2018) 131, jcs213579. doi:10.1242/jcs.213579 biology, we performed RNA interference (RNAi), in which PEDF influence PEDF or other factors have in the regulation of the TAG was decreased by >50%; pericentrin- and centrin-positive MTOC pathway in the stromal compartment of the prostate. PEDF is a 50 kD density was analyzed by immunofluorescence staining (Fig. 7E). glycoprotein that is expressed in almost all healthy (normal) cells. It Although there was no amplification of MTOC as observed in CAFs, has a broad spectrum of functions including anti-inflammatory, anti- PEDF-deficient NFs showed a significantly higher percentage of angiogenic and anti-tumorigenic activities, and acts as a Wnt inhibitor cells with double centrosomes (showing centrin- and pericentrin- (Dawson et al., 1999; Crawford et al., 2001; Chung et al., 2008; Filleur positive staining) when compared to the control group (62.8±3.5 vs et al., 2009; Becerra and Notario, 2013; Protiva et al., 2015). PEDF 13.5±2.9, P<0.0001) (Fig. 7F). These data suggest that PEDF influences systemic fatty acid metabolism by enhancing lipolysis, and deficiency results in the loss of a crucial inhibitory signal that is by promoting lipid accumulation in skeletal muscle and liver. The related to cell cycle progression or the early stages of centrosomal lipolytic activity of PEDF was discovered when PEDF null mice amplification. It is not surprising that deficiency of PEDF only was demonstrated hepatic steatosis, and when human hepatocytes required insufficient to induce amplification because other studies have interactions between PEDF and ATGL to regulate their lipid content shown that single knockdown of even was unable to trigger (Chung et al., 2008; Borg et al., 2011). In our study here, ATGL and amplification (Nigg and Holland, 2018). PEDF were found to be strongly expressed in NFs, an observation that was in contrast to the much reduced levels observed for the lipolysis DISCUSSION inhibitor protein G0S2. Prostate-derived CAFs had minimal to Metabolic adaptations are known to occur with high frequency in undetectable levels of PEDF and ATGL, which were lower than tumor epithelial cells to help meet the demands of their high those recovered in NFs. The limited availability of the key lipolysis- proliferative capacity (Levine and Puzio-Kuter, 2010; Satoh et al., regulating proteins PEDF and ATGL are likely to contribute to the net 2017). Cancer cells can revert to lipid stores to obtain the needed energy gain of stored lipid observed in CAFs, because restoration of PEDF and to alter cell cycle regulators, such as centrosomes, to fuel effectively decreased intracytoplasmic lipid content. In one study progression. Measurement of metabolites and lipids associated with investigating melanoma, CAFs expressed low levels of PEDF and loss the metabolic re-programming switch in prostate cancer has the of this protein contributed to tumor progression; however, lipid potential diagnostic use of distinguishing cancer from normal tissue metabolism was not explored (Nwani et al., 2016). Our data, (Banerjee et al., 2014). Much less is known about the spectrum of demonstrating modulation of PEDF, ATGL and G0S2 in CAFs, add metabolic changes made within stromal fibroblasts, specifically CAFs, new lipid mediators for consideration when evaluating aberrant lipid in the TME. In this study, we have demonstrated that human prostate- signaling in tumor–stroma crosstalk. derived CAFs store more neutral lipid in LDs than normal prostate LDs not only store lipid but they can also carry cargo and facilitate fibroblasts (NFs). Each cell type had a unique LD distribution pattern the movement of proteins to various locations within the cell. Studies and size range, which could impact signaling (Welte and Gould, 2017; involving Drosophila were some of the first to recognize that LD Bombrun et al., 2017). Normal fibroblasts had a distinct concentric ring movement is dependent on intact MTs. In Drosophila embryos and of neutral lipids residing close to the nucleus, whereas LDs in CAFs some normal human cells, kinesin 1 and cytoplasmic dynein, i.e. were dispersed throughout the cytoplasm. Intracellular lipid content MT-associated motor proteins, were shown to physically interact and LD size were sensitive to exogenous stimuli, such as lipid with LDs and direct LD motility (Welte, 2015). This cooperation challenge with OA, since both cell types augmented their lipid stores between LDs and MTs could have broader implications in the control and increased their mean LD size; however, the mean LD area was of cellular functions in the TME. MTOCs operate as sites in order to always greater in CAFs. To assess whether lipid storage in CAFs is locate MT minus ends, and function as the point of MT nucleation linked to growth, we blocked the lipogenesis enzyme DGAT1. Both and stabilization by using pericentriolar matrix proteins, such as growth and lipid stores of NFs and CAFs were suppressed, although the pericentrin (Dictenberg et al., 1998; Bornens, 2002). The plus-ends activation of CAFs was not affected. These results suggest that prostate of MTs are more dynamic