Loss of Corepressor PER2 Under Hypoxia Up-Regulates OCT1-Mediated EMT Gene Expression and Enhances Tumor Malignancy

Loss of corepressor PER2 under hypoxia up-regulates OCT1-mediated EMT gene expression and enhances tumor malignancy

Wendy W. Hwang-Versluesa, Po-Hao Changa, Yung-Ming Jengb, Wen-Hung Kuoc, Pei-Hsun Chianga, Yi-Cheng Changa, Tsung-Han Hsieha, Fang-Yi Sua, Liu-Chen Lina, Serena Abbondanted, Cheng-Yuan Yanga, Huan-Ming Hsue, Jyh-Cherng Yue, King-Jen Changf, Jin-Yuh Shewa, Eva Y.-H. P. Leea,d, and Wen-Hwa Leea,d,1

aGenomics Research Center, Academia Sinica, Taipei 115, Taiwan; Departments of bPathology and cSurgery, National Taiwan University Hospital, Taipei 100, Taiwan; dDepartment of Biological Chemistry, University of California, Irvine, CA 92697; eDivision of General Surgery, Department of Surgery, Tri-Service General Hospital, National Defense Medical Center, Taipei 114, Taiwan; and fCheng Ching General Hospital, Taichung 407, Taiwan

Edited by Gregg L. Semenza, Johns Hopkins University School of Medicine, Baltimore, MD, and approved June 14, 2013 (received for review December 28, 2012)

