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Heat shock protein 47 confers chemoresistance on pancreatic cancer cells by interacting with calreticulin and IRE1α

Abstract Pancreatic ductal adenocarcinoma (PDAC) is one of the most chemoresistant cancers. An understanding of the molecular mechanism by which PDAC cells have a high chemoresistant potential is important for improvement of the poor prognosis of patients with PDAC. Here we show for the first time that disruption of heat shock protein 47 (HSP47) enhances the efficacy of the therapeutic agent gemcitabine for PDAC cells and that the efficacy is suppressed by reconstituting HSP47 expression. HSP47 interacts with calreticulin (CALR) and the unfolded protein response transducer IRE1α in PDAC cells. Ablation of HSP47 promotes both the interaction of CALR with sarcoplasmic/endoplasmic reticulum Ca2+‐ATPase 2 and interaction of IRE1α with inositol 1,4,5‐triphosphate receptor, which generates a condition in which an increase in intracellular Ca2+ level is prone to be induced by oxidative stimuli. Disruption of HSP47 enhances NADPH oxidase‐induced generation of intracellular reactive oxygen species (ROS) and subsequent increase in intracellular Ca2+ level in PDAC cells after treatment with gemcitabine, resulting in the death of PDAC cells by activation of the Ca2+/caspases axis. Ablation of HSP47 promotes gemcitabine‐induced suppression of tumor growth in PDAC cell‐bearing mice. Overall, these results indicated that HSP47 confers chemoresistance on PDAC cells and suggested that disruption of HSP47 may improve the efficacy of chemotherapy for patients with PDAC.


| INTRODUC TI ON
Pancreatic cancer is one of the leading causes of cancer-related death worldwide. The median survival period of patients with pancreatic ductal adenocarcinoma (PDAC) is still only 5 to 8 mo and the 5-y survival rate is only c. 5%. 1,2 Currently available therapies are not sufficient for treatment of PDAC, and the development of novel therapeutic modalities for PDAC is urgently needed. 3,4 PDAC is also one of the most chemoresistant cancers, and the efficacy of therapeutic agents is hindered by multiple intrinsic and acquired mechanisms of resistance before and during chemotherapy. [5][6][7] Gemcitabine has been widely used as a first-line drug for PDAC. [8][9][10][11] Gemcitabine is a deoxycytidine nucleotide analog and induces inhibition of DNA synthesis and cell cycle arrest at the G1/S phase by its incorporation tients with PDAC. Here we show for the first time that disruption of heat shock protein 47 (HSP47) enhances the efficacy of the therapeutic agent gemcitabine for PDAC cells and that the efficacy is suppressed by reconstituting HSP47 expression. HSP47 interacts with calreticulin (CALR) and the unfolded protein response transducer IRE1α in PDAC cells. Ablation of HSP47 promotes both the interaction of CALR with sarcoplasmic/endoplasmic reticulum Ca 2+ -ATPase 2 and interaction of IRE1α with inositol 1,4,5-triphosphate receptor, which generates a condition in which an increase in intracellular Ca 2+ level is prone to be induced by oxidative stimuli. Disruption of HSP47 enhances NADPH oxidase-induced generation of intracellular reactive oxygen species (ROS) and subsequent increase in intracellular Ca 2+ level in PDAC cells after treatment with gemcitabine, resulting in the death of PDAC cells by activation of the Ca 2+ /caspases axis. Ablation of HSP47 promotes gemcitabine-induced suppression of tumor growth in PDAC cell-bearing mice. Overall, these results indicated that HSP47 confers chemoresistance on PDAC cells and suggested that disruption of HSP47 may improve the efficacy of chemotherapy for patients with PDAC.