compared to the slower remodeling CAFs are simultaneously keeping pace with their tumor cell partners minus ends (Voter and Erickson, 1984). Correct MTOC orientation by making pro-lipogenic metabolic adaptations and that the metabolic is crucial for several processes, including mitosis, cellular switch has a broader effect on the TME (Banerjee et al., 2014). In the differentiation and secretion (Vertii et al., 2016; Muroyama and TME of obese patients, where lipid flux is abnormal, it is possible that Lechler, 2017; Sanchez and Feldman, 2017). The centrosome is one CAFs acquire an even higher net lipid content to expand their cell of the best-studied MTOCs (Bornens, 2012). Normal cells have one – population and promote tumor progression (Engin, 2017). occasionally two, when dividing – centrosome, the cMTOC, close to Synthesis or mobilization of stored lipid, such as TAG, requires the nucleus. It is a non-membrane bound organelle surrounded by precise coordination of various enzymes to maintain lipid homeostasis pericentriolar matrix proteins, such as pericentrin and γ-tubulin, and (Zechner et al., 2017). The rate-limiting enzyme ATGL and its responsible for generating a radial array of MTs (Dictenberg et al., activator CGI-58 control lipolysis via the TAG pathway, and these 1998). Pericentrin controls the nucleation of MTs by anchoring the activities are counterbalanced by the lipogenesis enzyme DGAT1 γ-tubulin ring complex to the MTOC (Bornens, 2002). In the (Harris et al., 2011). Other TAG-regulating proteins that influence net cMTOC, precision regarding nucleation is essential for bipolar spindle lipid content include PEDF, which bindsto ATGL (Chung et al., 2008; formation and assembly during mitosis, in order to Borg et al., 2011) and G0S2, which acts as an inhibitor to ATGL ensure correct cell cycle progression. In support of the importance of (Schweiger et al., 2012; Cerk et al., 2014). Mutations in ATGL, CGI- pericentrin, its downregulation in peripheral blood leukocytes has 58 or PEDF can cause a range of human diseases, including systemic been shown to disrupt mitotic checkpoints and to cause arrest at the metabolic disorders or defects in bone development (Marini et al., G2/M checkpoint, leading to cell death (Unal et al., 2014). 2014). Recently, loss of CGI-58 in prostate cancer cells was found to Supernumerary centrosomes, represented by several pericentrin- promote an aggressive phenotype (Chen et al., 2017a,b). In many positive foci that were diffusely located in the cytoplasm, have also , including prostate cancer, there is a stepwise decrease in been associated with increased tumor aggression, raising the notion PEDF expression when tumors become more aggressive (Halin et al., that additional centrosomes are advantageous for tumor growth

2010; Becerra and Notario, 2013). Much less is known about the (Pihan et al., 2001; Chan, 2011; Rivera-Rivera and Saavedra, 2016). Journal of Cell Science

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In support of this concept, centrosome overexpression was found to 1–2 perinuclear MTOCs consistently observed in NFs. Additionally, increase MT nucleation and resulted in the formation of protrusions we observed that restoration of PEDF in CAFs not only normalized and cytoplasmic extensions that invaded the surrounding matrix, the number of MTOCs but also reduced MT density and levels of β- thus, enabling tumors to invade and metastasize (Godinho et al., catenin. A similar reduction in the number of cMTOCs and 2014). Cancers of the breast, ovary, liver and prostate were shown to ncMTOCs was found when cells of the prostate cancer cell lines have structural abnormalities of the centrosome regarding size, shape LNCaP and PC3 were treated with PEDF. One potential mechanism and number, although centrosomes have not been studied in the for these activities included the ability of PEDF to act as a potent Wnt stromal cell population within the TME (Lingle et al., 1998; Pihan inhibitor and reduce levels of activated β-catenin. Overexpression of et al., 2001; Kim et al., 2008). In our study, untreated control CAFs β-catenin in prostate cancer is well-documented (Kypta and Waxman, showed amplification of cMTOCs and ncMTOCs compared to the 2012). β-catenin has also been shown to localize to the centrosome,

Fig. 8. Proposed model of the lipid–MTOC axis. NFs have a single perinuclear centrosome (indicated by one single pericentrin-positive dot) with an organized radial MT network around the nucleus (see PCNT insert for NFs). In contrast, CAFs at baseline exhibit centrosome and/or MTOC amplification (mean MTOC number/cell: 69.1±10.6) in addition to a more complex MT network. Moreover, CAFs at baseline have more stored neutral lipids than NFs (mean LD number/cell in CAF vs NF: 188.5±17.8 vs 66.8±6.8). The addition of PEDF normalizes the number of MTOCs in CAFs and reduces the density of MTs (see the PCNT insert for CAFs treated with PEDF). One possible mechanism for these activities is that PEDF acts as a potent Wnt-signaling inhibitor and reduces the levels of activated β-catenin. Also, the treatment with a DGAT1 inhibitor results in a decrease in the number of both LDs and MTOCs, whereas addition of the lipogenic stimulus OA significantly increases their number (see Oil-Red-O and PCNT inserts for CAFs treated with DGAT1 inhibitor and OA, respectively). In CAFs at baseline, 30-40% of LDs switch to a MTOC-like phenotype and carry the key MTOC matrix proteins pericentrin and/or γ-tubulin on their surface (see