The circadian clock gene Period2 (PER2) has been suggested to be on γ-irradiation, these mice became cancer prone (20). In humans, a tumor suppressor. However, detailed mechanistic evidence has PER2 expression was found to be significantly reduced in both not been provided to support this hypothesis. We found that loss sporadic and familial primary breast cancers (21). A few breast of PER2 enhanced invasion and activated expression of epithelial- cancers had PER2 mutations (22). In cases where PER2 was not mesenchymal transition (EMT) genes including TWIST1, SLUG, and mutated, altered PER2 promoter methylation was observed (23). fi SNAIL. This finding was corroborated by clinical observation that Consistent with this, PER2 expression was signi cantly reduced in PER2 down-regulation was associated with poor prognosis in breast cancer stem cells (BCSCs) (24). Several studies have described a correlation between PER2 and cell cycle regulation or breast cancer patients. We further demonstrated that PER2 served – as a transcriptional corepressor, which recruited polycomb pro- DNA damage response gene expression (20, 25 30).However,the teins EZH2 and SUZ12 as well as HDAC2 to octamer transcription underlying mechanism by which PER2 serves as a tumor suppressor factor 1 (OCT1) (POU2F1) binding sites of the TWIST1 and SLUG remains to be elucidated. We found that PER2 was essential for assembly of a repressor promoters to repress expression of these EMT genes. Hypoxia, complex at OCT1 (POU2F1) sites on the promoters of the a condition commonly observed in tumors, caused PER2 degrada- epithelial-mesenchymal transition (EMT) genes TWIST and tion and disrupted the PER2 repressor complex, leading to activa- SLUG. In hypoxia, PER2 protein was degraded, and the PER2 tion of EMT gene expression. This result was further supported by suppressor complex dissociated from the EMT promoters. The fi clinical data showing a signi cant negative correlation between importance of this mechanism was supported by clinical correla- fi hypoxia and PER2. Thus, our ndings clearly demonstrate the tu- tion of decreased PER2 with hypoxia and poor prognosis in breast mor suppression function of PER2 and elucidate a pathway by cancer patients. Our data demonstrate a mechanism by which which hypoxia promotes EMT via degradation of PER2. PER2 suppresses breast tumor malignancy and elucidate a unique pathway by which hypoxia can release PER2 repression of EMT. HIF1alpha | breast cancer stem cell Results ircadian oscillation is a fundamental process that influences Loss of PER2 Is Associated with Breast Tumorigenesis. PER2 ex- Cmany physiological and biological processes. The master pression in breast ductal carcinoma (DC) was significantly sup- mammalian circadian clock, located in the suprachiasmatic nu- pressed compared with normal mammary tissues (N) (SI Appendix, clei (SCN) (1), coordinates peripheral circadian clocks in most Fig. S1A; www.oncomine.org). Consistent with these clinical tissues (2–5). These circadian rhythms are driven by a core set data, lower PER2 expression was observed in breast cancer of clock genes. In human, there are at least nine clock genes cell lines SKBR-3 and MDA-MB-231 (MB-231) compared with including Period 1 (PER1), Period 2 (PER2), Period 3 (PER3), normal mammary epithelial cells MCF-10A and H184B5F5/M10 Cryptochrome 1 (CRY1), Cryptochrome 2 (CRY2), CLOCK, (M10), both at the protein and mRNA levels (Fig. 1A; SI BMAL1, Casein kinase 1e (CK1«), and Timeless (TIM), which Appendix,Fig.S1B). Similar to PER2, PER1 expression was form a complex network of transcription-translation feedback low in ductal carcinoma; however, its protein level did not differ loops and posttranslational modifications. In peripheral tissues, between cancer and normal cell lines (Fig. 1B; SI Appendix, Fig. fi these circadian genes play important roles in tissue-specific S1C). Neither clinical samples nor cell lines showed signi cant responses to the circadian environment (6–8). difference in PER3 gene or protein expression between cancer Recent studies in animal models and epidemiological analyses and normal mammary epithelial cells (Fig. 1C; SI Appendix, Fig. fi have suggested that disruption of circadian rhythms is associated S1D). These data suggested a speci c role of PER2 in breast with increased incidence of various epithelial cancers (9–12). This tumorigenesis. association can be explained by roles of circadian genes in the cell cycle, cell proliferation, DNA damage response, tumorigenesis, and angiogenesis. For example, in human prostate cancer cells, Author contributions: W.W.H.-V., E.Y.-H.P.L., and W.-H.L. designed research; W.W.H.-V., PER1 overexpression caused significant growth inhibition and P.-H. Chang, P.-H. Chiang, T.-H.H., F.-Y.S., L.-C.L., S.A., and C.-Y.Y. performed research; W.-H.K., H.-M.H., J.-C.Y., K.-J.C., and J.-Y.S. contributed new reagents/analytic tools; apoptosis (13). Conversely, PER3 gene deletion in breast cancer W.W.H.-V., Y.-M.J., and Y.-C.C. analyzed data; and W.W.H.-V., E.Y.-H.P.L., and W.-H.L. was found to be associated with estrogen receptor-positive cancer wrote the paper. recurrence (14). PER2 is functionally distinct from PER1 and Conflict of interest statement: According to the UCI policy, W.-H.L. declares that he serves PER3 despite their structural similarity (15), and each PER family as a member of Board of Directors of the biotech company, GeneTex. This arrangement member regulates target gene signaling pathways by distinct has been reviewed and approved by UCI conflict of interest committee. mechanisms (16–19). Similar to PER1 and PER3, cancer associ- This article is a PNAS Direct Submission. ations have also been reported for PER2 (20–30). 1To whom correspondence should be addressed. E-mail: [email protected]. Additional lines of evidence suggest a role for PER2 in tumor This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. CELL BIOLOGY suppression. Per2-deficient mice had low tumor incidence. However, 1073/pnas.1222684110/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1222684110 PNAS | July 23, 2013 | vol. 110 | no. 30 | 12331–12336 Downloaded by guest on September 25, 2021 Fig. 1. PER2 suppression promoted tumorigenic ability. (A–C) Immunoblot analysis of PER2 (A), PER1 (B), and PER3 (C)in breast cancer cells (MB-231 and SKBR-3) and normal mammary epithelial cells (MCF-10A and M10). Tubulin was used as a loading control. Relative expression (RE) level of PER protein in SKBR-3, MCF-10A, and M10 relative to MB-231 is indicated. (D and E) Soft agar colony formation (SACF) assay using MCF- 10A (D) and SKBR-3 (E) cells infected with lentiviral-control shRNA (sh-Ctrl) or sh-PER2. (F) SACF assay using SKBR-3 cells transduced with lentiviral control (Ctrl) or PER2. PER2 expression in these cells was examined with immunoblot. Tubulin was used as a loading control. Data show means ± SD. *P < 0.05 (Student t test). (G) Tumorigenesis assay of NOD/SCID mice injected with SKBR-3 lentiviral Ctrl or PER2 overexpressing cells. Cell dose: 3 × 106 cells per fat pad. Five mice were used for each group. Data show means ± SD. *P < 0.05 (Student t test) (SI Appendix, Fig. S1).

