Heat Shock Protein 47 Maintains Cancer Cell Growth by Inhibiting

Published OnlineFirst February 26, 2020; DOI: 10.1158/1541-7786.MCR-19-0673

MOLECULAR CANCER RESEARCH | CELL FATE DECISIONS

Heat Shock Protein 47 Maintains Cancer Cell Growth by Inhibiting the Unfolded Protein Response Transducer IRE1a Akihiro Yoneda1, Kaori Sakai-Sawada1, Kenjiro Minomi1,2, and Yasuaki Tamura1

ABSTRACT ◥ HSP47 is a collagen-specific protein chaperone expressed in ROS induced accumulation of 4-hydroxy-2-nonenal-protein fibroblasts, myofibroblasts, and stromal cells. HSP47 is also adducts and activated two UPR transducers, PKR-like ER kinase expressed in and involved in growth of cancer cells in which collagen (PERK) and activating transcription factor 6a (ATF6a), resulting in levels are extremely low. However, its role in cancer remains largely impaired cancer cell growth. Our work indicates that HSP47 unclear. Here, we showed that HSP47 maintains cancer cell growth expressed in cancer cells relieves the ER stress arising from protein via the unfolded protein response (UPR), the activation of which is synthesis overload within these cells and tumor environments, such well known to be induced by endoplasmic reticulum (ER) stress. We as stress induced by hypoxia, low glucose, and pH. We also propose observed that HSP47 forms a complex with both the UPR trans- that HSP47 has a biological role that is distinct from its normal ducer inositol-requiring enzyme 1a (IRE1a) and ER chaperone BiP function as a collagen-specific chaperone. in cancer cells. Moreover, HSP47 silencing triggered dissociation of BiP from IRE1a and IRE1a activation, followed by an increase in Implications: HSP47 maintains cancer cell growth by inhibiting the intracellular level of reactive oxygen species (ROS). Increase in IRE1a.

Introduction In the tumor microenvironment, the extracellular matrix (ECM) secreted from stromal cells and cancer-associated fibroblasts (CAF) is HSP47 is a specific chaperone against collagens and is mainly required for tumor cell growth, invasion, and metastasis and is associated expressed in fibroblasts, myofibroblasts, and stromal cells that con- with resistance to chemotherapy (13–16). The proliferation and migra- stitutively produce and secrete several types of collagen (1–3). HSP47 is tion of cancer cells are promoted through degradation of the ECM by specifically localized in the endoplasmic reticulum (ER) by the pres- matrix metalloproteinases from themselves (17). Therefore, targeting ence of RDEL in the C-terminus of its molecule, and it transiently binds ECM-producing cells such as CAFs and targeting molecules such as to procollagens to prevent the aggregation and bundle formation of HSP47, which plays a crucial role in production of ECM in CAFs, are procollagens. Depletion of HSP47 induces a delay in the secretion of considered to be a promising modality for antitumor therapy (12, 18–22). collagen type 1 and type 4, leading to an accumulation of immature Intriguingly, some studies have shown that HSP47 is expressed in cancer procollagens in the ER (4–6). Excessive accumulation of immature cells and that an increased expression level of HSP47 is associated with a procollagens in the ER in Hsp47 / cells triggers dilation of the high malignant grade of gliomas, poor prognosis of breast cancer, and ER, causing impairment of cell proliferation accompanied by ER lymph node metastasis of colorectal cancer (23–28). Zhu and colleagues stress. Thus, it is widely accepted that HSP47 is indispensable for the demonstrated that HSP47 promotes proliferation of breast cancer cells homeostasis of collagen biosynthesis and maturation. Indeed, in vivo via augmented production of extracellular matrix components including silencing of HSP47 expression in collagen-producing cells dramati- collagen and fibronectin (27). However, how HSP47 directly or indirectly cally induced resolution of fibrosis in animal models of hepatic, regulatescancercellgrowthandwhetherHSP47servesasacollagen- pancreatic, skin, vocal fold mucosal, and cardiac fibrosis (7–11). specific chaperone in cancer cells remain largely unknown. An under- Recently, it has been demonstrated that silencing of HSP47 expression standing of the precise molecular mechanism by which HSP47 functions in pancreatic stellate cells induces reversal of pancreatic desmoplasia in cancer cells may provide a meaningful clue for practical therapy of and improves the efficacy of chemotherapy against pancreatic can- malignant tumors with poor prognosis. We here show that almost no cer (12), suggesting that targeted disruption of HSP47 is a possible collagen is synthesized in human cancer cells despite the fact that almost therapeutic modality for tissue fibrosis and tumors. all human cancer cells express HSP47. Silencing of HSP47 expression induced impaired cancer cell growth accompanied by the formation of an abnormal ultrastructure of the ER and activation of the unfolded protein 1 Department of Molecular Therapeutics, Center for Food and Medical Innovation, response (UPR), the activation of which is involved in the survival/death Institute for the Promotion of Business-Regional Collaboration, Hokkaido Uni- switch of cancer cells, tumor progression, and resistance to therapy and versity, Sapporo, Japan. 2Research & Development Department, Nucleic Acid Medicine Business Division, Nitto Denko Corporation, Osaka, Japan. plays a crucial role in the decision of cancer cell fate (29, 30). We also reveal the molecular mechanism by which silencing of HSP47 expression Corresponding Author: Akihiro Yoneda, Department of Molecular Therapeutics, Center for Food & Medical Innovation, Institute for the Business-Regional triggers activation of the UPR and suggests a biological role of HSP47, fi Collaboration, Hokkaido University, Nishi-11, Kita-21, Kita-ku, Sapporo 001- other than its role as a collagen-speci c chaperone, in cancer cells. 0021, Japan. Phone: 81-11-706-9464; Fax: 81-11-706-9463; E-mail: [email protected] Materials and Methods – Mol Cancer Res 2020;XX:XX XX Human cell lines doi: 10.1158/1541-7786.MCR-19-0673 Human cancer cell lines (MCF7, BT474, MDA-MB-231, HCT116, 2020 American Association for Cancer Research. SW480, HT29, PANC-1, MIA-PaCa-2, Suit2, Capan2, A549, H358,