Fig. 6E). These data suggest that lipid-laden CAFs can modulate MTOC numbers through a new PEDF-dependent lipid-MTOC axis. Journal of Cell Science

11 RESEARCH ARTICLE Journal of Cell Science (2018) 131, jcs213579. doi:10.1242/jcs.213579 and it appears to be important in mitotic spindle assembly and access to a MT matrix protein might facilitate modulation of MTs in a microtubule dynamics (Mbom et al., 2013). Its role in ncMTOCs has more efficient manner. We found both the MTOC matrix proteins not been explored. The ability of PEDF to modulate MTs has been pericentrin and γ-tubulin on the surface of a subset of LDs, distributed observed by another group, where PEDF-mediated disassembly of the throughout the cytoplasm in CAFs, thus, emphasizing the importance MT network was a mechanism responsible for increased permeability of testing the functional capacity of pericentrin-positive structures in in vascular endothelial cells (He et al., 2015). Our data, demonstrating the TME. It is unclear whether carrying these matrix proteins a new function of PEDF to normalize MTOC number in CAFs, indicates that LDs can acquire an ncMTOC-like phenotype within a highlight a previously unrecognized plasticity in cMTOCS and/or lipid-rich environment, such as in cells of obese individuals, or MTOC biology and a possible function of MTOCs as metabolic whether easy access of these proteins on the surface of LDs enables sensors, especially ncMTOCs. Furthermore, blockade of lipogenesis them to rapidly remodel MTs to facilitate movement. Additional in response to DGAT1 inhibitor reduced both lipid content and MTOC studies are required to assess the function of MTOC proteins on the number. This discovery of a new lipid–MTOC signaling axis (see LD surface and to investigate whether these proteins still perform model proposed in Fig. 8) that is highly responsive to PEDF and their usual MT-related functions. In summary, our data support DGAT1 inhibitors could prove to be an attractive therapeutic target to that prostate CAFs have acquired a tumor-like phenotype by stabilize tumor growth by simultaneously reducing stored lipid and the re-programming lipid metabolism and amplifying MTOCs. numbers of cMTOCS and ncMTOCs. Normalization of MTOCs by restoring PEDF or by blocking More recently, there has been more interest in characterizing lipogenesis in CAFs highlights a previously unrecognized the pericentrin- or γ-tubulin-stained structures distributed in the plasticity in cMTOC and ncMTOC biology. cytoplasm and peripheral regions of the cell. In addition to the well- described perinuclear centrosome, another type of structure – the MATERIALS AND METHODS ncMTOC, which can vary depending on the cell type –has been Cell culture and reagents described (Sanchez and Feldman, 2017). Studies are emerging to In this study the experiments were performed by using two primary human better characterize the structure and function of ncMTOCs. It normal fibroblast (NF) cultures and three primary cancer-associated appears that ncMTOCs contain proteins that interact with MT fibroblast (CAFs) cultures isolated, respectively, from normal prostate and radical prostatectomy specimens of different patients. CAFs were evaluated minus-ends and help to anchor proteins; however, details of their and tested in an in-vivo tissue recombination model to confirm their pro- function in cellular processes within the TME remain to be tumorigenic potential (Franco et al., 2011). Human prostate cancer cell lines elucidated. Cellular differentiation is one bioactivity that has been LNCaP and PC3 were purchased from ATCC (Manassas, VA). NFs, CAFs linked to activation of ncMTOCs (Sanchez and Feldman, 2017) and and LNCaP cells were cultured at 37°C under 5% CO2 in RPMI medium that is relevant to tumor aggressiveness. Some groups have reported (Thermo Fisher Scientific, Waltham, MA) containing 10% fetal bovine that other organelles, such as mitochondria or the Golgi complex, serum (FBS; Sigma, St Louis, MO) and 1% penicillin/streptomycin, can simulate or convert to ncMTOCs through modulation of various whereas PC3 cells were cultured in DMEM (Thermo Fisher Scientific, proteins (Zhu and Kaverina, 2013; Chen et al., 2017a,b). Recently, a Waltham, MA) containing again 10% FBS and 1% penicillin/streptomycin. – mouse embryonic study revealed a new function for ncMTOCs in After reaching 80 90% confluence, cells were harvested with 0.25% directing intracellular transport of molecules, such as E-cadherin trypsin-EDTA (Thermo Fisher Scientific, Waltham, MA) and passaged at a ratio of 1:2 (confluent cells in solution to fresh medium). After seeding (Zenker et al., 2017). In our current study of lipid metabolism in overnight (at ∼60% confluence), cells were exposed to