(GPX8)/IL-6/STAT3 Axis Is Essential in Maintaining an Aggressive Breast Cancer Phenotype

The glutathione peroxidase 8 (GPX8)/IL-6/STAT3 axis is essential in maintaining an aggressive breast cancer phenotype

Anees Khatiba, Balakrishnan Solaimuthua, Michal Ben Yosefa, Areej Abu Rmaileha, Mayur Tannaa, Gidi Orena, Michal Schlesinger Frischa, Jonathan H. Axelrodb, Michal Lichtensteina, and Yoav D. Shaula,1

aDepartment of Biochemistry and Molecular Biology, The Institute for Medical Research Israel–Canada, The Hebrew University–Hadassah Medical School, 91120 Jerusalem, Israel; and bGoldyne Savad Institute of Gene Therapy, Hadassah Hebrew University Hospital, 91120 Jerusalem, Israel

Edited by Michael Karin, University of California San Diego School of Medicine, La Jolla, CA, and approved July 17, 2020 (received for review May 24, 2020) One of the emerging hallmarks of cancer illustrates the importance EMT program and cancer stem cells (CSC) (8, 9). For example, of metabolic reprogramming, necessary to synthesize the building induction of the EMT program in epithelial cells results in the ex- blocks required to fulfill the high demands of rapidly proliferating pression of stemness markers such as CD44high/CD24low and their cells. However, the proliferation-independent instructive role of met- ability to form mammospheres (9–11). However, these CSCs are not abolic enzymes in tumor plasticity is still unclear. Here, we provide the outcome of the full execution of the EMT program but instead evidence that glutathione peroxidase 8 (GPX8), a poorly character- are at an intermediate state along the epithelial–mesenchymal ized enzyme that resides in the endoplasmic reticulum, is an essential spectrum (1). regulator of tumor aggressiveness. We found that GPX8 expression Tumors exhibit a distinct metabolic pattern relative to non- was induced by the epithelial–mesenchymal transition (EMT) pro- proliferative cells in order to satisfy the metabolic demands of the gram. Moreover, in breast cancer patients, GPX8 expression signifi- rapidly proliferating cells (reviewed in refs. 12, 13). This metabolic cantly correlated with known mesenchymal markers and poor remolding is mediated via the expression of a unique metabolic prognosis. Strikingly, GPX8 knockout in mesenchymal-like cells gene signature (14). However, cancer-dependent metabolic rewir- (MDA-MB-231) resulted in an epithelial-like morphology, down- ing is more complicated than initially described (15) as there are

regulation of EMT characteristics, and loss of cancer stemness fea- metabolic processes essential only for particular tumor types (16, CELL BIOLOGY tures. In addition, GPX8 knockout significantly delayed tumor initia- 17). These findings indicate that the cancer-dependent metabolic tion and decreased its growth rate in mice. We found that these rewiring is not only limited to support cell proliferation but is also GPX8 loss-dependent phenotypes were accompanied by the repres- required to satisfy other proliferation-independent cellular needs, sion of crucial autocrine factors, in particular, interleukin-6 (IL-6). In such as acquiring traits associated with high-grade malignancy. these cells, IL-6 bound to the soluble receptor (sIL6R), stimulating the To methodically identify the metabolic enzymes regulating JAK/STAT3 signaling pathway by IL-6 trans-signaling mechanisms, so tumor dynamics, we generated MERAV, a web-based tool to promoting cancer aggressiveness. We observed that in GPX8 knock- analyze human gene expression between different cancer types out cells, this signaling mechanism was impaired as sIL6R failed to and normal tissues (http://merav.wi.mit.edu/, ref. 18). By ana- activate the JAK/STAT3 signaling pathway. Altogether, we present lyzing MERAV, we characterized a set of 44 metabolic genes the GPX8/IL-6/STAT3 axis as a metabolic-inflammatory pathway that that are selectively present in high-grade tumors bearing acts as a robust regulator of cancer cell aggressiveness. Significance cancer metabolism | epithelial–mesenchymal transition | GPX8 | JAK/STAT3 signaling The cancer-dependent metabolic rewiring is mainly associated with the synthesis of building blocks that are needed to fulfill uring recent decades there is substantial progress in cancer the proliferating cell metabolic requirements. However, the Dtreatment due to a better understanding of tumor biology. proliferation-independent instructive role of metabolic en- However, despite these advances in therapy, the disease can re- zymes in tumor plasticity is still unclear. Here, we introduce lapse, as it acquires more aggressive traits, including resistance to glutathione peroxidase 8 (GPX8) as a metabolic enzyme that chemotherapeutic drugs (1). This plasticity is achieved through regulates cancer aggressiveness. We found that lack of GPX8 significant alterations in the tumor physiology, such as its ability to suppresses the aggressive phenotype and stemness features of transdifferentiate into a mesenchymal-like state (2), orchestrated by the tumor cells. Mechanistically, these cells express a nonfunc- the epithelial–mesenchymal transition (EMT) program (3). The tional IL-6 receptor, which fails to interact with IL-6. This impaired execution of the EMT program induces significant changes in the binding hinders the activation of the downstream JAK/STAT3 cellular phenotype as cells acquire chemoresistance, lose their po- signaling pathway, thereby inhibiting cancer cells transition to larity, eliminate their interactions with neighboring cells, and gain aggressive phenotypes. Thus, we present this GPX8/IL-6/STAT3 invasive properties (4). These shifts in the tumor characteristics axis as a prototype of metabolic enzymes regulating cancer provide a model describing how tumors gain the ability to detach aggressiveness-associated signaling pathways. from the primary site and promote the metastatic cascade (5). The EMT program is regulated by a core set of transcription Author contributions: A.K. and Y.D.S. designed research; A.K., B.S., M.B.Y., A.A.R., M.T., factors (EMT-TFs), which includes Twist family BHLH tran- G.O., M.S.F., and M.L. performed research; J.H.A. contributed new reagents/analytic tools; scription factor 1 (TWIST1), zinc finger E-box binding homeo- Y.D.S. analyzed data; and A.K., B.S., A.A.R., M.T., M.L., and Y.D.S. wrote the paper. box 1 and 2 (ZEB1 and ZEB2), Snail family transcriptional The authors declare no competing interest. repressor 1 and 2 (SNAI1 and SNAI2 [SLUG]) (6). The EMT- This article is a PNAS Direct Submission. TFs’ mechanism of action is directed by intracellular signaling Published under the PNAS license. pathways, including Wnt and Notch or by specific ligands such as 1To whom correspondence may be addressed. Email: [email protected]. chronic transforming growth factor-beta (TGFβ), mitogenic This article contains supporting information online at https://www.pnas.org/lookup/suppl/ growth factors (1), and inflammatory cytokines, such as IL-6 (7). doi:10.1073/pnas.2010275117/-/DCSupplemental. In recent years, many studies demonstrated a link between the