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and HeLa) and normal human dermal fibroblasts (NHDF) were ab48187, Abcam), anti-Xbp1s (1:2,000, 12782, Cell Signaling Tech- purchased from ATCC. A human hepatic stellate cell line (LX-2) was nology), anti-ASK1 (1:2,000, 3762, Cell Signaling Technology), anti- purchased from Millipore. Cancer cell lines other than PANC-1 cells phospho-ASK1 (1:1,000, 3765, Cell Signaling Technology), anti-JNK were cultured in DMEM (Sigma-Aldrich) supplemented with 10% (1:2,000, 9252, Cell Signaling Technology), anti-phospho-JNK heat-inactivated FBS (Thermo Fisher Scientific). PANC-1 cells were (1:1,000, 4668, Cell Signaling Technology), anti-Puma (1:1,000, cultured in RPMI1640 supplemented with 10% heat-inactivated FBS. 4976, Cell Signaling Technology), anti-Noxa (1:1,000, 14766, Cell All cell lines were authenticated using short-tandem repeat DNA Signaling Technology), anti-ATF6 (1:1,000, ab122897, Abcam), and profiling recommended by ATCC experts. These cells were passaged anti-GAPDH (1:5,000, ab8245, Abcam). After treatment with primary in our laboratory for fewer than 8 weeks after resuscitation. All cells antibodies, the membranes were incubated with the following sec- were tested for Mycoplasma using the Universal Mycoplasma Detec- ondary antibodies: horseradish peroxidase (HRP)-conjugated goat tion Kit (30-1012K; ATCC) and were negative for Mycoplasma within anti-rabbit IgG (1:10,000, 7074, Cell Signaling Technology) and 2 months of cell thawing and experimental work. HRP-conjugated goat anti-mouse IgG (1:10,000, 7076, Cell Signaling Technology). Proteins were detected by a chemiluminescent method qPCR and RT-PCR using ECL (GE Healthcare). GAPDH was used an internal control. Total RNAs from cancer cell lines were extracted using an RNeasy Mini Kit (Qiagen). Total RNA (1 mg) was used for reverse transcription Transfection of siRNA by High-Capacity RNA-to-cDNA Master Mix (Applied Biosystems). Cancer cells were transfected with control siRNA (siControl, qPCR was carried out using Power SYBR Green PCR Master Mix Thermo Fisher Scientific), HSP47-A siRNA (siHSP47-A, sense: 50- (Applied Biosystems) and primers against HSP47 [forward (F): 50- CUACGACGACGAGAAGGAAtt-30; antisense: 50-UUCCUUCUC- TGACCTGCAGAAACACCTGG-30; reverse (R): 50-AGGAAGAT- GUCGUCGUAGta-30), HSP47 siRNA-B (siHSP47-B, sense: 50-AG- GAAGGGGTGGTC-30], collagen type 1a1 (F: 50-GTGCTAAAG- CCCUCUUCUGACACUAAtt-30; antisense: 50-UUAGUGUCAGA- GTGCCAATGGT-30,R:50-ACCAGGTTCACCGCTGTTAC-30), col- AGAGGGCUgg-30) or HSP47-C siRNA (siHSP47-C, sense: 50-GGA- lagen type 1a2 (F: 50-GGTGTAAGCGGTGGTGGTTA-30,R:50-CT- CAGGCCUCUACAACUAtt-30; antisense: 50-UAGUUGUAGAGG- GGGTGGCTGAGTCTCAAG-30), collagen type 2a1 (F: 50-TCTAC- CCUGUCCtt-30) using Lipofectamine RNAiMAX (Thermo Fisher CCCAATCCAGCAAAC-30,R:50-GTTGGGAGCCAGATTGTCAT- Scientific) and were cultured for 5 hours at 37C in air. At 5 hours 30), collagen type 3a1 (F: 50-AGGGGAGCTGGCTACTTCTC-30,R:50- after transfection with siRNA, the cells were cultured in DMEM TAGGAGCAGTTGGAGGCTGT-30), collagen type 4a1 (F: 50-GGT- supplemented with 10% heat-inactivated FBS. ATTCCAGGATGCAATGG-30,R:50-TCTCACCTGGATCACCCT- TC-30), collagen type 4a2 (F: 50-CACCTTCCACCCAGATCAGT-30, Transmission electron microscopic analysis R: 50-CTCTGGCACCTTTTGCTAGG-30), ERdj4 (F: 50-AAAATAA- Cancer cells treated with siControl and siHSP47 were fixed with GAGCCCGGATGCT-30,R:50-CGCTTCTTGGATCCAGTGTT-30), 2.5% glutaraldehyde in 0.1 mol/L cacodylate buffer (pH 7.3). After p58IPK (F: 50-CTCAGTTTCATGCTGCCGTA-30,R:50-TTGCTGC- washing with 0.1 mol/L cacodylate buffer (pH 7.3), the cells were AGTGAAGTCCATC-30), EDEM (F: 50-TGGACTGCAGGTGCTGA- postfixed in 1% OsO4 and 1.5% potassium ferrocyanide in 0.1 mol/L TAG-30,R:50-GGCGTACCCACACTTGACTT-30), PDIA6 (F: 50- cacodylate buffer. Samples were stained with uranyl acetate for 2 hours CTCTGTGTTGTGGCTGTGCT-30,R:50-GCTCCCTGAGAAACT- at room temperature and then washed, dehydrated, and embedded in CGTTG-30), and GAPDH (F: 50-GAGTCAACGGATTTGGTCGT-30, Epon 812. Ultrathin sections were cut with a diamond knife and were R: 50-TTGATTTTGGAGGGATCTCG-30). RT-PCR was performed stained with lead citrate. Samples were examined with an electron using GoTaq DNA polymerase (Promega) and primer pairs against microscope (JEM-3200FS, JEOL) at an acceleration voltage of 300 kV. Xbp-1 (F: 50-GGAGTTAAGACAGCGCTTGG-30,R:50-GGAAGGG- CATTTGAAGAACA-30). After RT-PCR, samples were applied to 3% Establishment of HSP47 knockout cells agarose gel electrophoresis. The pCG SapI vector (gift from Dr. Takayuki Sakurai, Sinshu University, Matumoto, Japan) contains the Cas9 sequence and a Western blot analysis cloning site for targeted gRNA sequence insertion. The gRNA Cells were then washed with PBS and were lysed with a lysis buffer sequence (50-CCGACTGTACGGACCCAGCTCAG-30) specifically (50 mmol/L Tris, 250 mmol/L NaCl, 25 mmol/L EDTA, and 1% NP- designed for exon 2 of the human HSP47 genome was inserted into 40) supplemented with a protease inhibitor (Roche Diagnostics). the pCG SapI vector. The constructed targeting vector and pcDNA3.1 Equal amounts of proteins were separated by SDS-PAGE and trans- (þ)-Hygro were cotransfected into cancer cells using Lipofectamine ferred to polyvinylidene difluoride (PVDF) membranes (Millipore). 2000 (Thermo Fisher Scientific). The treated cells were cultured in After blocking with 5% skim milk in PBS containing 0.05% Tween-20, DMEM supplemented with 10% heat-inactivated FBS and hygromycin the membranes were probed with the following primary antibodies: (500 mg/mL, Thermo Fisher Scientific) to select cell clones stably anti-collagen type 1 (1:1,000, ab138492, Abcam), collagen type 4 silencing HSP47 expression. Single-cell clones were evaluated by DNA (1:10,00, ab6586, Abcam), HSP47 (1:2,000, ab77609, Abcam), ERdj4 sequencing analysis to detect indels (insertions/deletions) in the target (1:2,000, ab118282, Abcam), anti-p58IPK (1:1,000, 2940, Cell Signal- alleles. Furthermore, expression of HSP47 at the protein level in the ing Technology), anti-EDEM (1:1,000, E8159, Sigma), anti- selected clones was confirmed by Western blotting. The control cell PDIA6 (1:1,000, ab11432, Abcam), anti-PERK (1:2,000, sc-13073, clone (mock) was generated by transfecting the empty vector. Santa Cruz Biotechnology), anti-PERK (1:2,000, 5683, Cell Signaling Technology), anti-phospho-PERK (1:1,000, sc-32577, Santa Cruz Establishment of cells reconstituting HSP47 expression Biotechnology), anti-eIF2a (1:2,000, 9721, Cell Signaling Technolo- HSP47 knockout (KO) cancer cells were transfected with the gy), anti-phospho-eIF2a (1:1,000, ab48187, Abcam), anti-Chop pCMV6-HSP47 plasmid (Origene). At 48 hours after transfection (1:1,000, ALX-804-551, Enzo Life Sciences), anti-IRE1a (1:2,000, with the plasmid, the treated cells were cultured in DMEM supple- 3294, Cell Signaling Technology), anti-phospho-IRE1a (1:1,000, mented with 10% heat-inactivated FBS and G418 (800 mg/mL, Thermo