several treatments: CAFs, we noticed an excessive number of pericentrin-expressing 200 µM oleic acid (OA; Sigma) with or without 1 µM DGAT1 inhibitor cytoplasmic structures outside of the typical perinuclear location of (A-922500, Cayman Chemical, Ann Arbor, MI) for 24 h; 10 nM PEDF a centrosome. Centrin positivity and evidence of MT nucleation (BioProducts MD, Middletown, MD) with or without 200 µM OA for 48 h; were observed in four or more structures, supporting the concept and 10 nM PEDF with or without 1 µM DGAT1 inhibitor for 48 h. that both cMTOCs and ncMTOCs are amplified in CAFs. The group of ncMTOCs that did not show MT nucleation might participate in Oil-Red-O staining some of their other proposed – non-mitotic – functions, such as cell NFs and CAFs were grown on glass coverslips coated with poly-L-lysine differentiation and polarity. NFs contained endogenous lipid and, (Sigma) and cultured and treated as described above. Cells were then washed when stained for pericentrin, only one centrosome was found to be three times with PBS, fixed in 10% formalin (30 min at room temperature) localized close to the nucleus. Occasionally, a dividing cell would and stained with Oil-Red-O (Oil-Red-O Stain, propylene glycol; Newcomer Supply, Middleton, WI) to visualize neutral lipids. Coverslips were mounted have two centrosomes. When NFs were challenged with a lipid- on glass slides and sealed with permaslip. Pictures were taken of promoting stimulus, the centrosome number remained constant at representative fields for each treatment using a 100× objective to count 1. In contrast, untreated control CAFs harbored a significantly higher single intracellular LDs. number of cytoplasmic structures that stained positive for pericentrin and these increased in response to the lipid challenge. The mean Western blotting number of pericentrin-positive structures was more than three times NFs and CAFs cultured on dishes were treated as described above and after higher than the reported number of centrosomes in amplified tumors 24–48 h washed with PBS, scraped and lysed in M-PER+protease inhibitor (Lingle et al., 1998; Pihan et al., 2001). Our results prompted us to test buffer (Sigma). Cell lysates were centrifuged at 8050 g for 20 min at the hypothesis that crosstalk exists between a microenvironmental 4°C; the protein concentration was then determined using Pierce 660 nm lipid challenge and the modulation of centrosomal structures or Protein Assay Reagent (Thermo Fisher Scientific, Waltham, MA) and ncMTOCs. To test this, we blocked lipogenesis by using the DGAT1 compared with that of a standard. Proteins separated by pre-cast gels (Mini- inhibitor and found that this treatment not only reduced the lipid PROTEAN TGX Stain-Free Gel; Bio-Rad, Des Plaines, IL) were transferred onto 0.2 μm polyvinylidene difluoride (PVDF) membranes (Bio-Rad), content but, concurrently, also reduced pericentrin-positive blocked in 7% milk and incubated with primary and secondary antibodies. ncMTOCs in the cytoplasm. To further explore this signaling Antibodies against PEDF (1:1000, Cat. No. AB-PEDF4, BioProducts MD, network and test the hypothesis that a microenvironmental lipid Middletown, MD), ATGL (1:200, Cat. No. 10006409, Cayman Chemical), challenge elevates ncMTOC density, we asked whether pericentrin is CGI-58 (ABHD5, 1:250, Cat. No. sc-376931, Santa Cruz Biotechnology, one of the cargo proteins on LDs. Since LDs move along MTs, ready Dallas, TX), G0S2 (1:500, Cat. No. H00050486-B01P, Novus Biologicals, Journal of Cell Science

12 RESEARCH ARTICLE Journal of Cell Science (2018) 131, jcs213579. doi:10.1242/jcs.213579

Littleton, CO), total β-catenin (1 µg/ml, Cat. No. ab16051, Abcam, This analysis was performed using GraphPad Prism, version 7.03. ImageJ Cambridge, UK) and β-catenin phosphorylated at Y142 (0.25 µg/ml, Cat. software was used to analyze LD number and size and to quantify the protein No. ab83295, Abcam) were used. Antibody staining against GAPDH levels by densitometry. Microtubule density analysis was performed using (1:1000, Cat. No. 2118, Cell Signaling Technology, Danvers, MA) was for ImageJ by calculating the area (in %) occupied by MT. normalization. Membranes were incubated at room temperature with enhanced chemiluminescence (ECL) detection reagents (Bio-Rad) and the Acknowledgements protein bands were visualized by chemiluminescence using the ChemiDoc We acknowledge the technical expertise provided by Dr Lijun Huang NorthShore Touch Imaging System (Bio-Rad). University Research Institute, Evanston, IL.