www.pnas.org/cgi/doi/10.1073/pnas.2010275117 PNAS Latest Articles | 1of12 Downloaded by guest on September 25, 2021 mesenchymal markers, which we designated as the “mesenchy- cancer cell lines. To this end, we analyzed the GPX8 expression mal metabolic signature” (MMS) (11). To systematically deter- profile in breast cancer samples derived from the cancer genome mine the role of MMS in tumor progression, we developed a atlas (TCGA) project (34) available on the cBioportal web-based fluorescence-activated cell sorting (FACS)-based shRNA screen tool [https://www.cbioportal.org (34)]. Specifically, we correlated that identified 16 genes as essential for the EMT program (11). GPX8 expression to every gene in the genome and subjected the Among them is dihydropyrimidine dehydrogenase (DPYD), the obtained Spearman’s rank correlation coefficients to gene set rate-limiting enzyme of the pyrimidine degradation pathway (19), enrichment analysis (GSEA) (35, 36) which revealed a signifi- whose activity has been demonstrated to be vital for the proper cantly high correlation with the EMT markers (“hallmark execution of the EMT program (11). Having verified the essential epithelial–mesenchymal transition” [SI Appendix, Fig. S1A]). role of DPYD in the EMT, we were intrigued as to whether the This analysis was then individually validated by the mesenchymal second hit in the screen, glutathione peroxidase 8 (GPX8), is also markers ZEB1, ZEB2, and CDH11 which showed a significantly critical for tumor aggressiveness. high positive correlation with GPX8 expression (Spearman’s The primary function of the glutathione peroxidase (GPx) rank correlation coefficients = 0.55, 0.44, and 0.65, respectively) family of proteins is to limit the cellular accumulation of the and a negative correlation with the epithelial markers CDH1 reactive oxygen species (ROS) (20). These enzymes use gluta- (Spearman’s rank correlation coefficients = −0.19) (Fig. 1B). thione (GSH) as a reducing agent (21) to catabolize peroxides to Together, we systematically confirmed that GPX8 expression the corresponding alcohols. The specific activity of GPx is de- correlates with the more aggressive mesenchymal-like charac- termined by its particular amino acid composition, as most teristics of cancer cell lines and breast cancer patients. members contain the nonstandard amino acid selenocysteine in To validate these bioinformatics results, we determined GPX8 their active site (GPX1-4 and GPX6), whereas the others expression at both messenger RNA (mRNA) and protein levels (GPX5, GPX7-8) have a cysteine (21). GPX8, the last member in different cancer cell lines. We confirmed elevated GPX8 ex- of this family to be identified (21), is a type II transmembrane pression in basal B (mesenchymal-like) relative to luminal (epi- protein with high sequence similarity to the soluble GPX7 thelial)-derived cancer cell lines (Fig. 1 C and D). This GPX8 (NPGPx). These two enzymes share many characteristics, as they expression pattern is similar to other known mesenchymal contain a KDEL-like endoplasmic reticulum (ER) retrieval markers (CDH11, ZEB1, VIM, and ZEB2) and different to motif and are localized in the ER (22). Despite their name and epithelial markers (E-Cad [E-cadherin] [CDH1] and epithelial their similarity to the other members of the GPx family, both cell junction protein Occludin [OCLN]) (37) (Fig. 1 C and D). GPX7 and GPX8 demonstrate low GPx activity (22), as they lack We also found up-regulation of GPX8 expression in high-grade the GSH-binding domain (23). Thus, the proposed function of hepatocellular carcinoma cell lines (HCC) (SNU-387 and SNU- both GPX7 and GPX8 is associated with protein disulfide 423) (38) relative to the low-grade cell line (HepG2) (Fig. 1D). isomerase (PDI) peroxide-mediated oxidative protein folding Additionally, GPX8 expression in melanoma was up-regulated in (22). Specifically, they bind and clear the peroxides generated in the highly metastatic, mesenchymal-like cell line (A375-MA2) the ER by the endoplasmic reticulum oxidoreductase 1 Alpha (39) as compared to its less aggressive parental cell line (A375) (ERO1α) enzyme, which introduces disulfide bonds into PDI (SI Appendix, Fig. S1B). Altogether, these results confirm rela- (24). The physiological function of GPX8 is still unclear; how- tively high GPX8 expression in mesenchymal-like cancer sam- ever, it has been reported to be involved in diverse physiological ples, indicating its biological role in cancer cell aggressiveness. processes. For example, GPX8 protects against colitis (25), The elevated GPX8 expression in mesenchymal-like cells indi- serves as a cellular substrate to the hepatitis C virus NS3-4A cates that the EMT program regulates its expression. To investi- protease (26), induces ER stress in rat pancreatic β-cells (27), gate this further, we exploited the engineered human mammary and regulates calcium flux in HeLa cells (28). epithelial (HMLE) cells expressing Twist conjugated to the estro- Our knowledge about GPX8 regulation is still limited, al- gen receptor. Upon 4-hydroxytamoxifen (OHT) treatment, this though its expression was found to be regulated by hypoxia- ectopically expressed Twist translocates to the nucleus and grad- inducible factor (HIF1α) (29) and repressed by insulin-like ually induces the EMT-promoting factors (9). We found that OHT growth factor 1 receptor (IGF1R) in the lung (30). These stud- treatment stimulated GPX8 expression and simultaneously down- ies are only starting to reveal some of the physiological functions regulated the epithelial marker E-Cad (Fig. 1E). In addition, of GPX8, but its role in tumor biology is still unclear. Here, we GPX8 expression was up-regulated in the lung carcinoma cell line report that GPX8 robustly maintains cancer cells at their ag- A549 treated with transforming growth factor-beta 1 (TGFβ1) for gressive state via regulation of the IL-6/JAK/STAT3 signaling 9d(40)(Fig.1F). This treatment induced the expression of other pathway. mesenchymal markers (N-Cad [N-cadherin], ZEB1, and vimentin [VIM]) but repressed the epithelial marker (E-Cad). Together, Results these results confirm that the EMT program regulates GPX8 ex- GPX8 Expression Is Up-Regulated during the EMT Program. Previous pression, verifying its biological role in cancer cell aggressiveness. unsupervised hierarchical clustering analysis of cancer cell lines’ metabolic gene expression profile generated by the MERAV web GPX8 Expression Is Associated with Poor Patient Prognosis. To fur- portal (http://merav.wi.mit.edu/) (18) classified five distinct groups ther assess GPX8 role in tumor aggressiveness, we analyzed (11). These include the epithelial group comprising 378 cell lines whether GPX8 up-regulation is associated with poor patient out- originating from the epithelial tissues and the mesenchymal group comes. By utilizing the Kaplan–Meier (KM) Plotter tool (http:// consisting of 150 cell lines expressing a shared mesenchymal sig- kmplot.com/analysis/) (41), we found that high GPX8 expression nature. We compared the expression profile between these two was associated with reduced breast cancer patient overall survival groups and identified significant up-regulation of GPX8 in the (OS) (Fig. 2A), distance metastasis-free survival (DMFS), and mesenchymal group. We found this GPX8 expression pattern to relapse-free survival (RFS) (41) (SI Appendix,Fig.S2A). Moreover, be similar to that of other known mesenchymal markers such as when we classified the breast cancer samples to tumor subtypes DPYD (11), fibronectin (FN1)(10),ZEB1 (31), ZEB2 (32), and according to their aggressiveness, we found that high GPX8 ex- cadherin 11 (CDH11) (33) in contrast to the epithelial markers pression level correlates with poor patient outcomes. For example, cadherin 1 (CDH1 [E-cadherin]) (31), claudin (CLDN1) (10), and the effect of high GPX8 levels on the OS of patients with the less junction plakoglobin (JUP [ɣ-catenin]) (Fig. 1A). aggressive breast cancer subtype (luminal A) was insignificant (P = We then wanted to determine whether the GPX8 expression 0.2), in contrast to the effect on those with the aggressive breast pattern in patient-derived samples is similar to that seen in cancer subtype (basal) where it was significant (P = 0.00051)