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Fisher Scientific) for 2 weeks to select cell clones stably expressing Lipid staining and flow cytometric analysis HSP47. Expression of HSP47 protein in the selected cell clones was Intracellular lipid content was evaluated by flow cytometry using determined by Western blot analysis. 4,4-Difluoro-1,3,5,7,8-Pentamethyl-4-Bora-3a,4a-Diaza-s-Indacene (BODIPY 493/503; Thermo Fisher Scientific). Briefly, cancer cells at a Animal experiment concentration of 1 105 cells/60-mm dish were stained with BODIPY BALB/c nu/nu male mice at 6 to 8 weeks of age were purchased from 493/503 at 0.5 mg/mL in PBS for 15 minutes at room temperature in the CLEA Japan. All animal studies were reviewed and approved by the dark. BODIPY 493/503 staining was detected in the PE channel. Flow Experimental Animal Ethics Committee of Hokkaido University cytometric analysis was performed on a FACS CantoII (BD (approval number 15-0131). The feeding, maintenance, and use of Biosciences). the animals were performed in accordance with the guidelines of the Experimental Animal Ethics Committee of Hokkaido University Detection of intracellular 4-HNE–protein adducts (Hokkaido, Japan). BALB/c nu/nu male mice were subcutaneously Cell lysates from cells were applied to PVDF membranes. After injected with cancer cells (Mock), HSP47 KO cancer cells, and HSP47 blocking with 5% skim milk in PBS containing 0.05% Tween-20, the KO cancer cells with reconstituted HSP47 (reHSP47) at a concentra- membranes were probed with the following primary antibodies: tion of 1 106 cells/50 mL PBS/animal, respectively. Tumor growth 4-HNE (1:1,000, ab46545, Abcam) and GAPDH (1:5,000, ab8245, was measured once or twice a week for 5 weeks after transplantation Abcam). After treating with primary antibodies, the membranes were with cells. Tumor volumes (mm3) were calculated by using this incubated with the following secondary antibodies: HRP-conjugated formula: volume ¼ 3.14 (width2 length)/6. goat anti-rabbit IgG (1:10,000, 7074, Cell Signaling Technology) and HRP-conjugated goat anti-mouse IgG (1:10,000, 7076, Cell Signaling Immunoprecipitation of HSP47 and IRE1a Technology). Proteins were detected by a chemiluminescent method Cancer cells untreated or treated with control siRNA and with using ECL (GE Healthcare). Content of intracellular 4-HNE–protein HSP47 siRNA were washed with iced PBS and lysed with a lysis adducts in cancer cells was quantified by OxiSelect HNE Adduct buffer (50 mmol/L Tris, 250 mmol/L NaCl, 25 mmol/L EDTA, 1% competitive ELISA Kit (Cell Biolabs). NP-40) supplemented with a protease inhibitor. Immunoprecipi- tation of HSP47 and IRE1a from cell lysates was carried out using Statistical analysis anti-HSP47–immobilized and anti-IRE1a–immobilized Dynabeads All experiments in this study were carried out at an individually Protein G (Thermo Fisher Scientific), respectively. After elution triplicate. All data are shown as means SEM. Differences between with 0.1 mol/L glycine buffer (pH 2.8), samples were applied to groups were tested for statistical significance using Student t test or immunoblotting. ANOVA. Statistical significance was determined at P < 0.05.