Proliferation assays Competing interests Proliferation analysis was performed using two methods: the proliferating The authors declare no competing or financial interests. cell nuclear antigen (PCNA) staining and the Vibrant MTT Cell Proliferation Assay Kit (Molecular Probes, Eugene, OR). In the first case, Author contributions Conceptualization: F.N., O.E.F., S.W.H., M.A.W., S.E.C.; Methodology: F.N., P.F., NFs and CAFs were grown on glass coverslips and treated with DGAT1 O.E.F., M.A.W., S.E.C.; Validation: F.N., S.E.C.; Formal analysis: F.N., P.F., O.E.F., inhibitor for 24 h. After treatment, cells were washed three times with PBS, M.A.W., S.E.C.; Investigation: F.N., J.I., A.S., S.E.C.; Resources: O.E.F., S.W.H., fixed in 4% paraformaldehyde for 20 min and stained with PCNA antibody C.B.B., S.E.C.; Data curation: F.N., P.F., J.I., A.S., S.E.C.; Writing - original draft: (1:40, Cat. No. M0879, Dako, Denmark). After staining, coverslips were F.N., S.E.C.; Writing - review & editing: F.N., P.F., O.E.F., J.I., A.S., S.W.H., C.B.B., mounted on glass slides and the percentage of positive cells in the DNA M.A.W., S.E.C.; Supervision: S.E.C.; Project administration: F.N., S.E.C.; Funding synthesis phase was determined. Regarding the MTT Cell Proliferation acquisition: S.E.C., S.W.H., M.A.W. Assay, a total of 104 cells (CAFs) per well was seeded into 96-well culture plates and treated for 24 h with DGAT1 inhibitor. After 24 h, the medium Funding was replaced with 100 µl of fresh medium and 10 µl of 12 mM MTT was This work was supported by the National Institutes of Health (NIH) [grant added to each well. After incubation of 4 h at 37°C, 100 µl of 0.01 M SDS- number : NIH DK103483 (to S.W.H.), NIH GM102155 (to M.A.W.), NIH CA192701 (to S.E.C.)] and the Rob Brooks Fund for Precision Prostate Care.’ Deposited in HCl solution containing 0.1 g/ml SDS was added to solubilize the formazan PMC for release after 12 months. dye product for 4 h incubation at 37°C. The optical density was then determined using a spectrophotometer at a wavelength of 570 nm. Supplementary information Supplementary information available online at Immunofluorescence staining http://jcs.biologists.org/lookup/doi/10.1242/jcs.213579.supplemental Cells were grown and treated on glass coverslips. After the treatments, cells were washed three times with PBS, fixed in 4% paraformaldehyde for References 20 min, and permeabilized with 0.1% Triton X-100 for 15 min. Cells were Bahmanyar, S., Guiney, E. L., Hatch, E. M., Nelson, W. J. and Barth, A. I. M. first incubated for 20 min in 5% normal horse serum, and then with primary (2010). Formation of extra centrosomal structures is dependent on beta-catenin. and secondary antibodies. Mouse anti-γ-tubulin (1 µg/ml, Cat. No. J. Cell Sci. 123, 3125-3135. ab27074, Abcam), rabbit anti-α-tubulin (1:100, Cat. No. ab15246, Banerjee, J., Mishra, R., Li, X., Jackson, R. S., II, Sharma, A. and Bhowmick, N. A. (2014). A reciprocal role of prostate cancer on stromal DNA damage. Abcam), mouse anti-pericentrin (1 µg/ml, Cat. No. ab28144, Abcam) and Oncogene 33, 4924-4931. rabbit anti-centrin 1 (1:500, Cat. No. 12794-1-AP, Proteintech, Chicago, IL) Banerjee, S., Zare, R. N., Tibshirani, R. J., Kunder, C. A., Nolley, R., Fan, R., antibodies were used to observe the MT point of nucleation, MTs, MTOCs Brooks, J. D. and Sonn, G. A. (2017). Diagnosis of prostate cancer by desorption and centrosomes, respectively, whereas rabbit anti-β-catenin antibody electrospray ionization mass spectrometric imaging of small metabolites and (1:100, Cat. No. 95826, Cell Signaling Technology) was used to stain lipids. Proc. Natl. Acad. Sci. USA 114, 3334-3339. protein inside the cells. Intracellular LDs were stained using BODIPY for Bartolini, F. and Gundersen, G. G. (2006). Generation of noncentrosomal microtubule arrays. J. Cell Sci. 119, 4155-4163. 20 min (Thermo Fisher Scientific, Waltham, MA). After staining, the Becerra, S. P. and Notario, V. (2013). The effects of PEDF on cancer biology: coverslips were mounted on glass slides using ProLong Gold antifade mechanisms of action and therapeutic potential. Nat. Rev. Cancer 13, 258-271. reagent with DAPI (Invitrogen, Carlsbad, CA) and sealed with permaslip. Boeszoermenyi, A., Nagy, H. M., Arthanari, H., Pillip, C. J., Lindermuth, H., Cells were then imaged using confocal microscopy (Nikon Eclipse TE Luna, R. E., Wagner, G., Zechner, R., Zangger, K. and Oberer, M. (2015). 2000-U microscope equipped with NIS-Element version 4 software). Structure of a CGI-58 motif provides the molecular basis of lipid droplet anchoring. J. Biol. Chem. 290, 26361-26372. Bombrun, M., Gao, H., Ranefall, P., Mejhert, N., Arner, P. and Wählby, C. (2017). MT regrowth assay – Quantitative high-content/high-throughput microscopy analysis of lipid droplets in Cells were grown on glass coverslips and, after reaching 60 70% of subject-specific adipogenesis models. Cytometry A 91, 1068-1077. confluence, 5 µM nocodazole was added to the medium for 3 h at 37°C to Borg, M. L., Andrews, Z. B., Duh, E. J., Zechner, R., Meikle, P. J. and Watt, M. J. depolymerize the microtubules. After the incubation the cells were washed (2011). Pigment epithelium-derived factor regulates lipid metabolism via adipose with fresh medium and incubated at 37°C to allow regrowth. Cells were then triglyceride lipase. Diabetes 60, 1458-1466. fixed at intervals of 30 s, 1 min and 5 min in 4% paraformaldehyde for Bornens, M. (2002). Centrosome composition and microtubule anchoring immunofluorescence. MTs and MTOCs were stained using rabbit anti-α- mechanisms. Curr. Opin. Cell Biol. 14, 25-34. Bornens, M. (2012). The centrosome in cells and organisms. Science 335, tubulin and mouse anti-pericentrin antibody, respectively. 