2of12 | www.pnas.org/cgi/doi/10.1073/pnas.2010275117 Khatib et al. Downloaded by guest on September 25, 2021 GPX8 DPYD FN1 Pearson: 0.57 Pearson: 0.39 P<0.0001 P<0.0001 P<0.0001 ZEB1 ZEB2 A B Spearman: 0.55 Spearman: 0.44 3K 3K 12K P=2.03e-65 P=5.09e-39 4K 4K 2K 2K 3K 3K 8K ZEB1 2K 2K 1K ZEB2 1K 4K 1K 1K 0 0 0 0 0 0 1K 2K 3K 4K 0 1K 2K 3K 4K

ZEB1 ZEB2 CDH11 Pearson: 0.68 CDH11 CDH1 Pearson: -0.17 P<0.0001 P<0.0001 P<0.0001 Spearman: 0.65 Spearman: -0.19 20K P=2.88e-100 60K P=3.58e-8 800 600 4K 600 12K 40K 400 400

2K CDH1

CDH11 4K 20K 200 200 mRNA expression (RNA Seq V2 RSEM)

Relative expression Relative 0 0 0 0 0 CDH1 CLDN1 JUP 0 1K 2K 3K 4K 0 1K 2K 3K 4K

P<0.0001 P<0.0001 P<0.0001 GPX8, mRNA expression (RNA Seq V2 RSEM) 3K 12 Breast cancer cell lines HCC cell lines 12 D 2K 10 10 Luminal Basal B Low High 8 1K 8 6 6 GPX8 0 EM EM EM VIM

Luminal C E-Cad Basal B

GPX8 CDH11 Actin 800 3k CELL BIOLOGY

2k 500 MCF7 EVSA-T BT-474 Hs-578-T HEPG2 100 1k SNU-387SNU-423 MDA-MB-231MDA-MB-436 0 0 ZEB1 ZEB2 EF 3k OHT (days) 03691215 2k GPX8 2k GPX8 1k 1k E-Cad

0 0 E-Cad CDH1 OCLN N-Cad 2.0 Actin 4 ZEB1 1.5 TGFβ (days) 0 3 5 7 9 Relative mRNA expression levels 2 1.0 VIM 0.5 0 0 Actin MCF7 MCF7 EVSA-TBT-474 EVSA-TBT-474 Hs-578-T Hs-578-T MDA-MB-231MDA-MB-436 MDA-MB-231MDA-MB-436

Fig. 1. GPX8 expression is up-regulated in mesenchymal-like cells. (A) Elevated GPX8 gene expression in mesenchymal cell lines. Cancer cell lines were divided into epithelial (n = 378 cell lines) and mesenchymal (n = 150 cell lines) groups based on the expression of known mesenchymal markers. Box plots represent the expression levels of the indicated genes in each group. The P value was determined by Student’s t test. (B) GPX8 expression correlates with mesenchymal markers. Patients’ gene expression data were generated by the TCGA project and analyzed using the cBioportal web tool (https://www.cbioportal.org). GPX8 expression positively and significantly correlated with known mesenchymal markers (ZEB1, ZEB2, and CDH11) and negatively with the epithelial marker CDH1. The Pearson, Spearman correlation, and the P value, were calculated by the analysis tool. (C) GPX8 mRNA level is up-regulated in basal B breast cancer cells. The relative level of GPX8, as well as other indicated EMT markers in breast cancer cell lines, were determined by qPCR. The expression level of all cell lines is relative to that of the EVSA-T cell line. Each value represents the mean ± SD for n = 3. (D) GPX8 protein levels are up-regulated in high-grade breast and HCC cancer cell lines. Cells were lysed and subjected to immunoblotting using the indicated antibodies. (E) GPX8 expression is up-regulated during the EMT program. HMLE-Twist-ER cells were treated with OHT to induce EMT for a total of 15 d. Every 3 d, cells were collected, lysed, and subjected to immunoblotting using the indicated antibodies. (F) TGFβ1 induces GPX8 expression. A549 cells were treated with 5 ng/mL TGFβ1 to induce EMT for a total of 9 d. On each indicated day, cells were collected and subjected to immunoblotting using the indicated antibodies.

(Fig. 2B). Similarly, high GPX8 expression significantly affected the breast cancer, as it was also found in lung and gastric cancers DMS and RFS in basal breast cancer samples (P = 0.0049 and (Fig. 2C). To reinforce our findings regarding the direct role of 0.00059, respectively) relative to luminal A (P = 0.37 and 0.052, GPX8 in cancer aggressiveness, we demonstrate that other mes- respectively) (SI Appendix,Fig.S2B). This correlation between enchymal markers (ZEB1, DPYD,andVIM) did not show any GPX8 expression level and patient outcome was not restricted to significant effect on patient outcome (SI Appendix,Fig.S2C).

Khatib et al. PNAS Latest Articles | 3of12 Downloaded by guest on September 25, 2021 A Breast Cancer (626) B Luminal A (271) Luminal B (129)

1.0 1.0 1.0

0.8 0.8 0.8

0.6 0.6 0.6

0.4 0.4 0.4 Probability 0.2 HR=1.85(1.42-2.96) Probability 0.2 HR=1.45(0.84-2.32) 0.2 HR=3.12(1.59-6.14) logrank P=2.6e-05 logrank P=0.2 logrank P=0.00051 0.0 0.0 0.0

0 50 100 150 0 50 100 150 0 50 100 150 Time (months) Her2+ (73) Basal (153)

1.0 1.0

0.8 0.8

GPX8 Expression 0.6 0.6

low 0.4 0.4 Probability high 0.2 HR=1.84(0.83-4.07) 0.2 HR=3.06(1.58-5.93) logrank P=0.12 logrank P=0.00051 0.0 0.0 0 50 100 150 0 50 100 150 Time (months) Time (months)

C Lung cancer (1,145) Gastric cancer (631) 1.0 1.0 HR =1.22(1.03-1.44) HR=1.71(1.38-2.13) 0.8 0.8 logrank P=0.021 logrank P=9.2e-07

0.6 0.6

0.4 0.4

Probability 0.2 0.2

0.0 0.0 0 50 100 150 200 0 50 100 150

Time (months) Time (months)

Fig. 2. GPX8 expression is associated with poor patient prognosis. (A) KM survival plots for patients with breast cancer divided into high GPX8 expression (“high” [red]) and low (“low” [black]). The columns represent overall survival data of all breast cancers (Breast Cancer). Number in parentheses indicates the total number of patients. These plots were generated in the KM plotter website. The GPX8 228141_at Affymetrix ID symbol was used for all of the analyses. The P value (P), the hazard ratio (HR), and the number at risk were determined by the analysis tool. (B) GPX8 expression effects on patient overall survival are more profound in the aggressive breast cancers subtypes. KM plots represent the overall survival data from breast cancers; Luminal A, Luminal B, Her2+, and Basal subtypes. (C) Overall survival data of GPX8 expression levels in lung (Lung Cancer) and gastric cancers (Gastric Cancer).