Blue native-PAGE Cancer cells were washed with iced PBS and lysed with 1 Results NativePAGE sample buffer (50 mmol/L Bis-Tris buffer, 50 mmol/L Silencing of HSP47 induces impaired cancer cell growth NaCl, 6 N HCl, 10% glycerol, and 0.0001% Ponceau S; pH 7.2) accompanied by abnormal dilation of the ER supplemented with 1% digitonin. Samples were applied to blue We first examined the expression of HSP47 in 13 human cancer cell native-PAGE according to the instructions in the user manual of lines and found that HSP47 mRNA and protein were expressed in all of NativePAGE Novex Bis-Tris Gel System (Thermo Fisher Scientific) the human cancer cell lines (Fig. 1A and B). On the other hand, and immunoblotting. although it has been shown that the expression pattern of HSP47 is correlated with that of collagens in fibroblasts, myofibroblasts, and Establishment of cells stably expressing IRE1a and Xbp1 short stromal cells (1, 2) and we also confirmed the expression of collagen hairpin RNA type 1 and collagen type 4 proteins in the LX-2 and NHDF, the Cancer cells were transfected with plasmids containing a con- expression of collagen type 1 and collagen type 4 proteins was not trol short hairpin RNA (shRNA) sequence (Origene), IRE1a detected in any of the 13 cancer cell lines (Fig. 1B). Quantitative PCR shRNA sequence (50-TGAGGAAGTTATCAACCTGGTTGAC- also showed that the expression of collagen type 1a1, collagen type 1a2, CAGA-30), or Xbp1 shRNA sequence (50-GCTGGSACAG- collagen type 2a1, collagen type 3a1, collagen type 4a1, and collagen type CAAGTGGTAGATTTAGAAG-30). At 48 hours after transfection 4a2 mRNA was hardly detected in cancer cell lines (Fig. 1A). We with the plasmid, the treated cells were cultured in DMEM further performed cDNA microarray analysis of cancer cells (SW480 supplemented with 10% FBS and 2 mg/mL puromycin (Thermo and HCT116 cells) and found weak expression of some collagen Fisher Scientific) for 2 weeks to select cell clones stably expressing mRNAs (collagen type 2a1, collagen type 4a1, collagen type 4a2, control shRNA (shControl), IRE1a shRNA (shIRE1a), and Xbp1 collagen type 8a2, collagen type 9a1, collagen type 11a1, collagen type shRNA (shXbp1). 13a1, collagen type 16a1, collagen type 17a1, and collagen type 24a1), but the expression of these collagen mRNAs was not confirmed by Detection of intracellular ROS quantitative PCR (data not shown). These results suggest that HSP47 Cancer cells, shControl cells, shIRE1a cells, and shXbp1 cells were may have a functional role other than its role as a collagen-specific treated with CM-H2DCFDA (6-chloromethyl-20,70-dichlorodihydro- molecular chaperone in colorectal cancer cells. fluorescein diacetate, acetyl ester, Molecular Probes) at a concentration To explore the role of HSP47 in cancer cells, we examined cancer cell of 5 mmol/L for 10 minutes. Accumulation of intracellular ROS was growth when HSP47 expression was silenced by transfection with quantitatively detected by measuring a fluorescent intensity (495/527 siHSP47. The expression of HSP47 protein in cancer cells (SW480 and nm) of CM-H2DCFDA and was analyzed by NIH ImageJ software. HCT116 cells) was completely suppressed by transfection with three The number of cells examined for a quantification of ROS was 1,000 batches of siHSP47 (Fig. 2A), we used HSP47-C siRNA (at a con- cells. centration of 10 nmol/L) as siHSP47 for further experiments due to a

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Figure 1. Expression of HSP47 and collagens in human cancer cells. A, Expression of HSP47, collagen type 1a1, collagen type 1a2, collagen type 2a1, collagen type 3a1, collagen type 4a1,andcollagen type 4a2 mRNAs in 13 human cancer cell lines, human hepatic stellate cell line LX-2, and NHDF. B, Expression of HSP47, collagen type 1 (Col1), and collagen type 4 (Col4) proteins in the 13 human cancer cell lines, LX-2 cells, and NHDF.

high knockdown efﬁciency. As shown in Fig. 2B, silencing of HSP47 expression induced an abnormal dilation of the ER in cancer cells, expression suppressed the growth of cancer cells. We also generated being similar to the morphology of the dilated ER observed in cancer HSP47 KO cancer cells (HSP47 KO SW480 and HSP47 KO HCT116 cells treated with an ER stress inducer tunicamycin (Fig. 3A; ref. 31). cells; Fig. 2C), and investigated the in vitro proliferation and tumor Dilation of the ER is known to be induced by excessive accumulation of growth of HSP47 KO cancer cells. The in vitro proliferation rate and misfolded or unfolded proteins in the ER, causing ER stress that tumor growth of HSP47 KO cancer cells were signiﬁcantly lower than stimulates the UPR to maintain cell survival (29, 30). This study those of counterparts, which were recovered by the reconstitution of showed that silencing of HSP47 expression induced an abnormal HSP47 expression (Fig. 2D and E). These results suggest that HSP47 is dilation of the ER in cancer cells, suggesting activation of the UPR. involved in the cell growth and tumor growth of cancer cells. To clarify how silencing of HSP47 induces impaired cancer cell Silencing of HSP47 stimulates three branches of the UPR in growth, we performed transmission electron microscopic analysis to cancer cells examine the morphologic difference between siControl-transfected To address the hypothesis described above, we investigated whether and siHSP47-transfected cancer cells. Intriguingly, silencing of HSP47 silencing of HSP47 expression triggers activation of the UPR in cancer

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Figure 2. Silencing of HSP47 induces impaired cancer cell growth. A, Expression of HSP47 protein in cancer cells (SW480 and HCT116 cells) at day 3 after transfection with siControl and three batches of siHSP47. B, Proliferation of cancer cells after transfection with siControl and siHSP47 (siHSP47-A, siHSP47-B, siHSP47-C). C, Expression of HSP47 protein in HSP47 KO cancer cells [clone 1 (c1) and c2], HSP47 KO cells with reconstituted HSP47 (reHSP47; c1 and c2). D, Proliferation of HSP47 KO cancer cells (c1 and c2), HSP47 KO cancer cells with reHSP47 (c1 and c2). E, Tumor growth of cancer cells (mock, n ¼ 6), HSP47 KO cancer cells (c1 and c2, n ¼ 6, respectively), HSP47 KO cancer cells with reHSP47 (c1 and c2, n ¼ 6, respectively; , P < 0.05; n.s., not signiﬁcant). cells. Generally, there are three distinct branches of the UPR: further triggered the phosphorylation of IRE1a,ASK1,andJNK, inositol-requiring protein 1a (IRE1a), activation of transcription leading to increased expression of the proapoptotic factors Noxa factor 6a (ATF6a), and PKR-like ER kinase (PERK) axes (29, 30). and Puma (Fig. 3E). Silencing of HSP47 expression induced IRE1a activation initiates splicing of the mRNA encoding X-box- phosphorylation of PERK and eIF2a and expression of CHOP binding protein 1 (Xbp1) and activation of the ASK1/JNK axis. (Fig. 3F). Furthermore, cleaved ATF6a was clearly detectable in ATF6a becomes an active transcription factor after processing by cancer cells transfected with HSP47 siRNA (Fig. 3F). We examined the protease S1P/S2P. PERK directly phosphorylates the transcrip- the activation status of the UPR in HSP47 KO cancer cells. As tion factor eIF2a and induces expression of the transcription factor shown in Fig. 3G, the disruption of HSP47 induced phosphoryla- ATF4 and certain UPR genes such as Chop and Grp78/BiP.Inthis tion of IRE1a, expression of Xbp1s, phosphorylation of PERK, and study, a splicing of Xbp-1 mRNA was detected in cancer cells cleavage of ATF6a in cancer cells. On the other hand, activation of (SW480 and HCT116 cells) when HSP47 expression was silenced the UPR signaling pathways in HSP47 KO cancer cells was abol- (Fig. 3B). Silencing of HSP47 expression also induced mRNA ished by the reconstitution of HSP47 expression. Moreover, to expression of Xbp1s-targeted genes such as ERdj4, p58IPK,EDEM, clarify whether activation of the UPR after silencing of HSP47 and PDIA6 (32) and an increase in their protein levels in cancer cells expression is unique to cancer cells or not, we investigated activa- (Fig. 3C and D). Silencing of HSP47 expression in cancer cells tion of the UPR in NHDF, in which HSP47 and collagens were