Depletion of PER2 using shRNA increased colony-forming was observed in various breast cancer cells, the targets of PER2- ability in both normal mammary epithelial (Fig. 1D) and breast mediated EMT gene regulation were more cell type specific. cancer cells (Fig. 1E). In contrast, overexpression of PER2 re- Consistent with these results in culture, Twist1 and Snail were − − duced colony-forming ability of cancer cells (Fig. 1F), and tumors up-regulated in mammary glands of Per2-deficient (Per2 / ) derived from PER2 overexpressing cells grew less in nonobese mice compared with the WT (SI Appendix,Fig.S2G). Slug diabetic/severe combined immunodeficient (NOD/SCID) mice expression may also have been slightly higher, although the (Fig. 1G). These data indicated a tumor suppressor role of PER2 difference was not statistically significant. Together these data in tumorigenesis. indicated a new role of PER2 in EMT regulation.

Down-Regulation of PER2 Promotes Tumor Malignancy by Enhancing PER2 Is Recruited to EMT Promoters via OCT1 (POU2F1). PER2 does Invasion and Activating EMT Gene Expression. To test whether loss not contain a DNA binding domain. Therefore, it must regulate of PER2 contributes to tumor malignancy, we assayed invasion gene expression as a cofactor, which interacts with other tran- ability and found that PER2 depletion increased the invasion scription regulators. Using several promoter analysis databases ability of normal epithelial MCF-10A cells and breast cancer cell (34–37), we identified OCT1 as a transcription factor that may lines SKBR-3, MCF-7, MB-231, and MDA-MB-157 (MB-157) recruit PER2 to binding sites on the TWIST1, SLUG, and SNAIL (Fig. 2 A–E). In MCF-10A cells, down-regulation of PER2 promoters (Fig. 3A; SI Appendix, Fig. S4A). To test this hy- resulted in a 10-fold induction of invasion ability (Fig. 2A). In pothesis, we performed ChIP assays in MCF-10A cells using an less invasive SKBR-3 and MCF-7 cells, PER2 suppression pro- anti-PER2 antibody and primer sets amplifying 150- to 250-bp moted invasion ability by nearly fourfold (Fig. 2 B and C). Even fragments spanning the predicted OCT1 binding regions. PER2 in highly invasive MB-231 and MB-157 cells, down-regulation was associated with all predicted OCT1 sites of the TWIST1, of PER2 could further enhance invasion ability by twofold (Fig. 2 SLUG, and SNAIL promoters but not the 5′-UTR or the far site D and E). In contrast, overexpression of PER2 inhibited invasion control region (Fig. 3B; SI Appendix, Fig. S4B). ChIP assays using ability of SKBR-3 cells (Fig. 2F). However, PER2 overexpression an anti-OCT1 antibody further confirmed strong binding of did not suppress the invasion ability of MB-231 cells (SI Ap- OCT1 to the proximal OCT1 sites on the TWIST1 (Oct1-5) and pendix, Fig. S2 A and B), possibly due to multiple highly activated SLUG (Oct1-2) promoters (Fig. 3C; SI Appendix, Fig. S4C). A EMT pathways in these cells (31). weaker OCT1 binding to the TWIST1 Oct1-2 and Oct1-3 sites It has been previously reported that EMT is the driving force and the SLUG Oct1-1 site was also observed (Fig. 3C; SI Ap- of invasion and enhances stemness in epithelial cells (32, 33). pendix, Fig. S4C). However, ChIP assay failed to detect any Thus, we examined the expression of EMT markers (E-cadherin binding to the OCT1 site of the SNAIL promoter (SI Appendix, and N-cadherin), as well as key EMT regulators (SNAIL, SLUG, Fig. S4C) or to the TWIST1 Oct1-1 and Oct1-4 sites (Fig. 3C). and TWIST1). The elevated invasion ability of PER2-depleted Coimmunoprecipitation (Co-IP) showed a strong interaction MCF-10A cells was associated with up-regulation of N-cadherin, between endogenous PER2 and OCT1 in MCF-10A cells (Fig. SNAIL, SLUG, and TWIST1 and down-regulation of E-cadherin, 3D). Also, in vitro biotinylated oligonucleotide immunoprecipi- as well as a morphological change from epithelial phenotype to tation assays confirmed that binding of OCT1 and PER2 on both an elongated and spindle-like morphology (Fig. 2G; SI Appendix, the TWIST1 (Oct1-5) and SLUG (Oct1-2) promoters (Fig. 3E; Fig. S3). Conversely, decreased invasion ability of PER2-over- SI Appendix, Fig. S4D) involved the PER2–OCT1 interaction. expressing SKBR-3 cells was associated with repression of these OCT1 and PER2 binding was disrupted when the OCT1 binding EMT genes, with the exception of E-cadherin (Fig. 2H). In other sites were mutated (Fig. 3E; SI Appendix, Fig. S4D). These breast cancer cell lines, different EMT regulators were up-regulated results indicated that, although PER2 lacks a DNA binding do- by PER2 knockdown (SI Appendix,Fig.S2C–F). This observation main, it can be recruited to the TWIST1 and SLUG promoters indicated that, although invasion mediated by PER2 suppression through OCT1.