Therefore, our findings emphasize that association with the poor To this end, we knocked out GPX8 in the mesenchymal-like patient outcome is a unique feature of GPX8 expression and not a basal B breast cancer cell line MDA-MB-231, by applying the common characteristic of the EMT markers. Collectively, patient CRISPR-Cas9 gene knockout system. GPX8 knockout (KO) in outcome data support the clinical significance of GPX8 function in two different clones (GPX8-KO-1 and GPX8-KO-2) resulted in aggressive cancer subtypes. significant changes in cell morphology relative to wild-type (WT) cells. These KO cells became smaller, rounder, and clustered GPX8 Loss in MDA-MB-231 Induces Epithelial-Like Phenotype. We into an island-like morphology (Fig. 3A), phenocopying epithe- wanted to explore GPX8’s function in cancer cell aggressiveness. lial cell characteristics. Moreover, despite the differences in their

4of12 | www.pnas.org/cgi/doi/10.1073/pnas.2010275117 Khatib et al. Downloaded by guest on September 25, 2021 DNA deletion pattern (SI Appendix, Fig. S3A), both clones lost Next, we determined whether GPX8 plays a critical role in the GPX8 expression (Fig. 3B) without affecting cell proliferation proper execution of the EMT program. Thus, we induced this (SI Appendix, Fig. S3B). To eliminate the possibility of off-target program in HMLE-Twist-ER cells and found that as opposed to effects, we restored GPX8 expression in GPX8-KO-1 background control cells (shGFP), where OHT shifted the cell-surface high low by ectopically expressing GPX8 (GPX8-KO-1+GPX8-OE). This markers from an epithelial (CD24 /CD44 ) to a mesenchy- low high GPX8 SI rescue GPX8 construct is mutated in its PAM and guide recog- mal (CD24 /CD44 ) profile (42), -silenced cells ( Appendix D E nition sites but translates the same amino acid sequence. We ,Fig.S3 ) maintained their epithelial profile (Fig. 3 ). GPX8 DPYD found that GPX8 overexpression rescued the cellular morphology In addition, knocking down or (another MMS gene) A B resulted in high expression of the epithelial marker CDH1 and low changes induced by GPX8 loss (Fig. 3 and ), thus supporting ZEB1 CDH2 SI ’ ’ expression of the mesenchymal markers and ( GPX8 s role in maintaining the cells mesenchymal morphology. Appendix D Next, we subjected WT and GPX8-KO-1 cells to RNA-Seq ,Fig.S3 ). Collectively, these findings indicate that GPX8 is a critical component of the EMT program and an es- analysis (Dataset S1) to systematically determine the differential sential factor in maintaining the cell’s mesenchymal properties. gene expression profile (SI Appendix,Fig.S3C). GSEA confirmed “ As an in vitro functional readout for the EMT program, we that GPX8 loss caused a significant reduction in the hallmarks of determined the consequence of GPX8 loss on the migratory ca- – ” the epithelial mesenchymal transition gene set (false discovery pabilities of MDA-MB-231 cells using two methods, the Incucyte q < C rate [FDR] -value 0.0001; Fig. 3 ). Specifically, GPX8-KO-1 Live-Cell Analysis System to monitor in real time the rate of cell resulted in the down-regulation of the known mesenchymal migration in a scratch assay (SI Appendix,Fig.S4A) and the de- markers (FN1, SNAI2 [SLUG], and CDH11) along with the up- termination of the number of migrating cells in Boyden chamber regulation of the epithelial marker OCLN (Fig. 3D). This induc- with the transwell migration assay (SI Appendix,Fig.S4B). We tion of the mesenchymal–epithelial transition (MET) program by found that GPX8 loss in two different colonies resulted in a GPX8 loss was inhibited by reintroducing GPX8, implying that significant reduction in migration efficiency (SI Appendix, Fig. this gene has a role as a guardian of the mesenchymal state. S4 A and B). Furthermore, restoring GPX8 expression levels in

GPX8-KO-1 FDR q<0.0001 ABGPX8-WT +GPX8-OE C CELL BIOLOGY GPX8

Actin

GPX8-KO-1 GPX8-KO-2

GPX8-WT GPX8-KO-1GPX8-KO-2GPX8-KO-1 +GPX8-OE E shGFP-OHT shGFP+OHT 4 4 10 1.83% 94.5% 10 78.35% 20.5% 3 3 10 (±0.07) 10 (±4.31) VC (±3.67) (±6.57)

D 2 2 GPX8-OE 10 10

1 1 10 10 1.5 1.5 FN1 SNAl2 0 0 10 10 0 1 2 3 4 0 1 2 3 4 10 10 10 10 10 10 10 10 10 10 1.0 1.0 shDPYD_1+OHT shGPX8_1+OHT CD44

4 4 0.5 10 31.66% 10 10.74% 0.5 3 60.5% 3 85.16% 10 (±4.66) 10 (±0.89) (±2.11) (±0.23)

2 2 0 0 10 10

1 1 CDH11 OCLN 10 10 3 3 0 0 10 10 0 1 2 3 4 0 1 2 3 4 10 10 10 10 10 10 10 10 10 10 2 2 CD24 1 1

Relative mRNA expression levels Relative 0 0

GPX8-WT GPX8-WT GPX8-KO-1GPX8-KO-2GPX8-KO-1 GPX8-KO-1GPX8-KO-2GPX8-KO-1

Fig. 3. GPX8 loss results in epithelial-like characteristics. (A) Silencing of GPX8 expression in MDA-MB-231 cells, using the CRISPR-Cas9 system, induces epithelial-like morphology. (Scale bar, 100 μm.) (B) GPX8 protein levels in the different clones. Cells were separated into single clones, and for each, GPX8 levels were measured by immunoblot using a specific antibody against GPX8. GPX8-KO-1+GPX8-OE: GPX8 was reintroduced in the background of GPX8-KO-1. (C) GPX8 loss leads to reduced gene expression of EMT markers. MDA-MB-231 WT cells and GPX8-KO-1 were subjected to RNA-Seq analysis. The expression ratio between all genes (∼22,000) was calculated and ranked based on the relative expression between the GPX8 WT and KO. The samples were subjected to GSEA. GSEA computed FDR q-value. (D) KO of GPX8 in MDA-MB-231 cells reduces the expression of known mesenchymal markers. The RNA was isolated from the different clones as described above (A) and subjected to qPCR analysis. Each value represents the mean ± SD for n = 9. (E) GPX8 KO inhibits the EMT program. HMLE-Twist-ER cells were infected with the indicated hairpins. The cells were either left untreated or treated with OHT for 15 d, followed by FACS analysis of the cell-surface markers CD24 and CD44 to separate the epithelial and mesenchymal populations. The percentage of cells in each gate is presented. Each value represents the mean ± SD for n = 3.