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Figure 3. Silencing of HSP47 stimulates the UPR in cancer cells. A, Transmission electron microscopy in cancer cells at 48 hours after treatment with siControl (SW480, n ¼ 25; HCT116, n ¼ 28) and siHSP47 (SW480, n ¼ 31; HCT116, n ¼ 29). As a control, cancer cells were treated with tunicamycin (Tm). Red lines indicate the dilated ER. N, nucleus; scale bars, 2 mm. Quantiﬁcation of area (pixels/ER) was determined by NIH ImageJ software. B, Expression of unspliced Xbp-1 (Xbp1u) mRNA and spliced Xbp1 (Xbp1s) mRNA in cancer cells treated with or not treated with siControl or siHSP47. C, Expression of mRNAs of UPR-associated genes (ERdj4, p58IPK, EDEM,and PDIA6) in cancer cells at 48 hours after transfection with siControl and siHSP47. D, Expression of ERdj4, EDEM, p58IPK, and PDIA6 proteins in cancer cells at day 2 after transfection with siControl or siHSP47. E, Activation status of IRE1a signaling pathways in cancer cells at 48 hours after transfection with siControl and siHSP47. F, Activation of the PERK pathway and cleavage of ATF6a in cancer cells at 96 hours after transfection with siControl and siHSP47. G, Activation status of IRE1a and PERK and cleavage of ATF6a in HSP47 KO cancer cells (c1 and c2), HSP47 KO cancer cells with reconstituted HSP47 (reHSP47, c1, and c2). H, Activation status of IRE1a and PERK and cleavage of ATF6a in NHDF at 48 hours (for RE1a) and at 96 hours (for PERK and ATF6a) after transfection with siControl and siHSP47 (, P < 0.05; n.s., not signiﬁcant).

highly expressed, after silencing of HSP47 expression. As shown HSP47 showed that HSP47 bound to IRE1a,butnottoPERKor in Fig. 3H, silencing of HSP47 expression hardly induced activation ATF6a, in siControl-transfected cancer cells (Fig. 4A). We further of the UPR (phosphorylation of IRE1a and PERK, and cleavage of performed immunoprecipitation of IRE1a and found that IRE1a ATF6a) in NHDF. These results indicated that silencing of HSP47 interacted with HSP47 and BiP in untreated and siControl- expression stimulates the three branches of the UPR in cancer cells, transfected cancer cells (Fig. 4B). On the other hand, in cancer and suggest that activation of the UPR after silencing of HSP47 cells treated with siHSP47, phosphorylation of IRE1a and dissoci- expression is unique to cancer cells and that HSP47 may control the ation of BiP from IRE1a were detected (Fig. 4B), indicating ER stress sensors IRE1a, PERK, and ATF6a in cancer cells. activation of IRE1a. To further determine whether HSP47 forms acomplexwithIRE1a and BiP in cancer cells, we carried out blue HSP47 interacts with IRE1a, but not with PERK or ATF6a,in native PAGE and found that HSP47 forms a trimer with IRE1a and cancer cells BiP in both whole-cell lysates and anti-HSP47 immunoprecipitants To determine how silencing of HSP47 expression activates the from SW480 cells (Fig. 4C). The results suggested that HSP47 forms UPR in cancer cells, we examined the interactions of HSP47 with a complex with IRE1a and BiP in cancer cells and regulates the IRE1a, PERK, and ATF6a in cancer cells. Immunoprecipitation of activation of IRE1a in cancer cells.

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Figure 4. HSP47 forms a complex with IRE1a and BiP in cancer cells. A, Immunoprecipitation of HSP47 in cancer cells (SW480 and HCT116 cells) not treated or treated with siControl and siHSP47 was carried out, and then immunoblotting against HSP47, IRE1a, PERK, and ATF6a was performed. B, Interaction of IRE1a with HSP47 in cancer cells at 72 hours after transfection with siControl or siHSP47 was determined by immunoprecipitation (IP), and phosphorylated IRE1a (p-IRE1a), IRE1a, HSP47, and BiP were detected by immunoblotting. C, Complex formation of HSP47 with IRE1a and BiP in SW480 cells was determined by native-PAGE and immunoblotting (IB). Arrowheads indicate a complex of HSP47 with IRE1a and BiP.