K E Y W O R D S
calreticulin, chemoresistance, HSP47, IRE1α, PDAC into DNA during DNA replication. Gemcitabine has also been reported to trigger oxidative stress by the generation of reactive oxygen species (ROS) by the activity of NAD(P)H oxidase (NOX), which is a major source of the generation of ROS, resulting in induction of apoptosis. 12,13 Although gemcitabine remains the therapeutic agent of choice that is used either alone or in combination with nabpaclitaxel in patients with PDAC, treatment with gemcitabine has not resulted in a marked improvement in survival of patients with PDAC due to the presence of innate or acquired chemoresistant mechanisms in PDAC. [8][9][10][11] It has been widely accepted that reduction of anti-cancer drug uptake, alteration of the drug target, induction of drug-detoxifying mechanisms, repair of drug-induced damage and insensitivity to drug-induced cell death are responsible for the chemoresistance of PDAC cells. 7,14,15 However, current therapies targeting the chemoresistance-associated molecular pathways in PDAC cells have not provided satisfactory results, due in part to rapid upregulation of alternative compensatory pathways. Therefore, an understanding of the precise molecular mechanism by which PDAC cells have tolerance against chemotherapies is an urgent issue for improvement of the poor survival of patients with PDAC.
Heat shock protein 47 (HSP47), a molecular chaperone of collagen, is localized in the endoplasmic reticulum (ER) and it is widely accepted to play a crucial role in correct folding of collagen. 16,17 Disruption of HSP47 triggers the accumulation of unfolded procollagen and subsequent activation of ER stress, resulting in delayed cell growth and apoptosis. [18][19][20] HSP47 is ubiquitously expressed in several types of cancer cells in which the expression level of collagen is extremely low, and increased expression of HSP47 is associated with a high malignant grade of glioma and low overall survival of patients with breast cancer and colon cancer. [21][22][23][24][25][26][27] It has also been reported that the overall survival rate of patients with PDAC in which the expression level of HSP47 is low is higher than that of patients with PDAC in which the expression level of HSP47 is high. 21,28 Furthermore, induced expression of HSP47 promotes the ability of cancer cells for tumor growth and metastasis to other organs. 25,27,29,30 Recently, we showed that HSP47 interacts with the unfolded protein response (UPR) transducer IRE1α and maintains cancer cell growth by inhibiting IRE1α. 29,31 Furthermore, interaction of HSP47 with IRE1α stimulates the activity of myosin IIA, augmenting the metastatic potential of breast cancer cells. 30 Although the involvement of HSP47 in the malignancy and chemoresistance of cancer cells has been suggested, the precise molecular mechanism by which HSP47 contributes to the tolerance of cancer cells against treatment with therapeutic agents remains unclear. Here we show that disruption of HSP47 improves the efficacy of the therapeutic agent gemcitabine against PDAC cells, while overexpression of HSP47 confers chemoresistance to PDAC cells by regulation of calcium homeostasis by inhibiting interaction of HSP47 with the ERresident Ca 2+ -binding protein calreticulin (CALR) and with IRE1α, both of which are associated with chemoresistance of PDAC cells.
The results of the present study also indicated the possibility of HSP47 being a candidate for a therapeutic target against PDAC.

| Western blot analysis
The cells were then washed with PBS and lysed with a lysis buffer (50 mmol/L Tris, 250 mmol/L NaCl, 25 mmol/L EDTA and 1% NP-40) supplemented with a protease inhibitor (Roche). Equal amounts of proteins were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes (Millipore). After blocking with 5% skim milk in PBS containing 0.05% Tween-20, the membranes were probed with the following primary antibodies:

| Establishment of gene knockout (KO) cells
The gRNA sequence (5′-TGCGGAGAAGTTGAGCCCCA-3′) de- The treated cells were cultured in RPMI1640 medium supplemented with 10% FBS and G418 (1000 μg/mL, Thermo Fisher Scientific) to select cell clones stably silencing IRE1α or CALR expression. Singlecell clones were evaluated by DNA sequencing analysis to detect insertions or deletions in the target alleles and to confirm expression of IRE1α or CALR at the protein level by western blotting.  containing SDS-PAGE sample buffer. Eluents were immediately transferred to a fresh tube containing 5 μL of 1 M Tris-HCl buffer (pH 7.5) in advance to neutralize sample pH.

| Proteomic analysis
To identify co-immunoprecipitated proteins, immunoprecipitated proteins after concentration by centrifugal filtration (3000 molecular weight cut-off, PALL Life Science) were subjected to tryptic digestion. Peptides were combined and dried with a centrifugal evaporator (Thermo Fisher Scientific) and then finally re-dissolved

| Measurement of intracellular Ca 2+ level
At 72 h after treatment with gemcitabine (10 μmol/L for PANC1

| Detection of NAD(P)H oxidase activity
The treated cells were lysed in an NADH oxidase lysis buffer (BioVision). All samples were stored at −80°C until use. NAD(P)H oxidase (NOX) activity was determined using the NADH Oxidase Activity Assay kit (BioVision).

| Immunohistological analysis
Mice were perfused with 10 N formalin and fixed pancreatic tissues were embedded in paraffin. Paraffin-embedded pancreatic tissue sections (7 μm in thickness) were deparaffinized with xylene and ethanol. Pancreatic tissue sections were stained by hematoxylin and eosin. For immunohistological staining, after permeabilization with HistoVT-ONE (Nacalai Tesque) or 10 mmol/L citrate buffer (pH 6.0), tissue sections were blocked with 5% goat serum in PBS. The sections were incubated with an antibody for Ki67 (ab15580, Abcam) and secondary antibodies conjugated with horseradish peroxidase (ab6721, Abcam).

| Statistical analysis
All data are shown as means ± SEM. All experiments in this study were performed in duplicate or triplicate. Differences between groups were tested for statistical significance using Student t test or ANOVA. Statistical significance was determined at P < .05.