Khatib et al. PNAS Latest Articles | 5of12 Downloaded by guest on September 25, 2021 clone-1(GPX8-KO-1+GPX8-OE) rescued these observed effects our RNA-Seq data, we limited our secretome only to genes that are in both the scratch assay and the transwell assay (Fig. 4 A and B). expressed in MDA-MB-231 cells. These restrictions limited our These results demonstrate that GPX8 has a vital role in maintaining secretome to 50 genes, which we designated as the cytokines gene cellular mesenchymal features, such as the ability to migrate. set (CGS, Dataset S2). By further analyzing our RNA-Seq data, we found a significant down-regulation of the CGS in GPX8-KO-1 GPX8 Regulates the Stemness Properties of Cancer Cells. Previous relative to the WT cells (Fig. 6B and SI Appendix,Fig.S6B). To studies connected the EMT program and the elevation of CSC validate this GPX8 loss-dependent expression pattern, we focused markers (9, 43). Indeed, several of the basal B breast cancer cell on interleukine-15 (IL-15), interleukine-1β (IL1B), C-X-C motif lines, including MDA-MB-231, have stem-like properties as they chemokine ligand 10 (CXCL10), colony-stimulating factor 3 express specific markers and can initiate tumors in mice (44, 45). (CSF3), and interleukine-6 (IL-6) as they were all significantly We identified that in addition to the EMT markers, GPX8 loss SI Appendix resulted in the down-regulation of the CSC markers such as down-regulated in the RNA-Seq analysis by twofold ( , C CD44 (42) (Fig. 5A), [NGFR, p75NTR (46)] (SI Appendix, Fig. Fig. S6 ). This GPX8 loss-dependent expression pattern was then S5A), and integrin-β4 (ITGB4) (47) (Fig. 5B and SI Appendix, validated by qPCR. Moreover, the down-regulation of these se- Fig. S5B). To further determine the role of GPX8 as a regulator lected cytokines in KO cells was rescued by GPX8 overexpression of the stemness state, we ectopically expressed GPX8 (GPX8- (Fig. 6C). OE) both in GPX8-KO-1 cells and in the basal epithelial breast Among these selected cytokines, we then focused our study on cancer cell line MDA-MB-468 (48), in which GPX8 expression is IL-6, as it has been reported to play a crucial role in cancer relatively low (SI Appendix, Fig. S5C). We found that GPX8-OE in GPX8-KO-1 cells rescued both CD44 and ITGB4 expression levels (Fig. 5 A and B). In addition, introducing GPX8 in MDA-MB-468 A Scratch Assay resulted in CD44 up-regulation (Fig. 5C), demonstrating GPX8 suf- 100 ficiency for inducing this CSC/mesenchymal marker expression. GPX8-WT As an in vitro functional readout for the stemness properties of 80 * the cells, we determined the effect of GPX8 loss on mammosphere GPX8-KO-1+ formation ability in MDA-MB-231 cells. We found that in com- 60 GPX8-OE parison to the WT cells, GPX8 KO clones (GPX8-KO-1 and 40 GPX8-KO-2) formed significantly fewer mammospheres (Fig. 5D GPX8-KO-1 and SI Appendix,Fig.S5D and E). In contrast, GPX8-OE in 20 GPX8-KO-1 cells resulted in a significant increase in the number of D SI Appendix E 0

mammospheres (Fig. 5 and ,Fig.S5 ), thus demon- conﬂuency (%) Wound strating the specific role of GPX8 in mammosphere formation 0 102030 capabilities. Time (hour) We then determined the effect of GPX8 loss on the ability of the MDA-MB-231 to form tumors in mice. Accordingly, we injected cells originating from WT, GPX8-KO-1, or GPX8-KO-1+GPX8- B Transwell Migration Assay OE lines into the mammary fat pad of female NOD-SCID mice P<0.0001 and monitored weekly the number and size of the generated tu- 200 mors for up to 7 wk. Interestingly, only three out of seven mice 150 VC injected with GPX8-KO-1 developed tumors, whereas tumors GPX8-OE formed in all mice injected with WT or GPX8-KO-1+GPX8-OE 100 cells (Fig. 5E). Moreover, tumors generated from GPX8-KO-1 P < SI Appendix F cells weighed significantly less ( 0.005) ( ,Fig.S5 ) 50 andweresmallerinsize(Fig.5F and SI Appendix,Fig.S5G). Al- (per well) together, we found that GPX8 regulates the stemness character- 0 istics of these cancer cells as it plays an essential role in their

initiation ability and tumor growth. Number of migrated cells GPX8 GPX8 WT KO-1 GPX8 Regulates the Secretion of Cytokines. Our next goal was to define the cellular mechanism by which GPX8 regulates the aggressiveness of cancer cells. Analysis of our RNA-Seq data followed by GSEA revealed that GPX8 loss does not only affect the C GPX8-KO-1 hallmark of the EMT (Fig. 3C) but also modulates the cellular GPX8-WT GPX8-KO-1 +GPX8-OE inflammatory response (“hallmark inflammatory response”)(SI Appendix,Fig.S6A). To experimentally verify these global changes in the production of secreted factors, we cultured GPX8-KO-1 cells for 3 d in the growth media from WT cells (WT-conditioned media), which includes all of the desired cytokines/chemokines. While GPX8-KO-1 cells in regular media demonstrated a reduction in CD44, ITGB, and fibronectin (FN1) expression, the ad- Fig. 4. GPX8 loss inhibits cell migration in the MDA-MB-231 breast cancer dition of WT-conditioned media rescued the expression of these cell line. (A) Quantification of scratch confluence during 24 h for the indi- EMT and CSC markers (Fig. 6A). cated WT, GPX8-KO-1, and GPX8-KO-1+ GPX8-OE cells. Each value represents ± = = ’ We then aimed to systematically identify the GPX8-regulated the mean SD for n 3. The P value 0.01 (*) was determined by Student s secreted factors that modulate cancer cell aggressiveness. Using t test. (B) GPX8 loss inhibits the MDA-MB-231 migration capabilities. The “ ” migration capability of the different samples was determined in a transwell the Human Protein Atlas database (https://www.proteinatlas.org), assay. The data are reported as the number of migrated cells per 10,000 we generated a list of all of the predicted secreted proteins seeded cells; each value represents the mean ± SD for n = 6. The P value was (secretome). From the 2,249 listed genes, we chose 78 genes determined by Student’s t test. (C) Representative cell migration images of encoding for cytokines and 44 for chemokines. Then, by analyzing each sample. (Scale bar, 200 μm.)

6of12 | www.pnas.org/cgi/doi/10.1073/pnas.2010275117 Khatib et al. Downloaded by guest on September 25, 2021 GPX8-WT AB200 C GPX8-KO-1 ITGB4 CD44 150 GPX8-KO-1 +GPX8-OE SLUG FLAG 100 Count GPX8 GPX8 50 Actin 0 Actin 103 104 105 CD44 -Fluorescence Intensity VC

GPX8-OE GPX8-WTGPX8-KO-1GPX8-KO-1 D Mammosphere Formation Assay +GPX8-OE P<0.001 25 20 VC E 15 GPX8-OE Number of mice 10 Sample with tumors

(per well) 5 es mmospher a 0 GPX8-WT 8/8 M GPX8 GPX8 GPX8-KO-1 3/7 * WT KO-1 GPX8-KO-1 Tumor Growth 7/7 F +GPX8-OE GPX8-WT