Silencing of HSP47 first stimulates the IRE1a/Xbp1 axis followed inhibitor of IRE1a,endoribonuclease4m8, silencing of HSP47 by activation of the PERK and ATF6a pathways expression scarcely induced phosphorylation of PERK or cleavage This study demonstrated that silencing of HSP47 expression of ATF6a (Fig. 5F). We further found that silencing of HSP47 stimulates the UPR in cancer cells. HSP47 formed a complex with expression reduced the phosphorylation level of PERK and did IRE1a,butnotwithPERKorATF6a, and its silencing induced not induce cleavage of ATF6a in shXbp1 SW480 cells in which activation of the IRE1a axis in cancer cells. However, how silencing expression of Xbp1 was abolished (Fig. 5D and E). We investigated of HSP47 expression triggers activation of the PERK and ATF6a the in vitro proliferation of shIRE1a cancer cells and shXbp1 axes in cancer cells has remained unclear. Interactions among cancer cells after silencing of HSP47 expression, and found that IRE1a, PERK, and ATF6a signaling pathways in cancer cells are silencing of HSP47 expression hardly suppressed the in vitro pro- largely unknown. To uncover their relationship, we examined liferation of shIRE1a SW480 cells and shXbp1 SW480 cells (Fig. 5G temporal changes in the activation of IRE1a,PERK,andATF6a and H). These results suggest that silencing of HSP47 expression in cancer cells after silencing of HSP47 expression. Intriguingly, first stimulates the IRE1a/Xbp1 axis followed by activation of the phosphorylation of IRE1a was observed from 48 to 120 hours PERK and ATF6a pathways, resulting in inhibition of cancer after transfection of SW480 cells with siHSP47, while expression of growth. Xbp1s was transiently detected at 48 and 72 hours (Fig. 5A). Phosphorylation of PERK, expression of ATF4 and CHOP, and Silencing of HSP47 increases ROS via activation of the cleavage of ATF6a were clearly detectable at 96 and 120 hours after IRE1a/Xbp1 axis transfection of SW480 cells with siHSP47. These results suggest that As mentioned above, activation of the IRE1a/Xbp1 axis by silencing silencing of HSP47 expression first activates the IRE1a axis fol- of HSP47 expression is thought to be required for activation of the lowed by activation of the PERK and ATF6a axes in cancer cells. PERK and ATF6a pathways in cancer cells. However, how the IRE1a/ To determine whether activation of the IRE1a/Xbp1 axis is Xbp1 axis activates both the PERK and ATF6a pathways in cancer cells required for activation of the PERK and ATF6a pathways in cancer is unclear. We showed that silencing of HSP47 expression triggered the cells after silencing of HSP47 expression, we investigated whether expression of Xbp1s-targeted genes and that their protein levels were silencing of HSP47 expression induces activation of PERK and increased in cancer cells (Fig. 3B and C). It has been reported that ATF6a in shIRE1a cancer cells and in shXbp1 cancer cells (Fig. 5B activation of the IRE1a/Xbp1s axis induces the expression of PDI and C). In shIRE1a SW480 cells, expression of Xbp1s was family and the subsequent progression of protein folding (33). Oxi- completely abrogated even though HSP47 expression was silenced dation–reduction reaction in a protein folding process, such as disul- (Fig. 5D). Interestingly, phosphorylation of PERK and cleavage of fide bond formation, generates ROS (34–36). Activation of the IRE1/ ATF6a were hardly detectable in shIRE1a SW480 cells treated with Xbp1s axis also induces generation of ROS in macrophages (37). ROS HSP47 siRNA (Fig. 5D and E). In SW480 cells treated with an triggers activation of the UPR (34, 38, 39). Therefore, we hypothesized

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Figure 5. Silencing of HSP47 induces activation of the IRE1a/Xbp1 axis followed by activation of PERK and ATF6a. A, Temporal changes in activation status of the three branches of the UPR in cancer cells (SW480 cells) at each indicated time point after transfection (TF) with siControl and siHSP47. B, Immunoblot of IRE1a protein in shControl SW480 cells (c1 and c2) and shIRE1a SW480 cells (c1, c2, and c3). C, Expression of Xbp1u and Xbp1s mRNAs in shControl SW480 cells (c1 and c2) and shXbp1 SW480 cells (c1 and c2) treated with tunicamycin (Tm, 250 ng/mL). D, Activation status of the UPR in shControl SW480 cells, shIRE1a SW480 cells and shXbp1 SW480 cells at 96 hours after transfection with siControl and siHSP47. E, Phosphorylation level of PERK in shControl SW480 cells, shIRE1a SW480 cells, and shXbp1 SW480 cells at 96 hours after transfection with siControl and siHSP47 were determined by calculating the ratio of phosphorylated PERK to total PERK. F, Immunoblots of HSP47, Xbp1s, phosphorylated PERK (p-PERK), PERK, and cleaved ATF6a in SW480 cells treated with 4m8 (10 mmol/L) at day 2 (for Xbp1s) and at day 4 after transfection with siControl and siHSP47. Proliferation of shControl SW480 cells (siControl and siHSP47), shIRE1a SW480 cells (siControl and siHSP47; G) and shXbp1 SW480 cells (siControl and siHSP47; H) after transfection with siRNA (, P < 0.05; n.s., not signiﬁcant).

that ROS are generated in cancer cells after silencing of HSP47 of HSP47 expression increases the ROS level via activation of the expression via activation of the IRE1a/Xbp1s axis. To determine IRE1a/Xbp1 pathway and subsequent protein synthesis in cancer the validity of our hypothesis, the level of ROS in cancer cells after cells. silencing of HSP47 expression was examined by using a ROS To further uncover whether silencing of HSP47 expression indicator CM-H2DCFDA. As expected, silencing of HSP47 expres- induces activation of the UPR via increment of ROS in cancer sion in cancer cells (SW480 cells and HCT116 cells) induced the cells, we examined activation of IRE1a, PERK, and ATF6a when increase of ROS level (Fig. 6A). We next examined whether HSP47-silenced cancer cells were treated with the ROS scavenger treatment with an inhibitor of protein synthesis, cycloheximide, NAC or cycloheximide. We conﬁrmed that treatment with NAC ameliorates the increment of ROS level in cancer cells after silenc- markedly inhibited the increase of ROS in HSP47-silenced cancer ing of HSP47 expression. Treatment with cycloheximide inhibited cells (Fig. 6D). In HSP47-silenced SW480 cells treated with NAC or the increase of ROS level in cancer cells treated with siHSP47 cycloheximide, phosphorylation of IRE1a and expression of Xbp1s (Fig. 6B). We also found that silencing of HSP47 expression were detected, while phosphorylation of PERK and cleavage of scarcely triggered increment of the ROS level in shIRE1a or shXbp1 ATF6a were hardly observed (Fig. 6E and F). These results suggest cancer cells (Fig. 6C). Therefore, the results suggest that silencing that under the condition of silencing of HSP47 expression, the

