Mutations and epigenetic inactivation of the tumor suppressor gene von Hippel-Lindau (VHL) are major causes of clear-cell renal cell carcinoma (ccRCC) that may originate from chronic inflammation. However, the role of VHL loss of function in the development of ccRCC via inflammation remains poorly understood. VHL-mutant cells exhibit metabolic abnormalities that can cause chronic endoplasmic reticulum (ER) stress and unfolded protein response. We hypothesize that unresolved ER stress induces the inflammatory responses observed in ccRCC. ER stress markers including BiP and XBP1s were significantly increased in cultured and primary VHL loss-of-function kidney cells. In epithelial cells, the kinase activity of IRE1α was required for the induction of NF-κB and JNK and for the recruitment of macrophages. IRE1α kinase activity was also important for the development of fibrotic phenotype in conditional Vhlh knockout mice. Our results offer insights into the therapeutic potential against ccRCC development by relieving metabolic stress. Such cancer prevention strategy may be critical for high-risk cohorts such as the familial VHL disease patients. Cancer Res; 77(13); 3406–16. ©2017 AACR.

Genomic mutations and epigenetic inactivation of the VHL tumor suppressor gene underlie the development of a majority of clear-cell renal cell carcinoma (ccRCC; refs. 1, 2). VHL encodes the substrate-recognition component of an E3 ubiquitin ligase. Its most prominent target is the alpha subunit of the hypoxia-inducible factor (HIF-α). HIF is a master transcription factor stabilized in low oxygen tension, which induces a metabolic switch from oxidative phosphorylation to glycolysis (3, 4). HIF also promotes angiogenesis through expression of cytokines such as VEGF (5). In VHL-mutant ccRCC cells, HIF function is activated even in normal oxygen conditions (6, 7), exhibiting heightened pseudohypoxic responses. As such, ccRCC is an excellent model for studying tumor metabolism and the interaction between tumor cells and the microenvironment (8).

The association of tumor with inflammation has been proposed in mid-19th century by Rudolf Virchow, and the concept was reintroduced by H.F. Dvorak more than a hundred years later (9). Since then, accumulated evidence has indeed implicated inflammation in the development of various cancers (10, 11). Recently, the development of ccRCC has also been linked to tissue inflammation (12, 13), and kidney disease has been identified as an important risk factor for renal cancer (14, 15). Inflammation can promote tumor initiation and progression in various ways. It can stimulate cell proliferation (10, 16) and importantly, induce genome instability (11, 17, 18). Consistent with these observations, our recent study shows that inactivation of the mouse VHL tumor suppressor gene (designated Vhlh) in a subpopulation of kidney distal tubule cells, using the HOXB7-Cre-GFP driver, resulted in Hif-1α–dependent hyperplastic clear-cell lesions and severe inflammation and fibrosis (19). This model thus provides a good system for studying the relationship between inflammation and tumorigenesis.

In this study, we ask the critical question of how VHL inactivation in a subpopulation of renal epithelial cells can elicit systemic responses such as inflammation. It has been shown that in VHL-mutant cells, the load of translation is significantly increased (20), probably as a result of PI3K signal transduction and mTOR pathway activation (21). HIF activation in VHL-defective cells also generates excessive reactive oxygen species (ROS; ref. 22). This is partly because HIF indirectly inhibits conversion of pyruvate to acetyl-CoA that is the carbon source feeding into the tricarboxylic acid (TCA) cycle (4, 23). Therefore, the TCA cycle is slowed while the oxygen supply is normal in these VHL-mutant cells. Slowed TCA cycle causes electron relay to stall; the electrons are then prone to be captured by the oxygen, resulting in the generation of reactive oxygen species (ROS; refs. 24, 25).

We hypothesize that such metabolic abnormalities can disrupt normal proteostasis in at least two ways: protein overload in the endoplasmic reticulum (ER) compounded by protein disulfide bond malformation resulting from excessive ROS. These conditions have been shown to cause ER stress (26, 27). ER stress activates the unfolded protein response (UPR) that enables the cell to either reduce the load of unfolded proteins or, if unresolved, initiate apoptosis (28). The cellular response to ER stress is complex, but mainly involves three pathways that lead to production of three active transcription factors: ATF6, ATF4 via PERK activation, and XBP1 via IRE1 activation.