) 1500 3 * P=0.0256 vs. WT GPX8-KO-1+GPX8-OE

1000 GPX8-KO-1

P=0.005 CELL BIOLOGY 500

Tumor volume (mm Tumor 0 02468 Weeks

Fig. 5. GPX8 loss in MDA-MB-231 cells affects cancer stemness. (A) Loss of GPX8 results in CD44 cell surface expression reduction. The different indicated samples were subjected to FACS analysis of the cell-surface markers CD44. The histogram represents CD44 fluorescence intensity values. n = 3. (B) GPX8 expression levels correlate with the stem cell markers. Cells were subjected to immunoblotting with the indicated antibodies. (C) GPX8 overexpression in MDA-MB-468 cells induces CD44 expression. Cells were subjected to immunoblotting with the indicated antibodies. VC, vector control; GPX8-OE, GPX8 overexpression. (D) GPX8 expression in MDA-MD-231 cells correlates with the cells’ ability to form mammospheres. Quantification of in vitro mammosphere formation by cells from the different clones was performed. The data are reported as the number of mammospheres formed per 600 seeded cells; each value represents the mean ± SD for n = 5. The P value was determined by Student t test. (E) GPX8 loss affects tumor formation in mice. Female NOD-SCID mice were injected with 106 cells generated from the different clones. After 7 wk, the proportion of animals bearing tumors was assessed and presented. The P value was determined by Fisher’s exact test. (F) GPX8 expression affects tumor formation and growth rate in mice. During the in vivo time course, the tumor volume of each group was measured weekly and presented in a graph; each value represents the mean ± SD. For GPX8-WT, n = 8; GPX8-KO-1, n = 3; GPX8-KO-1+GPX8- OE, n = 7. The P value was determined by Student t test.

aggressiveness and the EMT program (49, 50). We validated IL- readout for IL6R function and the downstream JAK/STAT3 6’s selective up-regulation expression pattern in mesenchymal signal activation. relative to epithelial cell lines using the MERAV database (SI Comparative gene expression analysis followed by GSEA Appendix, Fig. S6D). Accordingly, GPX8-KO-1 cells demonstrated revealed that GPX8 loss resulted in a significant reduction in a significant reduction in the IL-6 secretion relative to WT cells. “hallmark of IL-6/JAK/STAT3 signaling” (Fig. 7A), indicating This effect on cytokine secretion was rescued by GPX8 over- that GPX8 is a key regulator of this signaling pathway. In D expression (Fig. 6 ). Together, these findings indicate that GPX8 is addition, we performed an mRNA stability assay that dem- a global regulator of fundamental cancer-associated cytokines such onstrated that IL-6 down-regulation was due to reduced as IL-6. transcription rate in GPX8-KO-1 cells (SI Appendix,Fig.S7A) and not due to posttranscriptional regulation by mRNA sta- GPX8 Regulates IL6R, an Essential Player in the IL-6/STAT3 Signaling Pathway. The Janus kinase (JAK)-signal transducer and activator bility factors (55). of transcription 3 (STAT3) signaling pathway provides a critical We then determined whether GPX8 regulation of cytokine link between inflammation and cancer (51). In tumors, STAT3 production (Fig. 6) is mediated by the IL6/JAK/STAT3 signaling activation is associated with the induction of the EMT program cascade. We found that treating GPX8-KO-1 cells with WT (52) and enhancing stem-like properties (53). Activation of the growth media (WT-conditioned media) resulted in a dramatic JAK/STAT3 signaling pathway induces the expression of multi- increase in STAT3 phosphorylation on tyrosine 705 (Fig. 7B). ple cancer aggressiveness promoting factors such as IL-6 (54). However, supplementing this conditioned media with neutraliz- IL-6 is stimulated by an autocrine loop, whereby the cytokine ing IL-6 antibodies, which specifically blocked the activity of this interacts with the IL6 receptor (IL6R), resulting in JAK/STAT3 cytokine (SI Appendix, Fig. S7B), resulted in JAK/STAT3 sig- signal transduction (54). Therefore, IL-6 levels serve as a naling pathway inhibition (Fig. 7B). These findings highlight the

Khatib et al. PNAS Latest Articles | 7of12 Downloaded by guest on September 25, 2021 GPX8 GPX8 All Genes Cytokines/Chemokines IL−6 A WT KO-1 B WT 300 conditioned --+ media 250 CD44 200

ITGB4 150

FN1 100 Adjusted p−value 10 50

GPX8 −Log

0 Actin −10 −5 0 5 10 Log Fold Change C 2

IL-15 IL-1B CSF3 CXCL10 IL-6 1.0 1.0 1.0 1.0 1.0

0.5 0.5 0.5 0.5 0.5

0 0 0 0 0 Relative mRNA Expression Levels GPX8 GPX8 GPX8 GPX8 GPX8 GPX8 GPX8 GPX8 GPX8 GPX8 WT KO-1 WT KO-1 WT KO-1 WT KO-1 WT KO-1

P<0.05 D 200 VC GPX8-OE 150 100 50 IL-6 (pg/ml) 0 GPX8 GPX8 WT KO-1

Fig. 6. GPX8 KO impairs cytokine production in MDA-MB-231 cells. (A) Conditioned media from WT cells can rescue the expression of CSC and EMT markers. Conditioned media from the WT were added to GPX8-KO-1cells for 3 d. The cells were lysed and subjected to immunoblot using the indicated Abs. (B) GPX8 loss induces a global reduction in cytokine expression. The expression level in WT and GPX8 KO cells is presented in a volcano plot. Cytokines and chemokines are presented as a blue triangle apart from IL-6 expression, which is presented in red. (C) GPX8 loss in MDA-MB-231 cells reduces the expression of selected cytokines. The RNA was isolated from WT, GPX8-KO-1, and GPX8-KO-1+GPX8-OE cells, and the expression of the selected genes was determined by qPCR. Each value represents the mean ± SD for n = 3. (D) IL-6 level is reduced in GPX8 KO cells growth media. Cell growth media were collected from each of the indicated samples after 24 h. The level of IL-6 was determined using a specific ELISA kit (n = 3). The P value was determined by Student t test.

importance of GPX8 as a regulator of IL-6 production and its lesser extent, in the GPX8-KO-1 cells (Fig. 7C). Moreover, long- subsequent impact on the downstream JAK/STAT3 cascade. term treatment with Hyper-IL-6 rescued the expression of the The proper protein folding and maturation of both IL-6 and EMT markers SLUG and CD44 in the GPX8-KO-1 cells (Fig. 7D). IL6R is mediated by disulfide bonds (56, 57) that can be po- Since sIL6R was sufficient to induce STAT3 phosphoryla- tentially regulated by GPX8. Thus, we wanted to understand the tion, these results indicate the endogenous IL-6 is active and exact cellular mechanisms by which GPX8 regulates JAK/STAT3 thus not affected by the GPX8 loss. Therefore, these results signaling. Since IL-6 failed to induce STAT3 phosphorylation reveal that GPX8 mediates the expression of mesenchymal (Fig. 7C) in both WT and GPX8-KO-1 cells, we speculated that markers by regulating the IL-6 receptor and not the cytokine GPX8 function is mediated through the IL-6 receptor (IL6R) itself. regulation. IL6R can induce its cellular effect by two mecha- Next, we determined the cellular mechanisms by which GPX8 nisms; the “IL-6 classic signaling”, which is composed of secreted regulates IL6R activity. Surprisingly, we found that when com- IL-6 and transmembrane IL6R, or the alternative IL-6 trans- pared to WT, KO cells up-regulate IL6R expression (SI Ap- signaling (58). In this alternative mechanism, activation of the pendix,Fig.S7E) and secrete higher levels of sIL6R (Fig. 7E). coreceptor IL-6 signal transducer (IL6ST or GP130) is mediated Importantly, as demonstrated in Fig. 7F, IL6R produced in via a complex of IL-6 with the soluble form of IL6R (sIL6R). We GPX8-KO cells cannot activate the JAK/STAT3 signaling found that WT, GPX8-KO-1, andGPX8-KO-1+GPX8-OEcells cascade, and therefore its elevation can be presumably at- expressed GP130 at similar levels (SI Appendix,Fig.S7C); however, tributed to compensation machinery. Moreover, the high IL6R expression is absent from the surface of all these cells (SI Ap- secretion levels of sIL6R in GPX8-KO-1 cells indicates that pendix,Fig.S7D). Accordingly, we propose that GPX8 regulates the cellular trafficking machinery is unaffected by GPX8 IL6R, which consequently activates the JAK/STAT3 signaling through activity. the IL-6 trans-signaling mechanism. Soluble IL6R (sIL6R) can be generated by two mechanisms; To provide further support for this IL-6 trans-signaling mecha- either through alternative splicing, which skips the exon encod- nism, we treated both WT and GPX8-KO-1 cells with IL-6, soluble ing the transmembrane domain or by shedding of the full-length IL-6 receptor (sIL6R), or IL-6/sIL6R fusion protein called Hyper- receptor by ADAM metallopeptidase domain 17 (ADAM17) IL6 (59). In contrast to recombinant IL-6 treatment, sIL6R and (58). We excluded the first possibility as we detected a reduction Hyper-IL6 induced STAT3 phosphorylation in WT cells and, to a in the transcript levels of the sIL6R form in the GPX8-KO-1