12332 | www.pnas.org/cgi/doi/10.1073/pnas.1222684110 Hwang-Verslues et al. Downloaded by guest on September 25, 2021 histone modiﬁcations are associated with repression of transcription. Using Co-IP assays, we found a PER2 interaction with EZH2, SUZ12, and HDAC2, but not with other corepressors such as Sin3A and CoREST, which are known to complex with HDACs (40, 41) (Fig. 3H). ChIP assays indicated colocalization of PER2 and EZH2/SUZ12 on the OCT1-binding regions of the TWIST1 and SLUG promoters (Fig. 3 I and J; SI Appendix, Fig. S4 F and G). ChIP assays also detected the repressive trimethylated histone 3 lysine 27 (H3K27me3) mark on all of the OCT1/PER2/EZH2/SUZ12 binding regions (Fig. 3K; SI Appen- dix, Fig. S4H). HDAC2 was also found associated with all of these promoter regions (Fig. 3L; SI Appendix, Fig. S4I). Taken together, these data suggested a mechanism by which PER2 suppressed TWIST1 and SLUG transcription by recruitment of a repressor complex containing EZH2, SUZ12, and HDAC2.

Hypoxia Down-Regulates PER2 Posttranslationally to De-Repress EMT Gene Expression. Hypoxia promotes EMT, invasion, and metastasis (38, 42), whereas our data indicated that PER2 re- presses EMT genes. To test whether hypoxia promotion of EMT involves a change in PER2, we examined PER2 protein levels of

Fig. 2. PER2 suppression promoted invasion and EMT gene expression. (A–E) Invasion assay for MCF-10A (A), SKBR-3 (B), MCF-7 (C), MB-231 (D), and MB-157 (E) cells transduced with lentiviral sh-Ctrl or sh-PER2. (F) Invasion assay for SKBR-3 cells transduced with lentiviral control (Ctrl) or PER2. (G and H) mRNA expression of EMT markers (E-cadherin and N-cadherin) and regulators (SLUG, SNAIL, TWIST1) determined by qRT-PCR in MCF-10A infected with sh-Ctrl or sh-Per2 (G) and in Ctrl or PER2 overexpressing SKBR-3 (H). Data are means ± SD. *P < 0.05 (Student t test). (SI Appendix,Figs.S2andS3).