422-426. Bostrom, P., Rutberg, M., Ericsson, J., Holmdahl, P., Andersson, L., Frohman, Silencing of PEDF by transfection of siRNA into normal M. A., Boren, J. and Olofsson, S. O. (2005). Cytosolic lipid droplets increase in fibroblasts size by microtubule-dependent complex formation. Arterioscler. Thromb. Vasc. To reduce PEDF levels in human normal fibroblasts, we transfected normal Biol. 25, 1945-1951. fibroblasts according to the manufacturer’s instructions (Thermo Fisher Cerk, I. K., Salzburger, B., Boeszoermenyi, A., Heier, C., Pillip, C., Romauch, M., Schweiger, M., Cornaciu, I., Lass, A., Zimmermann, R. et al. (2014). A Scientific, Waltham, MA) with commercial small interfering RNA (siRNA) peptide derived from G0/G1 switch gene 2 acts as noncompetitive inhibitor of constructs targeting PEDF (Cat. No. 4392420). After 72 h of transfection, adipose triglyceride lipase. J. Biol. Chem. 289, 32559-32570. cells were harvested and cultured for 48 h in fresh medium lacking siRNA in Chan, J. Y. (2011). A clinical overview of centrosome amplification in human a dish or on poly-L-lysine-coated coverslips to perform western blotting or cancers. Int. J. Biol. Sci. 7, 1122-1144. immunofluorescence staining, respectively. Chen, G., Zhou, G., Aras, S., He, Z., Lucas, S., Podgorski, I., Skar, W., Granneman, J. G. and Wang, J. (2017a). Loss of ABHD5 promotes the aggressiveness of prostate cancer cells. Sci. Rep. 7, 13021. Statistical analysis Chen, J. V., Buchwalter, R. A., Kao, L. R. and Megraw, T. L. (2017b). A splice To determine differences between groups we used Student’s t-test, where variant of centrosomin converts mitochondria to microtubule-organizing centers. the differences were considered statistically significant when P<0.05. Curr. Biol. 27, 1928-1940 e6. Journal of Cell Science

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Chung, C., Doll, J. A., Gattu, A. K., Shugrue, C., Cornwell, M., Fitchev, P. and Lord, C. C., Ferguson, D., Thomas, G., Brown, A. L., Schugar, R. C., Burrows, Crawford, S. E. (2008). Anti-angiogenic pigment epithelium-derived factor A., Gromovsky, A. D., Betters, J., Neumann, C., Sacks, J. et al. (2016). regulates hepatocyte triglyceride content through adipose triglyceride lipase Regulation of hepatic triacylglycerol metabolism by CGI-58 does not require ATGL (ATGL). J. Hepatol. 48, 471-478. co-activation. Cell Rep. 16, 939-949. Crawford, S. E., Stellmach, V., Ranalli, M., Huang, X., Huang, L., Volpert, O., De Marini, J. C., Reich, A. and Smith, S. M. (2014). due to Vries, G. H., Abramson, L. P. and Bouck, N. (2001). Pigment epithelium-derived mutations in non-collagenous genes: lessons in the biology of bone formation. factor (PEDF) in neuroblastoma: a multifunctional mediator of Schwann cell Curr. Opin. Pediatr. 26, 500-507. antitumor activity. J. Cell. Sci. 114, 4421-8442. PMID: 11792807 Mbom, B. C., Nelson, W. J. and Barth, A. (2013). Beta-catenin at the centrosome: Dawson, D. W., Volpert, O. V., Gillis, P., Crawford, S. E., Xu, H., Benedict, W. and discrete pools of beta-catenin communicate during mitosis and may co-ordinate Bouck, N. P. (1999). Pigment epithelium-derived factor: a potent inhibitor of centrosome functions and cell cycle progression. BioEssays 35, 804-809. angiogenesis. Science 285, 245-248. Muroyama, A. and Lechler, T. (2017). Microtubule organization, dynamics and Dictenberg, J. B., Zimmerman, W., Sparks, C. A., Young, A., Vidair, C., Zheng, functions in differentiated cells. Development 144, 3012-3021. Y., Carrington, W., Fay, F. S. and Doxsey, S. J. (1998). Pericentrin and gamma- Murphy, D. J. (2001). The biogenesis and functions of lipid bodies in animals, plants tubulin form a protein complex and are organized into a novel lattice at the and microorganisms. Prog. Lipid Res. 40, 325-438. centrosome. J. Cell Biol. 141, 163-174. Nielsen, J., Christensen, A. E., Nellemann, B. and Christensen, B. (2017). Lipid Du, H. and Che, G. (2017). Genetic alterations and epigenetic alterations of cancer- droplet size and location in human skeletal muscle fibers are associated with associated fibroblasts. Oncol. Lett. 13, 3-12. insulin sensitivity. Am. J. Physiol. Endocrinol. Metab. 313, E721-E730. Eichmann, T. O., Grumet, L., Taschler, U., Hartler, J., Heier, C., Woblistin, A., Nieman, K. M., Kenny, H. A., Penicka, C. V., Ladanyi, A., Buell-Gutbrod, R., Pajed, L., Kollroser, M., Rechberger, G., Thallinger, G. G. et al. (2015). ATGL Zillhardt, M. R., Romero, I. L., Carey, M. S., Mills, G. B., Hotamisligil, G. S. et al. and CGI-58 are lipid droplet proteins of the hepatic stellate cell line HSC-T6. (2011). Adipocytes promote ovarian cancer and provide energy for J. Lipid Res. 56, 1972-1984. rapid tumor growth. Nat. Med. 17, 1498-1503. Engin, A. (2017). Fat cell and fatty acid turnover in obesity. Adv. Exp. Med. Biol. 960, Nigg, E. A. (2006). Origins and consequences of centrosome aberrations in human 135-160. cancers. Int. J. Cancer 119, 2717-2723. Filleur, S., Nelius, T., de Riese, W. and Kennedy, R. C. (2009). Characterization of Nigg, E. A. and Holland, A. J. (2018). Once and only once: mechanisms of centriole PEDF: a multi-functional family protein. J. Cell. Biochem. 