| HSP47 interacts with calreticulin and IRE1α in PDAC cells
To uncover the mechanism by which HSP47 is involved in the chemoresistance of PDAC cells, we investigated HSP47-interacting proteins in PDAC cells. We purified HSP47-interacting proteins from the HSP47-Myc-overexpressing PDAC cell line PANC1 using anti-Myc antibody-conjugated magnetic microbeads and we performed LC-MS/MS analysis of purified HSP47-interacting proteins. As shown in Table 1, we identified 6 molecules (IRE1α, β-subunit prolyl 4-hydroxylase (P4HB), endoplasmin, CALR, translocon-associated protein subunit α (SSR1) and mesencephalic astrocyte-derived neurotrophic factor (MANF)) as HSP47-interacting proteins localized in the ER. Among the identified HSP47-interacting proteins, we focused on 2 proteins, the Ca 2+ -binding protein CALR and the UPR transducer IRE1α, as both CALR and IRE1α have been reported to be associated with cancer progression. 32 Figure 3C).

| HSP47 interferes with both the interaction of CALR with SERCA2 and the interaction of IRE1α with IP 3 R in PDAC cells
Over 50% of Ca 2+ stored in the ER is bound to CALR and CALR serves as a regulator of Ca 2+ homeostasis in the ER. 35  Immunoprecipitation showed that IRE1α interacted with IP 3 R in HSP47 KO PANC1 cells but not in Mock:PANC1 cells regardless of treatment or no treatment with gemcitabine ( Figure 3H). The interaction of IRE1α with IP 3 R in HSP47 KO PANC1 cells was completely inhibited by reconstitution of HSP47-GFP expression ( Figure 3I).
These results indicated that HSP47 inhibits both the interaction of CALR with SERCA2 and the interaction of IRE1α with IP 3 R in PDAC cells.

| Disruption of HSP47 enhances the increase in intracellular Ca 2+ level in PDAC cells after treatment with gemcitabine
We examined whether disruption of HSP47 affected the level It has been reported that an increase in intracellular Ca 2+ concentration triggers activation of the caspase 12/caspase 9/caspase 3 axis, causing apoptosis of cells. 40 Therefore, we investigated the activation of the calcium/caspases axis in HSP47 KO PDAC cells after treatment with gemcitabine. As expected, cleaved caspase 12, cleaved caspase 9 and cleaved caspase 3 were detected in HSP47 KO PANC1 cells treated with gemcitabine ( Figure 4E). The activation of the calcium/caspases axis in HSP47 KO PANC1 cells treated with gemcitabine was inhibited by treatment with BAPTA-AM. These results indicated that gemcitabine promotes the increase in intracellular Ca 2+ level, resulting in activation of the calcium/caspase axis and subsequent death of HSP47-deleted PDAC cells.

| Ablation of HSP47 promotes NOX-induced generation of intracellular ROS in PDAC cells after treatment with gemcitabine
Treatment with gemcitabine induces the generation of ROS by the activity of NOX. 12,13 Recently, we showed that silencing of HSP47 promoted the generation of intracellular ROS in cancer cells. 29 Therefore, we investigated the level of intracellular ROS in HSP47 KO PDAC cells after treatment with gemcitabine. The level of intracellular ROS in HSP47 KO PDAC cells was higher than that in its counterparts, and the level of ROS was increased by treatment with gemcitabine ( Figure 5A). It has been reported that the expres-

| Gemcitabine-induced generation of ROS triggers an excess increase of intracellular Ca 2+ level in HSP47-depleted PDAC cells
It has been reported that ROS lead to an increase in intracellular Ca 2+ concentrations by stimulation of IP 3 R and inhibition of SERCA2 activity. [42][43][44][45] We showed that disruption of HSP47 induced both the interaction of CALR with SERCA2 and the interaction of IRE1α with IP 3 R in PDAC cells (Figure 3), and these interactions have been reported to induce inactivation of SERCA2 and stimulation of IP 3 R, respectively. 36,39 Therefore, we hypothesized that depletion of HSP47 generated a condition that is prone to increase intracellular Ca 2+ concentration in response to oxidative stimuli such as ROS.
Therefore, we examined the effects of NAC and DPI on the increase in intracellular Ca 2+ level in HSP47-depleted PDAC cells treated with gemcitabine. Treatment with gemcitabine induced an increase in intracellular Ca 2+ level in HSP47 KO PANC1 cells, which was clearly inhibited by treatments with NAC and DPI ( Figure 6A). We also in-

| Disruption of HSP47 enhances the efficacy of chemotherapy with gemcitabine against pancreatic tumors
To

| D ISCUSS I ON
We demonstrated that silencing and ablation of HSP47 improved the efficacy of the therapeutic agent gemcitabine against PDAC cells. In addition to the function of HSP47 as a collagen-specific chaperone, 16 homeostasis. 34,39 In the present study, we showed that depletion of Akt. 40,57,58 It has been reported that treatment with a Ca 2+ chelator and inhibition of CamK and Akt improved the efficacy of therapeutic agents for tumors. 50 Conversely, an excess increase in intracellular Ca 2+ concentration triggered cell death by activation of the calcium/ caspases axis. 40 The present study showed that the increase in in-

D I SCLOS U R E
The authors declare no conflict of interest.