PER2 Suppresses OCT1-Mediated TWIST1 and SLUG Promoter Activity. Transient reporter assays using reporter vectors containing −139 to +48 (Oct1-5 site) of the TWIST1 proximal promoter (38) or −908 to −2 (including the Oct1-2 binding site) of the SLUG promoter region (39) demonstrated that PER2 acted as a transcriptional corepressor. OCT1 alone acted as a transactivator and induced both TWIST1 and SLUG promoter activities. PER2 alone had no significant effect, consistent with its lack of DNA binding domain (Fig. 3F; SI Appendix, Fig. S4E). However, when PER2 and OCT1 were coexpressed, both promoter activities were suppressed in a dose-dependent manner (Fig. 3 F and G; SI Appendix, Fig. S4E). These results indicated that PER2 could be recruited and suppress TWIST1 or SLUG expression via association with OCT1. To confirm these results, the EMT gene levels in SKBR-3 cells with perturbed OCT1 or PER2 expres- Fig. 3. PER2 recruited corepressors to suppress OCT1-mediated TWIST1 sion was evaluated. OCT1 depletion reduced EMT gene ex- promoter activity. (A) Diagram showing the five predicted OCT1 sites (Oct1- pression, confirming its transactivator role (SI Appendix,Fig. 1–Oct1-5) on the TWIST1 promoter. (B and C) ChIP of PER2 (B) or OCT1 (C)on S5A). PER2 overexpression repressed these EMT genes, but the TWIST1 promoter in MCF-10A cells. Normal IgG (IgG) and a site in 5′-UTR OCT1depletioninthePER2-overexpressing cells abolished were used as controls for PER2-promoter association. (D) Coimmunopreci- PER2-mediated EMT suppression (SI Appendix,Fig.S5B). These pitation (Co-IP) of PER2 and OCT1 in MCF-10A cells. Normal IgG was used as data suggested that OCT1 is required for PER2-mediated EMT a control. (E) Immunoprecipitation of OCT1 or PER2 using MCF-10A nuclear extract (N.E.) and biotin-labeled oligonucleotides containing WT or mutated gene suppression. (mut) Oct1-5 site. (F and G) Relative fold change in luciferase activity of the TWIST1 SLUG TWIST1 reporter construct in HEK-293T cells transiently cotransfected with PER2 Suppresses and Transcription by Recruiting EZH2, OCT1 or/and PER2 expression plasmids. Data show means ± SD. *P < 0.05 SUZ12, and HDAC2. Corepressor complexes can be large multi- (Student t test). (H) Co-IP of PER2 and EZH2, SUZ12, HDAC2, Sin3A, or CoREST protein complexes that include polycomb repressive complex 2 in MCF-10A cells. (Left) EZH2 and HDAC2 immunoblots were input images (PRC2), which catalyzes trimethylation of histone H3 lysine 27 from the same blots with different exposure time. (I–L) ChIP of SUZ12 (I), (H3K27me3), as well as histone deacetylases (HDACs), which EZH2 (J), trimethylated Histone 3 lysine 27 (H3K27me3) (K), and HDAC2 (L)in CELL BIOLOGY deacetylate lysine residues on the core histones. Both of these MCF-10A cells (SI Appendix, Figs. S4 and S5).