106, 769-775. duplication and their deregulation in disease. Nat. Rev. Mol. Cell Biol. 19, 297-312. Franco, O. E. and Hayward, S. W. (2012). Targeting the tumor stroma as a novel Nwani, N. G., Deguiz, M. L., Jimenez, B., Vinokour, E., Dubrovskyi, O., Ugolkov, therapeutic approach for prostate cancer. Adv. Pharmacol. 65, 267-313. A., Mazar, A. P. and Volpert, O. V. (2016). Melanoma cells block PEDF Franco, O. E., Jiang, M., Strand, D. W., Peacock, J., Fernandez, S., Jackson, production in fibroblasts to induce the tumor-promoting phenotype of cancer- R. S., II, Revelo, M. P., Bhowmick, N. A. and Hayward, S. W. (2011). Altered associated fibroblasts. Cancer Res. 76, 2265-2276. TGF-beta signaling in a subpopulation of human stromal cells promotes prostatic Orlicky, D. J., Monks, J., Stefanski, A. L. and McManaman, J. L. (2013). . Cancer Res. 71, 1272-1281. Dynamics and molecular determinants of cytoplasmic lipid droplet clustering and Gandellini, P., Andriani, F., Merlino, G., D’Aiuto, F., Roz, L. and Callari, M. dispersion. PLoS ONE 8, e66837. Park, K., Lee, K., Zhang, B., Zhou, T., He, X., Gao, G., Murray, A. R. and Ma, J.-X. (2015). Complexity in the tumour microenvironment: Cancer associated fibroblast (2011). Identification of a novel inhibitor of the canonical Wnt pathway. Mol. Cell. patterns identify both common and unique features of tumour- Biol. 31, 3038-3051. stroma crosstalk across cancer types. Semin. Cancer Biol. 35, 96-106. Pavlides, S., Whitaker-Menezes, D., Castello-Cros, R., Flomenberg, N., Gazi, E., Gardner, P., Lockyer, N. P., Hart, C. A., Brown, M. D. and Clarke, N. W. Witkiewicz, A. K., Frank, P. G., Casimiro, M. C., Wang, C., Fortina, P., (2007). Direct evidence of lipid translocation between adipocytes and prostate Addya, S. et al. (2009). The reverse Warburg effect: aerobic glycolysis in cancer cancer cells with imaging FTIR microspectroscopy. J. Lipid Res. 48, 1846-1856. associated fibroblasts and the tumor stroma. Cell Cycle 8, 3984-4001. Godinho, S. A., Picone, R., Burute, M., Dagher, R., Su, Y., Leung, C. T., Polyak, Pihan, G. A., Purohit, A., Wallace, J., Malhotra, R., Liotta, L. and Doxsey, S. J. K., Brugge, J. S., Théry, M. and Pellman, D. (2014). Oncogene-like induction of (2001). Centrosome defects can account for cellular and genetic changes that cellular invasion from centrosome amplification. Nature 510, 167-171. characterize prostate cancer progression. Cancer Res. 61, 2212-2219. Gonzalez, C. D., Alvarez, S., Ropolo, A., Rosenzvit, C., Bagnes, M. F. and Protiva, P., Gong, J., Sreekumar, B., Torres, R., Zhang, X., Belinsky, G. S., Vaccaro, M. I. (2014). Autophagy, Warburg, and Warburg reverse effects in Cornwell, M., Crawford, S. E., Iwakiri, Y. and Chung, C. (2015). Pigment human cancer. Biomed. Res. Int. 2014, 926729. epithelium-derived factor (PEDF) inhibits Wnt/beta-catenin signaling in the liver. Grace, S. A., Meeks, M. W., Chen, Y., Cornwell, M., Ding, X., Hou, P., Rutgers, Cell Mol. Gastroenterol Hepatol 1, 535-549 e14. J. K., Crawford, S. E. and Lai, J. P. (2017). Adipose triglyceride lipase (ATGL) Qiu, W., Hu, M., Sridhar, A., Opeskin, K., Fox, S., Shipitsin, M., Trivett, M., expression Is associated with adiposity and tumor stromal proliferation in patients Thompson, E. R., Ramakrishna, M., Gorringe, K. L. et al. (2008). No evidence with pancreatic ductal adenocarcinoma. Anticancer Res. 37, 699-703. of clonal somatic genetic alterations in cancer-associated fibroblasts from human ̈ Halin, S., Rudolfsson, S. H., Doll, J. A., Crawford, S. E., Wikstrom, P. and Bergh, breast and ovarian carcinomas. Nat. Genet. 40, 650-655. A. (2010). Pigment epithelium-derived factor stimulates tumor macrophage Rivera-Rivera, Y. and Saavedra, H. I. (2016). Centrosome - a promising anti-cancer recruitment and is downregulated by the prostate tumor microenvironment. target. Biologics 10, 167-176. Neoplasia 12, 336-345. Sachdev, V., Leopold, C., Bauer, R., Patankar, J. V., Iqbal, J., Obrowsky, S., Harris, C. A., Haas, J. T., Streeper, R. S., Stone, S. J., Kumari, M., Yang, K., Han, Boverhof, R., Doktorova, M., Scheicher, B., Goeritzer, M. et al. (2016). Novel X., Brownell, N., Gross, R. W., Zechner, R. et al. (2011). DGAT enzymes are role of a triglyceride-synthesizing enzyme: DGAT1 at the crossroad between required for triacylglycerol synthesis and lipid droplets in adipocytes. J. Lipid Res. triglyceride and cholesterol metabolism. Biochim. Biophys. Acta 1861, 52, 657-667. 1132-1141. He, T., Zhao, L., Zhang, D., Zhang, Q., Jia, J., Hu, J. and Huang, Y. (2015). Sanchez, A. D. and Feldman, J. L. (2017). Microtubule-organizing centers: from Pigment epithelium-derived factor induces endothelial barrier dysfunction via p38/ the centrosome to non-centrosomal sites. Curr. Opin. Cell Biol. 44, 93-101. MAPK phosphorylation. Biomed. Res. Int. 2015, 791825. Satoh, K., Yachida, S., Sugimoto, M., Oshima, M., Nakagawa, T., Akamoto, S., Hu, M., Yao, J., Cai, L., Bachman, K. E., van den Brule, F., Velculescu, V. and Tabata, S., Saitoh, K., Kato, K., Sato, S. et al. (2017). Global metabolic Polyak, K. (2005). Distinct epigenetic changes in the stromal cells of breast reprogramming of colorectal cancer occurs at adenoma stage and is induced by cancers. Nat. Genet. 