8of12 | www.pnas.org/cgi/doi/10.1073/pnas.2010275117 Khatib et al. Downloaded by guest on September 25, 2021 A B GPX8-KO-1 C GPX8-WT GPX8-KO-1 FDR q<0.0001 WT conditioned media -++ IL-6 --+-- +-- Neutralized IL6-Ab - - + SIL6R --+---+- Hyper-IL6 -+-- -- +- pSTAT3(Y705) pSTAT3(Y705) STAT3

STAT3 Actin

GPX8

Actin

DEGPX8-WT GPX8-KO-1 F GPX8-WT GPX8-KO-1 P<0.0001 Hyper-IL6 - +-+ SIL6R-OE -++-- - IL6R-OE- -+ - - + SLUG 400 pSTAT3(Y705) 300 CD44 200 STAT3 GPX8 100 IL6R (pg/ml) CELL BIOLOGY FLAG Actin 0 GPX8 GPX8 WT KO-1 GPX8

G Actin

WT cells GPX8-KO cells

IL6R IL6R sIL6R sIL6R

Feedback Feedback loop loop IL6R IL6R

GP130 GP130 sIL6R

sIL6R GPX8 GPX8 IL-6 STAT3 sIL6R IL-6 STAT3

sIL6R IL-6 IL-6 IL-6 signaling signaling Nucleus IL-6 GP130 IL-6 GP130 IL-6

IL-6 sIL6R IL-6 IL-6 IL-6 sIL6R Secretion Secretion Nucleus IL-6

sIL6R Cancer ER Cancer ER Cytosol stemness Cytosol stemness

Fig. 7. GPX8-KO cellular effect is mediated by IL-6/JAK/STAT3 signaling pathway. (A) GPX8 loss leads to a reduction in the gene expression pattern for “hallmark IL-6/JAK/STAT3 signaling.” MDA-MB-231 WT cells and GPX8-KO-1 were subjected to RNA-Seq analysis. The expression ratio of all genes was calculated and ranked based on the relative expression in GPX8 WT and KO. The samples were subjected to GSEA. The FDR q-value was computed by GSEA. (B) STAT3 signaling is reduced by the addition of neutralizing IL-6 antibodies to the conditioned media. Conditioned media from the WT cultured for 3 d were added to GPX8-KO-1 cells in the absence or presence of neutralizing IL-6 antibodies (800 ng/mL) for 1 h. The cells were lysed and subjected to immunoblot using the indicated antibodies. (C) GPX8 WT and KO cells respond to soluble IL6 receptor and Hyper IL-6 but not to IL-6 treatment. GPX8-WT and GPX8-KO-1 cells were starved with 0.1% FBS medium for 24 h and treated with 20 ng/mL IL-6, 300 ng/mL soluble IL-6 receptor, and 50 ng/mL Hyper IL-6 for 1 h. Cells were subjected to immunoblot using the indicated Abs. (D) Hyper-IL6 induces EMT markers expression in GPX8-KO cells. WT and GPX8 KO cells were stimulated with Hyper IL-6 (50 ng/mL) for 3 d. The cells were then lysed and subjected to immunoblot using the indicated antibodies. (E) The soluble IL-6 receptor level is affected by the expression level of GPX8 in cell growth media. Cell growth media were collected from each of the indicated samples after 24 h starvation. The level of sIL6R was determined using a specific ELISA kit (n = 4). The P value was determined by Student t test. (F) Ectopic expression of full-length IL-6 receptor activates the STAT3 signaling pathway only in WT cells. The soluble IL6R and the full-length IL6R were overexpressed (SIL6R-OE, IL6R-OE, respectively, both FLAG-tag) in both GPX8-WT and GPX8-KO-1 cells, were lysed after 24 h, and subjected to immunoblot using the indicated antibodies. (G) A scheme rep- resenting the role of GPX8 in IL-6/JAK/STAT3 activation. Green arrows represent IL-6 secretion, and dark blue arrows represent intracellular IL-6 signaling. Dashed arrows and reduced font size represent impaired secretion and intracellular signaling. In WT cells, IL-6 and IL6R are processed in the ER; gradient color represents proteins before maturation and solid color after maturation.