Hwang-Verslues et al. PNAS | July 23, 2013 | vol. 110 | no. 30 | 12333 Downloaded by guest on September 25, 2021 MCF-10A cells at various times under hypoxia (1% O2) (Fig. 4A). A significant decrease of PER2 protein was observed within 6 h after transfer to 1% O2 along with induction of the hypoxia marker HIF1α (Fig. 4A). Cycloheximide treatment and the pulse-chase assay showed that the half-life of PER2 was reduced under hypoxia (Fig. 4B; SI Appendix, Fig. S6). Such a rapid decrease in PER2 half-life suggested that a hypoxia-dependent posttranslational modification such as phosphorylation may promote PER2 degradation (43). It has been reported that phosphorylation of PER2 S662 facilitates phosphorylation of other PER2 serines by casein kinase-1 δ/e (CK1δ/e) and sub- sequently triggers proteasomal degradation of the hyperphosphorylated PER2 (44, 45). Consistent with this, an increase in hyperphosphorylated PER2 on hypoxia was detected by a PER2-S662 phospho-specific antibody (Fig. 4C), and PER2 translocation from nucleus to cytoplasm was observed after 6 h of hypoxia (Fig. 4D; SI Appendix, Fig. S7A). A phospho-null PER2 mutant (S662A) could not be degraded in hypoxia (SI Appendix, Fig. S8). Also, treatment with proteasome inhibitors MG132 and lactacystin prevented PER2 degradation (Fig. 4E) and increased ubiquitinated PER2 (SI Appendix, Fig. S9). These data indicated that hypoxia induced PER2 posttranslational modification, leading to proteasome degradation. Transient reporter assays showed that both the TWIST1 and SLUG promoter activities were induced on hypoxia treatment (Fig. 4F; SI Appendix, Fig. S7B). ChIP analysis of MCF-10A cells under 0–24 h of hypoxia demonstrated that promoter-bound PER2 could no longer be detected after the first 6 h of hypoxia (Fig. 4G; SI Appendix, Fig. S7 C and D). Correspondingly, strong Fig. 4. Hypoxia-induced PER2 degradation resulted in suppressor complex binding of EZH2 and HDAC2 was only detected at the begin- dissociation and de-repression of TWIST1 promoter transactivation. (A) Time ning of hypoxia treatment when PER2 was still associated with course assay using MCF-10A cells cultured in normoxia or hypoxic conditions the promoter (Fig. 4G; SI Appendix, Fig. S7 C and D). Later in (1% oxygen). PER2 levels were determined by Immunoblotting. HIF1α ac- the hypoxia time course when PER2 binding was no longer cumulation was used to verify the activation of hypoxic response. (B) Time detected, the repressive epigenetic mark of H3K27 trimethyla- course assay using MCF-10A treated with cycloheximide (CHX) (100 μg/mL) in tion decreased, whereas the activating mark of lysine acetylation normoxia or hypoxia. PER2 expression was determined by immunoblotting. increased (Fig. 4G; SI Appendix, Fig. S7 C and D). Importantly, (C) Phosphorylated PER2 species in MCF-10A cells cultured in hypoxia were a strong HIF1α binding signal on the TWIST1 promoter was detected using immunoblotting with PER2-S662 phospho-specific antibody. observed at 24 h on hypoxia treatment (Fig. 4G). These data (D) Immunofluorescence (IF) of PER2 in MCF-10A cells at 6 h in normoxia or suggested a progression by which hypoxia activates EMT by first hypoxia. Nuclear matrix protein p84 and DAPI were used as nuclear staining removing PER2-mediated repression and then transactivating controls. Arrows indicate PER2 staining. (Scale bar, 10 μm.) (E) Time course EMT gene expression by HIF1α. assay using MCF-10A treated with proteasome inhibitors MG132 (20 μM) or To confirm these in vitro findings, we obtained a cohort of 101 lactacystin (20 μM) in hypoxia. PER2 expression was determined by immu- breast cancer specimens. The characteristics of these cases are noblotting. (F) Time course reporter assay using MCF-10A transfected with the TWIST1 promoter construct in hypoxia. Relative fold change in luciferase given in SI Appendix, Table S1. Immunohistochemistry (IHC) of ± < serial paraffin embedded sections of 57 breast cancer cases found activity was shown. Data shown is means SD. *P 0.05 (Student t test). (G) fi Time course ChIP of PER2, OCT1, EZH2, SUZ12, HeK27me3, HDAC2, HIF1α, a signi cant negative correlation between PER2 expression and and acetylated lysine (Ac-lysine) on Oct1-5 region of the TWIST1 promoter in hypoxia using CAIX (carbonic anhydrase IX), a HIF1α down- – = MCF-10A cells cultured in hypoxia. For all immunoblot assays, tubulin was stream target, as a hypoxia marker (46) (Fig. 5 A G; P 0.03). used as a loading control. All experiments were performed at least two times Severely hypoxic samples (high CAIX membrane staining) with similar results. One representative result is shown (SI Appendix, Figs. showed low or nondetectable PER2 nuclear staining (Fig. 5 S6–S9). A–C). In contrast, samples with no detectable CAIX membrane staining showed high PER2 nuclear staining (Fig. 5 D–F). Con- sistent with this result, PER2 gene expression in MCF-10A cells correlation, r = −0.22, P = 0.02), and histological grading (P < treated with hypoxia was down-regulated (SI Appendix, Fig. 0.001; Fig. 6B). This correlation indicated an association be- S10A). Data from human breast tumor gene expression profiling tween reduction of PER2 expression and tumor malignancy. (31) also found a significant negative correlation between CAIX Moreover, a lower level of PER2 expression was associated with and PER2 expression (SI Appendix, Fig. S10B; r = −0.35, P = poor prognosis in breast cancer patients (P = 0.01; Fig. 6C). 0.03). These results indicated that hypoxia not only promoted The hazard ratio of patients with high PER2 expression was PER2 protein degradation, but also triggered PER2 gene suppres- 0.37-fold (95% CI: 0.14–0.99; P = 0.04) of those with low PER2 sion by an as yet unknown mechanism. Our in vitro experiments and expression after adjusting for age, tumor size, lymph node clinical data all support a model whereby PER2 down-regulation status, and estrogen receptor and progesterone receptor ex- is a key step in induction of EMT gene expression under hypoxia pression (SI Appendix,TableS2). Further analysis showed that (Fig. 5H). patients with a low level of PER2 and high level of TWIST1 (PER2low/TWIST1high) had worse prognosis compared with those Suppression of PER2 Is Associated with Tumor Malignancy and Poor with PER2high/TWIST1low (group 1 vs. group 2, P = 0.03; Fig. 6D). Clinical Outcome in Breast Cancer Patients. To explore whether In high TWIST1 expression cases, low PER2 expression was still PER2 can influence clinical outcomes, we analyzed PER2 ex- associated with worse survival rate (group 1 vs. group 4, P = 0.04; pression in the 101 breast cancer tissue specimens. Consistent Fig. 6D), indicating that other PER2 target genes in addition to with our molecular mechanism, PER2 expression was inversely TWIST1 may contribute to this clinical outcome. These results correlated with tumor size (P = 0.002; Fig. 6A), tumor stage using indicated that down-regulation of PER2 enhances tumor ma- the TNM (tumor, node, metastasis) staging system (Pearson’s lignancy in breast cancer patients.