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Figure 6. Activation of the IRE1a/Xbp1 axis by silencing of HSP47 induces increment of ROS in cancer cells. A, Levels of ROS in cancer cells (SW480 and HCT116 cells) treated with siControl or siHSP47 was determined by calculating the ﬂuorescent intensity of CM-H2DCFDA per cell. Representative images and quantiﬁcation were shown in left panel and right panel, respectively. B, Levels of ROS in cancer cells not treated (siControl and siHSP47) or treated with cycloheximide (CHX; 10 mmol/L; siControl and siHSP47). C, Levels of ROS in shControl cancer cells (siControl and siHSP47), shIRE1a cancer cells (siControl and siHSP47), and shXbp1 cancer cells (siControl and siHSP47). D, Levels of ROS in cancer cells not treated or treated with NAC (50 mmol/L) at 48 hours after transfection with siControl and siHSP47. E, Phosphorylation of PERK and cleavage of ATF6a in SW480 cells treated with or without NAC at 48 hours (for p-IRE1a, IRE1a, and Xbp1s) and at 96 hours after transfection with siControl and siHSP47. F, Immunoblots of HSP47, phosphorylated PERK (p-PERK), PERK, and cleaved ATF6a in SW480 cells treated with cycloheximide at day 4 after transfection with siControl and siHSP47 (, P < 0.05). increment of intracellular ROS levelisresponsibleforactivationof accumulation of intracellular lipid and increment in the content of the PERK and ATF6a pathways. 4-HNE–protein adducts in cancer cells (SW480 and HCT116 cells) treated with siHSP47 (Fig. 7A and B). To determine whether the 4-Hydroxy-2-nonenal–protein adducts by hydrogen peroxide increment of ROS level by silencing of HSP47 expression is involved in stimulate the PERK and ATF6a axes in cancer cells the increase in 4-HNE–protein adducts in cancer cells, we examined How ROS triggers activation of the PERK and ATF6a pathways in the content of 4-HNE–protein adducts in HSP47-silenced cancer cells cancer cells after silencing of HSP47 expression remains unclear. treated with NAC. Treatment with NAC markedly alleviated the Activation of the IRE1a/Xbp1 pathway has been reported to enhance increase in the content of 4-HNE–protein adducts in cancer cells lipid biosynthesis (31, 40). Intracellular lipid oxidation by H2O2 treated with siHSP47 (Fig. 7B). generates multiple byproducts such as the unsaturated aldehyde To further determine whether accumulation of 4-HNE–protein 4-hydroxy-trans-2-nonenal (4-HNE), which induce protein-folding adducts in cancer cells by silencing of HSP47 expression results in stress by forming stable adducts with ER-resident chaperones (41, 42). activation of the UPR pathways, we examined activation of IRE1a, Accumulation of 4-HNE–mediated protein adducts triggers activation PERK, and ATF6a when HSP47-silenced cancer cells were treated of the UPR, suggesting that silencing of HSP47 expression in cancer with a sequestering molecule of 4-HNE, hydralazine. We found that cells induces the generation of 4-HNE–protein adducts through treatment with hydralazine ameliorated the accumulation of 4-HNE– increment of the ROS level. Thus, we examined the accumulation of protein adducts (Fig. 7C and D) and inhibited the phosphorylation of intracellular lipid and the content of 4-HNE-protein adducts in cancer PERK and cleavage of ATF6a in SW480 cells treated with siHSP47 cells after silencing of HSP47 expression. As expected, we found despite activation of the IRE1a/Xbp1 axis (Fig. 7E). These results

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Figure 7. Silencing of HSP47 stimulates the PERK and ATF6a axes via increment of ROS-mediated 4-HNE–protein adducts. A, Accumulation of intracellular lipid in cancer cells (SW480 and HCT116 cells) after transfection with siControl and siHSP47. B, Content of 4-HNE–protein adducts in cancer cells not treated or treated with NAC (siControl and siHSP47). C, Dotblot of 4-HNE–protein adducts in SW480 cells not treated or treated with hydralazine after transfection with siControl and siHSP47. D, Content of 4-HNE–protein adducts in SW480 cells not treated or treated with hydralazine (siControl and siHSP47). E, Phosphorylation of PERK and cleavage of ATF6a in SW480 cells not treated or treated with hydralazine at 48 hours (for p-IRE1a, IRE1a, and Xbp1s) and at 96 hours after transfection with siControl and siHSP47 (, P < 0.05; n.s., not signiﬁcant).

suggest that under the condition of silencing of HSP47 expression, the molecular chaperone for collagen but prevents activation of the UPR increment of intracellular ROS level in cancer cells is responsible for via inhibition of IRE1a activity and maintains the growth of cancer accumulation of 4-HNE–protein adducts followed by activation of the cells. PERK and ATF6a pathways. Although the expression level of HSP47 in tumors has been reported to correlate with the malignant grade of glioma and poor prognosis of breast cancers and colorectal cancers (23–28), the role of HSP47 in Discussion cancer cells had remained unclear. This study demonstrated that In this study, we demonstrated that HSP47 is expressed in almost all HSP47 acts as a regulator of IRE1a activity, which is controlled by cancer cell lines in which collagen is hardly expressed and that complex formation of HSP47 with IRE1a and BiP. IRE1a, one of the silencing of HSP47 expression induces suppression of cancer cell three branches of the UPR, is localized on the membrane of the ER and growth accompanied by an abnormal dilation of the ER and activation its luminal region binds to BiP. IRE1a contributes to the maintenance of the UPR. HSP47 bound to IRE1a and formed a complex with IRE1a of cell survival via the production of Xbp1s transcription factor by and BiP in cancer cells under basal conditions. Silencing of HSP47 splicing Xbp1 mRNA to alleviate cell damage caused by ER stress, while expression enhanced the expression of UPR-associated genes, protein it triggers activation of the proapoptotic signaling pathway under the synthesis, and lipid accumulation via activation of the IRE1a/Xbp1 condition of persistent ER stress (29, 30, 32). Under a condition of ER axis, causing an increase of the intracellular ROS level in cancer cells. stress such as an excessive accumulation of misfolded and unfolded The increment of intracellular ROS level by silencing of HSP47 proteins, BiP dissociates from IRE1a, leading to activation of the expression induced accumulation of 4-HNE–protein adducts, result- IRE1a endoribonuclease and IRE1a kinase. Intriguingly, this study ing in phosphorylation of PERK and cleavage of ATF6a in cancer cells. showed that silencing of HSP47 expression triggered the dissociation The results suggest that HSP47 does not function as a speciﬁc of BiP from IRE1a, resulting in splicing of Xbp1 mRNA via stimulation