Khatib et al. PNAS Latest Articles | 9of12 Downloaded by guest on September 25, 2021 cells relative to WT cells (SI Appendix, Fig. S7F). However, we derived cell lines expressing mesenchymal markers such as ZEB1 found ADAM17 to be an active metalloprotease in all of the (70). In addition, persistent cancer cells, which are resistant to samples, including GPX8-KO (SI Appendix, Fig. S7G). We con- lapatinib treatment and up-regulate mesenchymal as well as stem clude that the shedding of the full-length receptor in cell markers, are vulnerable to GPX4 inhibition by RSL3 (71). GPX8-KO-1 is the central mechanism in the generation of This mutual mesenchymal cell-dependent function of GPX4 and sIL6R. GPX8 suggests a conserved role for this subfamily of GPx in To gain insight into GPX8 activity, we overexpressed sIL6R aggressive cancer cells. and IL6R in WT and GPX8-KO-1 cells and assessed the impact The maturation process of cytokines and their respective re- on STAT3 signaling. We found that overexpression of the sIL6R ceptors, such as IL-6 and IL6R, occurs in the ER. In this organ- induced STAT3 phosphorylation in both WT and GPX8-KO-1 elle, these proteins acquire their proper structural conformation cells. Remarkably, overexpression of the full-length receptor in via modifications such as disulfide bond formation (56, 57). GPX8-KO-1 cells failed to activate this signaling cascade F Specifically, the extracellular soluble domain of IL6R, which (Fig. 7 ). Thus, GPX8 preferentially regulates full-length IL6R interacts with the ligand, contains four conserved cysteines prior to its cleavage, as the function of the ectopically expressed that form disulfide bonds (72). Here, we determined that sIL6R was unaffected. GPX8 modulates IL6R, as the addition of recombinant IL6R Discussion rescues the GPX8 loss cellular phenotype. Thus, we suggest a We have found that the MMS gene GPX8 is a guardian of the potential mechanism whereby the ER-localized GPX8 and its mesenchymal state in aggressive cancer cells. GPX8 demon- function in protein disulfide bond formation (22) regulate the strated a significant correlation with known mesenchymal maturation of IL6R, essential for its ability to transmit the markers in both cell lines and patients. Moreover, high GPX8 signaling downstream. expression was associated with poor patient prognosis in ag- We suggest an overall model in which the EMT program el- gressive breast cancer samples. We show that GPX8 expression evates GPX8 expression central to the activity of cytokines and was up-regulated during the EMT program, and its loss con- their receptors. These fully functional autocrine cytokines are ferred epithelial characteristics in the mesenchymal-like breast secreted and induce the aggressive mesenchymal-like properties cancer cells, MDA-MB-231. Specifically, knocking out GPX8 in these tumors via the activation of signaling cascade such as resulted in a profound inhibition of cytokine production, in- JAK/STAT3 (54) (Fig. 7G). Finally, we have revealed a GPX8/ cluding the EMT-promoting factor, IL-6. In these cells, IL-6 is IL-6/STAT3 axis, essential for the maintenance of the aggressive produced through an autocrine loop, which activates the JAK/ mesenchymal-like state in cancer cells. We hypothesize that STAT3 signaling pathway through a trans-signaling mechanism. further studies evaluating GPX8 function in posttranslational We demonstrated that GPX8 modulated the ability of IL-6 and modification, such as disulfide bond formation, would identify its receptor to activate this signaling cascade. Thus, we propose a new mechanisms essential for the regulation of cancer cell model in which GPX8 regulates the full-length IL6R activity, aggressiveness. probably by affecting its interaction with its ligand, resulting in impaired JAK/STAT3 signaling (Fig. 7G). Materials and Methods We determine GPX8 up-regulation in mesenchymal-like ag- Cell Lysis and Immunoblotting. Cells were rinsed once with ice-cold PBS and gressive cancer cells and in two different EMT-induction system lysed with RIPA lysis buffer (20 mM Tris [pH 7.4]), 137 mM NaCl, 10% glycerol models (TGFβ and Twist) (Fig. 1). HIF1α plays a regulatory role (vol/vol), 1% Triton X-100, 0.5% (wt/vol) deoxycholate, 0.1% (wt/vol) SDS, in the EMT program (60) as it represses E-cadherin expression 2.0 mM ethylenediaminetetraacetic acid (EDTA) (pH 8.0), and one tablet of (61) or activates the expression of the key EMT transcription EDTA-free protease inhibitor (Roche) and phosphatase inhibitor mixture factors such as TWIST (62), ZEB1, and ZEB2 (3). Moreover, mixes A and B (100X) (Bimake). The protein concentration was determined this factor in triple-negative breast cancer promotes tumorigen- by Bradford (BioRad, 500-0006). The supernatants were separated by a 10% μ esis and adaptation to hypoxia (63). A previous study has shown SDS/PAGE, transferred onto a 0.45 m PVDF membrane (Merck), and probed that GPX8 expression is transcriptionally regulated by HIF1α in with the appropriate antibodies. HeLa cells (29), as GPX8 promotor contains two hypoxia- α Antibodies. Antibodies were obtained from the following sources: GPX8 response elements. Thus, we suggest HIF1 as a key regulator (HPA036720) from Sigma Aldrich, CDH1 (3195), CDH2 (13116), β-actin of GPX8 expression in breast cancer. (4970)\(3700), ZEB1 (3396), VIM (5741), ITGB4 (14803), CD44 (for immuno- The EMT program was first reported in embryonic develop- blotting [3570]), p-sSTAT3-Tyr705 (9145), STAT3 (9139), from Cell Signaling ment when selected cells change their epithelial identity and gain Technology; FITC-labeled anti-CD24 (555427), APC-labeled anti-CD44 mesenchymal-like characteristics (3). Further studies expanded (559942) from BD Bioscience; IL6-R anti-mouse (sc-373708) HRP-labeled the role of the EMT program to be included in wound healing, anti-mouse and anti-rabbit secondary antibodies from Santa Cruz Bio- fibrosis, and cancer. Fibrosis is a complex disease associated with technology. GP130 anti-rabbit (06-291) from Upstate Biotechnology. For reduced organ function (64), which is caused by cellular IL-6 neutralization assay, we used goat anti-human IL-6 (500-P26G; mechanisms reminiscent of oncogenesis (65). Interestingly, Peprotech). fibrosis, like cancer aggressiveness, is driven by ROS (65) and selected cytokines such as IL-6 (66). Since both of these fac- Cancer Sample Analysis. KM analysis of the data from breast and other cancer tors are associated with GPX8, this enzyme is a potential samples were analyzed and generated by the KM Plotter website (http:// druggable candidate to inhibit cancer aggressiveness and kmplot.com/analysis/) (73). GPX8 was searched as a gene symbol, and the fibrosis. 228141_at Affymetrix ID was chosen. The autoselect best cutoff was selected The eight-member GPx family of enzymes is divided into two as well. The obtained KM plots are presented. The statistics were generated by the website itself. groups based on the presence of the nonstandard amino acid, selenocysteine (GPX1-4, and GPX6) or of cysteine (GPX5, Animal Studies. MDA-MB-231 WT, GPX8-KO-1, and GPX8 rescue cells were GPX7-8) in their active site. Unambiguous phylogeny analysis injected into the mammary fat pad of female NOD-SCID mice (1 × 106 cells further subdivides this family into three groups, whereby GPX4, per mouse). The tumors were measured weekly. After 7 wk, the tumors were GPX7, and GPX8 belong to the same evolutionary branch (67). removed and weighed. All mouse experiments were carried out under He- GPX4 was found to be one of the primary regulators of fer- brew University Institutional Animal Care and Use Committee-approved roptosis (68), a form of apoptotic cell death (69). A selective protocol MD-16-14939-5. Hebrew University is Association for Assessment GPX4 inhibitor, RSL3, induces cell death in epithelial cancer- and Accreditation of Laboratory Animal Care-approved.

10 of 12 | www.pnas.org/cgi/doi/10.1073/pnas.2010275117 Khatib et al. Downloaded by guest on September 25, 2021 Cytokine and Receptor Measurement. Measurement of IL-6 levels was per- ACKNOWLEDGMENTS. We thank the members of the Y.D.S. laboratory. This formed by sandwich ELISA using the human IL-6 mini ABTS ELISA develop- work was supported by the Israel Science Foundation (Grant 1816/16), the ment kit (Peprotech). Human soluble IL-6 receptor levels in the medium were Israel Cancer Association (Grant 20180062, Abraham Rutstein funds), and the measured by the in vitro ELISA kit (Abcam, ab46029). WT and GPX8-KO-1 Hebrew University start-up funds. B.S. is supported by the Lady Davis cells were starved with serum-free media for 36 h. The media were col- Fellowship for postdoctoral researchers at The Hebrew University of Jerusalem. The Genomic Applications Laboratory of the Core Research lected and concentrated by centrifugation using 10 KDa cutoff Spin filter Facility, The Faculty of Medicine, The Hebrew University of Jerusalem, (Amicon). Israel, performed the RNA-Seq data analysis. Prof. Rotem Karni, Hebrew University, designed the primers for IL6R alternative splice isoform Data Availability. All relevant data in the paper are entirely available through analysis. Prof. Irit Sagi, Weizmann Institute of Science, assisted the both text and figures, in the main text and SI Appendix. ADAM17 activity assay.

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