12334 | www.pnas.org/cgi/doi/10.1073/pnas.1222684110 Hwang-Verslues et al. Downloaded by guest on September 25, 2021 Discussion Our results provide a unique mechanism describing how PER2 serves as a tumor suppressor to restrict cancer development. First, we demonstrated that loss of PER2 in breast epithelial cells, either normal or cancerous cells, was able to modulate EMT, an important part of cancer stem cell formation as well as the ﬁrst step in conversion of early stage tumors into invasive malignancies. Consistent with all of these mechanistic data, a positive relationship between loss of PER2 expression and breast cancer malignancy was observed. Second, we demonstrated that PER2 serves as a corepressor to assemble a repression complex at OCT1 binding sites for this tumor suppressor function. This PER2-OCT1 interaction effectively converted OCT1 sites, which normally activate expression, into repressor sites by recruitment of a polycomb repressor complex including EZH2 and SUZ12, as well as HDAC2. The repression was released in response to hypoxia, which caused PER2 protein degradation and thus destroyed the connection between OCT1 sites and the repressor complex (Fig. 5H). Importantly, this mechanism appears to be speciﬁc to PER2, because PER1 and PER3 were not degraded on hypoxia treatment (SI Appendix, Fig. S11). Also, hypoxia-induced PER2 protein degradation is HIF1α in- dependent, because PER2 level did not change when a non- α degradable HIF1 was overexpressed (SI Appendix, Fig. S12). Fig. 6. PER2 suppression is associated with tumor malignancy and poor prognosis. (A) Regression analysis of PER2 mRNA expression vs. tumor size in 101 breast cancer patients. (B) Trend test of PER2 expression and histological grading in breast cancer patients. (C) Comparison of the overall survival periods of patients with different levels of PER2 gene expression using the Kaplan-Meier method. (D) Comparison of the overall survival periods of patients with different patterns of PER2 and TWIST1 gene expression using the Kaplan–Meier method. (E) Schematic diagram of the central role of PER2 in tumorigenesis promoted by hypoxic microenvironments.