37, 899-905. MYC. Proc. Natl. Acad. Sci. USA 114, E7697-E7706. Kalluri, R. (2016). The biology and function of fibroblasts in cancer. Nat. Rev. Schweiger, M., Paar, M., Eder, C., Brandis, J., Moser, E., Gorkiewicz, G., Grond, Cancer 16, 582-598. S., Radner, F. P. W., Cerk, I., Cornaciu, I. et al. (2012). G0/G1 switch gene-2 Khor, V. K., Shen, W.-J. and Kraemer, F. B. (2013). Lipid droplet metabolism. Curr. regulates human adipocyte lipolysis by affecting activity and localization of Opin Clin. Nutr. Metab. Care 16, 632-637. adipose triglyceride lipase. J. Lipid Res. 53, 2307-2317. Kim, J., Choi, Y.-L., Vallentin, A., Hunrichs, B. S., Hellerstein, M. K., Peehl, D. M. Tokumaru, Y., Yamashita, K., Osada, M., Nomoto, S., Sun, D.-I., Xiao, Y., Hoque, and Mochly-Rosen, D. (2008). Centrosomal PKCbetaII and pericentrin are M. O., Westra, W. H., Califano, J. A. and Sidransky, D. (2004). Inverse critical for human prostate cancer growth and angiogenesis. Cancer Res. 68, correlation between cyclin A1 hypermethylation and p53 mutation in head and 6831-6839. neck cancer identified by reversal of epigenetic silencing. Cancer Res. 64, Kypta, R. M. and Waxman, J. (2012). Wnt/beta-catenin signalling in prostate 5982-5987. cancer. Nat. Rev. Urol. 9, 418-428. Unal, S., Alanay, Y., Cetin, M., Boduroglu, K., Utine, E., Cormier-Daire, V., Levine, A. J. and Puzio-Kuter, A. M. (2010). The control of the metabolic switch in Huber, C., Ozsurekci, Y., Kilic, E., Simsek Kiper, O. P. et al. (2014). Striking cancers by oncogenes and tumor suppressor genes. Science 330, 1340-1344. hematological abnormalities in patients with microcephalic osteodysplastic Li, H., Fan, X. and Houghton, J. M. (2007). Tumor microenvironment: the role of the primordial dwarfism type II (MOPD II): a potential role of pericentrin in tumor stroma in cancer. J. Cell. Biochem. 101, 805-815. hematopoiesis. Pediatr. Blood Cancer 61, 302-305. Lingle, W. L., Lutz, W. H., Ingle, J. N., Maihle, N. J. and Salisbury, J. L. (1998). Vertii, A., Ivshina, M., Zimmerman, W., Hehnly, H., Kant, S. and Doxsey, S. Centrosome hypertrophy in human breast tumors: implications for genomic (2016). The centrosome undergoes Plk1-independent interphase maturation

stability and cell polarity. Proc. Natl. Acad. Sci. USA 95, 2950-2955. during inflammation and mediates cytokine release. Dev. Cell 37, 377-386. Journal of Cell Science

14 RESEARCH ARTICLE Journal of Cell Science (2018) 131, jcs213579. doi:10.1242/jcs.213579

Voter, W. A. and Erickson, H. P. (1984). The kinetics of microtubule assembly. Zechner, R., Kienesberger, P. C., Haemmerle, G., Zimmermann, R. and Lass, A. Evidence for a two-stage nucleation mechanism. J. Biol. Chem. 259, (2009). Adipose triglyceride lipase and the lipolytic catabolism of cellular fat 10430-10438. stores. J. Lipid Res. 50, 3-21. Wang, Y., Zhang, Y., Zhu, Y. and Zhang, P. (2015). Lipolytic inhibitor G0/G1 switch Zechner, R., Madeo, F. and Kratky, D. (2017). Cytosolic lipolysis and lipophagy: gene 2 inhibits reactive oxygen species production and apoptosis in endothelial cells. Am. J. Physiol. Cell Physiol. 308, C496-C504. two sides of the same coin. Nat. Rev. Mol. Cell Biol. 18, 671-684. Warburg, O. (1956). On the origin of cancer cells. Science 123, 309-314. Zehmer, J. K., Huang, Y., Peng, G., Pu, J., Anderson, R. G. W. and Liu, P. (2009). Welte, M. A. (2015). As the fat flies: the dynamic lipid droplets of Drosophila A role for lipid droplets in inter-membrane lipid traffic. Proteomics 9, 914-921. embryos. Biochim. Biophys. Acta 1851, 1156-1185. Zenker, J., White, M. D., Templin, R. M., Parton, R. G., Thorn-Seshold, O., Welte, M. A. and Gould, A. P. (2017). Lipid droplet functions beyond energy Bissiere, S. and Plachta, N. (2017). A microtubule-organizing center directing storage. Biochim. Biophys. Acta 1862, 1260-1272. intracellular transport in the early mouse embryo. Science 357, 925-928. Yamagishi, S. and Matsui, T. (2014). Pigment epithelium-derived factor (PEDF) Zhang, H., Sun, T., Jiang, X., Yu, H., Wang, M., Wei, T., Cui, H., Zhuang, W., Liu, and cardiometabolic disorders. Curr. Pharm. Des. 20, 2377-2386. Z., Zhang, Z. et al. (2015). PEDF and PEDF-derived peptide 44mer stimulate Young, S. G. and Zechner, R. (2013). Biochemistry and pathophysiology of intravascular and intracellular lipolysis. Genes Dev. 27, 459-484. cardiac triglyceride degradation via ATGL. J. Transl. Med. 13, 68. Zagani, R., El-Assaad, W., Gamache, I. and Teodoro, J. G. (2015). Inhibition of Zhu, X. and Kaverina, I. (2013). Golgi as an MTOC: making microtubules for its own adipose triglyceride lipase (ATGL) by the putative tumor suppressor G0S2 or a good. Histochem. Cell Biol. 140, 361-367. small molecule inhibitor attenuates the growth of cancer cells. Oncotarget 6, Zyss, D. and Gergely, F. (2009). Centrosome function in cancer: guilty or innocent? 28282-28295. Trends Cell Biol. 19, 334-346. Journal of Cell Science

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