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of the IRE1a endoribonuclease and activation of the ASK1/JNK PERK, and ATF6a) of the UPR had not been sufficiently investigated signaling pathway via activation of the kinase domain of IRE1a. even though the UPR has been studied for many years (49). In this HSP47 has an RDEL motif at the C-terminus of its molecule and is regard, this study demonstrated for the first time that under the also localized in the lumen of the ER (1, 2), suggesting that HSP47 condition of silencing of HSP47 expression in cancer cells, activation binds to the luminal region of IRE1a in the ER and regulates IRE1a of the IRE1a/Xbp1 axis is a priming event for stimulation of the PERK activity by a complex formation with IRE1a and BiP in cancer cells. In and ATF6a pathways via production of ROS and accumulation of fact, Sepulveda and colleagues recently demonstrated that HSP47 4-HNE–protein adducts. physically interacts with the luminal domain of IRE1a in mouse Cancer cells usually arise and progress in stressful microenviron- embryonic fibroblasts and plays a role as a regulator of the UPR (43). ments such as hypoxia, glucose shortage, and nutrient fluctuations, This study showed that silencing of HSP47 expression in colorectal and many studies have demonstrated that activation of the UPR plays a cancer cells induced an abnormal dilation of the ER accompanied by critical role in the development and progression of cancer cells (29, 30). activation of the IRE1a/Xbp1 axis, enhanced expression of ER stress– Therefore, UPR pathway–associated molecules such as PERK, IRE1a, associated proteins, and subsequent increment of intracellular ROS HSP90, GRP94, and BiP could be candidate compounds as therapeutic level. Activation of the IRE1a/Xbp1s axis induces transcription of targets for tumors. It is also a fact that prolonged or sustained UPR UPR-associated genes such as PDI family, while it triggers progressive activation fails to restore ER homeostasis and induces UPR-mediated lipid biosynthesis, resulting in an increased surface area and volume of cell death of cancer cells (29, 30, 50). Although the mechanisms of the ER (33, 40, 41). Oxidative protein folding such as disulfide bond prevention of cell death or induction of apoptosis have investigated in formation during protein synthesis induces the production of H2O2 in detail at the molecular level, the nature and function of the real switch the ER (34–36). Activation of the IRE1a/JNK pathway stimulates remain elusive. This study demonstrated that HSP47 acts as an expression of the ER-localized nicotinamide adenine dinucleotide inhibitor of IRE1a activity as well as its role as a collagen-specific phosphate oxidase 4 (Nox4), which produces H2O2 in the ER (44, 45). chaperone and maintains cancer cell growth by preventing UPR This study showed that treatment with an inhibitor of protein syn- activation that is induced by tumor environments such as a hypoxia thesis inhibited the increment of intracellular ROS level in cancer cells and a low level of glucose. Therefore, targeted disruption of HSP47 in despite the silencing of HSP47 expression. We further demonstrated cancer cells might become a powerful approach for therapeutic that silencing of HSP47 expression caused very little increase in the modalities against tumors. ROS level in IRE1a-silenced and Xbp1-silenced cancer cells. These results suggest that silencing of HSP47 expression induces an abnor- Disclosure of Potential Conflicts of Interest mal dilation of the ER and increment of ROS level through IRE1a/ No potential conflicts of interest were disclosed. Xbp1-dependent lipid biosynthesis and the process of oxidative protein folding, such as disulfide bond formation, during protein synthesis Authors’ Contributions in cancer cells. Conception and design: A. Yoneda, Y. Tamura This study confirmed that silencing of HSP47 expression caused Development of methodology: A. Yoneda, K. Sakai-Sawada, Y. Tamura Acquisition of data (provided animals, acquired and managed patients, provided activation of the IRE1a/Xbp1 axis and enhanced intracellular lipid facilities, etc.): A. Yoneda, K. Sakai-Sawada, K. Minomi accumulation and increment of the H2O2 level in colorectal cancer Analysis and interpretation of data (e.g., statistical analysis, biostatistics, cells. Intracellular lipid oxidation by ROS such as H2O2 generates computational analysis): A. Yoneda, K. Sakai-Sawada multiple byproducts such as the unsaturated 4-HNE, which induce Writing, review, and/or revision of the manuscript: A. Yoneda, K. Minomi, protein folding stress by forming stable adducts with ER-resident Y. Tamura chaperones (41, 42). Treatment of endothelial cells with 4-HNE Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): A. Yoneda activates the three branches of the UPR (41). This study showed that Study supervision: A. Yoneda silencing of HSP47 expression in cancer cells increased the content of 4-HNE–protein adducts and activation of the UPR (phosphorylation Acknowledgments of PERK and cleavage of ATF6a), which was markedly alleviated by The authors thank Dr. Takayuki Sakurai (Sinshu University, Matumoto, Japan) treatment with the ROS scavenger and an inhibitor of 4-HNE. An and Dr. Norio Takei (Hokkaido University, Sapporo, Japan) for providing pCG-Sap I increase in 4-HNE–protein adducts in malignant and nonmalignant vector. This work was supported by Nitto Denko Corporation and a Grant-in-Aid fi cells inhibits cell proliferation by a p53-independent increase of p21 (KAKENHI 18K07287) for Scienti c Research from the Ministry of Education, expression, decrease of cyclin D1 synthesis, suppression of the MAPK Culture, Sports, Science and Technology, Japan. pathway, and downregulation of c-myc expression (46–48). These The costs of publication of this article were defrayed in part by the payment of page results suggest that silencing of HSP47 expression stimulates the charges. This article must therefore be hereby marked advertisement in accordance a IRE1 /Xbp1 axis followed by increment of the ROS level and content with 18 U.S.C. Section 1734 solely to indicate this fact. of 4-HNE–protein adducts that trigger activation of the PERK and ATF6a pathways, resulting in the growth arrest of colorectal cancer Received July 2, 2019; revised October 6, 2019; accepted February 21, 2020; cells. Intriguingly, communication among the three branches (IRE1a, published first February 26, 2020.

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OF12 Mol Cancer Res; 2020 MOLECULAR CANCER RESEARCH

Heat Shock Protein 47 Maintains Cancer Cell Growth by Inhibiting the Unfolded Protein Response Transducer IRE1 α

Akihiro Yoneda, Kaori Sakai-Sawada, Kenjiro Minomi, et al.

Mol Cancer Res Published OnlineFirst February 26, 2020.

Updated version Access the most recent version of this article at: doi:10.1158/1541-7786.MCR-19-0673

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