These observations further indicate that PER2 has a distinct function in tumor progression and that PER2 degradation under hypoxia is a specific mechanism to reactivate EMT gene expression. Taken together, our results provide a mechanistic un- derpinning for a central role of PER2 in tumorigenesis promoted by hypoxic microenvironments (Fig. 6E). The data indicated that PER2 regulates EMT genes in a cellular-specific manner, whereas PER2 down-regulation could promote tumorigenicity and invasion ability independently of cellular context. Thus, there could be a more complex role of PER2 in EMT involving additional PER2 target genes. PER2 was the core component of a repressor complex assembled at OCT1 sites, and lack of PER2 led to dissolution of the repressor complex and reversal of the repressive epigenetic marks (Fig. 4). We did observe differences between promoter sites that indicate that additional transcription factors may modify PER2 binding or that there may be heterogeneity in the repressor complex anchored by PER2 (Figs. 2 and 3; SI Appendix, Figs. S2 and S13). Study of these cell type– and promoter-specific differences will further elucidate the role of PER2. The connection between EMT and cancer stemness is also of interest. For example, BMI1, an important factor for maintain- ing cancer stem cell self-renewal ability, was directly up-regulated by TWIST1 and played an essential role for hypoxia- Fig. 5. Hypoxia induces PER2 suppression. (A–F) Representative pictures mediated EMT (33). Also, SLUG expression, along with SOX9, of the IHC serial sections of PER2 nuclear and CAIX membrane staining in could convert differentiated luminal cells into mammary stem a CAIX-positive/PER2-low case (A–C) and a CAIX-negative/PER2-high case cells and promote BCSC phenotypes (47). These observations (D–F). Boxes show the enlarged area. (A and D) Original magnification, 100×. fi μ fi × μ are consistent with our ndings that PER2 lies upstream of (Scale bar, 100 m.) B and E, original magni cation, 400 . (Scale bar, 100 m.) TWIST1 and SLUG (Fig. 2) and that PER2 suppression in- C and F, original magnification, 1,600×. (Scale bar, 10 μm.) (G) Correlation of χ2 creases the prevalence of two different types of BCSCs (44) CAIX with PER2 gene expression in 57 breast cancer cases. test was used. fl (H) Diagram of PER2 function as a repressor in EMT regulation. PER2 recruits (SI Appendix,Fig.S14). Because PER2 has in uence on BCSC a corepressor complex including EZH2, SUZ12, and HDAC2 to the promoters prevalence and, most importantly, their EMT property, it rep- of EMT genes via interaction with OCT1. Hypoxia induces PER2 protein resents a potential target for therapeutic development. proteasomal degradation and PER2 gene suppression and causes dissocia- Epidemiological studies have suggested that altered circadian tion of corepressors, which leads to reactivation of EMT gene expression rhythms may be a crucial risk factor contributing to tumorigen- CELL BIOLOGY (SI Appendix,Fig.S10). esis, but the underlying mechanisms are unknown (11, 48, 49).

Hwang-Verslues et al. PNAS | July 23, 2013 | vol. 110 | no. 30 | 12335 Downloaded by guest on September 25, 2021 Many physiological functions are modulated by the circadian Hospital. Animal care and experiments were approved by the Institutional system via chromatin remodeling (50–52). Disruption of circa- Animal Care and Utilization Committee of Academia Sinica (IACUC#080085). dian outputs may promote tumorigenesis via epigenetic modifi- All data points of cell line experiments were performed in at least trip- cation. Thus, a disrupted circadian rhythm, such as in shift licate, and all experiments were performed at least three times with workers and frequent flyers, may contribute to tumor initiation, similar results. One representative result is shown. Detailed materials and and then the hypoxic tumor microenvironment further induces methods used in this research are described in SI Appendix, Materials PER2 degradation and gene suppression to promote tumor and Methods. growth and malignancy. Overall, our findings provide a mechanistic link between the circadian clock and tumorigenesis and ACKNOWLEDGMENTS. We thank Dr. Paul E. Verslues (Institute of Plant and a basis to further elucidate the central role of PER2 and its Microbial Biology, Academia Sinica) for critical reading of the manuscript clinical significance. and Ms. Meng-Han Wang, Kai-Lin Peng, Mr. Chun-Chin Chen, Wen-Ting Lo, Alfie Chen, Dr. Li-Jung Juan, Ruey-Hwa Chen, Hungwen Chen, Shiaw- Materials and Methods Wei Tyan, Heng-Hsiung Wu, and Shih-Chia Huang for kind assistance throughout this study. This work was supported by an internal research All breast tissue specimens are from BioBank of the Tri-Service General grant of Academia Sinica and an Academia Sinica Postdoctoral Research Hospital (Taipei, Taiwan). All patients were given informed consent, which Fellowship (to W.W.H.-V.). E.Y.-H.P.L. is supported by National Cancer was approved by the institutional review board of the Tri-Service General Institute Grant CA137102 and the BCRF-35127 fund.

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