Among these pathways, IRE1 is the most ancient and has been linked to inflammatory response (29, 30). IRE1 is an ER transmembrane protein (31). Two isoforms of IRE1 exist: IRE1α is ubiquitously expressed while the β isoform is mainly expressed in the gastrointestinal epithelia (32). It has recently been shown that one of the sensors that detects mitochondrial ROS and translates the signal to induce ER stress is IRE1α, which is frequently found at the junction between mitochondria and the ER lumen (27). IRE1α contains the dual activity of kinase and endonuclease (RNase; refs. 33, 34). Canonically, ER stress activates IRE1α via autophosphorylation. Activated IRE1α then induces specific alternative splicing (via its RNase activity) that generates a functional “spliced” XBP1 transcription factor (XBP1s; for clarity, we will use XBP1 to denote activated XBP1 in this article). The IRE1α kinase activity is required for the oligomerization that precedes the activation of the RNase activity (34–36). In a separate function, autophosphorylation of IRE1α also results in binding to the adaptor protein TNFα receptor-associated factor 2 (TRAF2) and activation of c-Jun N-terminal kinase (JNK) via ASK1 (37). The IRE1α−TRAF2 complex can also recruit and activate IκB kinase (IKK), which phosphorylates IκB, leading to the degradation of IκB and nuclear translocation of NFκB (38). NFκB and the JNK-activated transcription factor AP1 are two major inducers of the expression of inflammatory genes (39, 40). Therefore, it is possible that the metabolic abnormalities in VHL loss-of-function cells trigger cellular inflammatory pathways via ER stress and IRE1α signaling. In this study, we set out to test this hypothesis.

Animal protocol and mouse strains

All procedures were conducted in accordance with the US Public Health Service Policy on Humane Care and Use of Laboratory Animals. Mice were maintained in National Central University, Taiwan, animal housing facility according to protocols approved by the Institutional Animal Care and Use Committee. Mouse strains used were in C57BL/6 background as described previously (19). The Vhlh conditional knockouts were generated by intercross of Hoxb7-Cre-GFP/+; Vhlhfl/+. Littermates with the genotype Hoxb7-Cre-GFP/+ served as wild-type control. Hoxb7-Cre-GFP is a Hoxb7 promoter driven Cre line with a GFP reporter. All triplicate experiments were performed using three different animals.

Cell culture

HK-2, a human renal proximal tubular epithelial cell line, and HEK293, a human cell line of kidney origin, were purchased from Bioresource Collection and Research Center, Taiwan, in 2014 with identity certification. Each batch of cells were cultured for <10 passages in DMEM/Ham F12 (Gibco-Thermo Fisher) supplemented with 10% FBS, and penicillin–streptomycin (50 U/mL; Sigma-Aldrich) at 37°C in 5% CO2/95% air. Culture medium was replaced on alternate days. Once the cells reached 60%–70% confluency, they were collected or passaged (via trypsinization) for the subsequent experiments. When appropriate, cells were treated with solvent [DMSO:PBS = 1:3, PBS, or DMSO], tunicamycin (at 1 μg/mL in DMSO/PBS; Sigma-Aldrich), APY29 (at 0.2 μmol/L in DMSO; Tocris Bioscience), or 4-phenylbutyric acid (4-PBA at 1 mmol/L in PBS; Sigma-Aldrich) for 24 hours.

The murine monocyte/macrophage cell line RAW264.7 was kindly provided by Dr. N.H. Ma (Institute of Systems Biology and Bioinformatics, National Central University, Jhongli, Taiwan) and was originally obtained from ATCC in 2012. The cells were cultured in DMEM (Gibco-Thermo Fisher) supplemented with 10% FBS, 4 mmol/L l-glutamine, 4,500 mg/L glucose, 1 mmol/L sodium pyruvate, 1,500 mg/L sodium bicarbonate, and penicillin–streptomycin (50 U/mL; Sigma-Aldrich) at 37°C in 5% CO2/95% air, for no more than 5 passages. Culture medium was replaced on alternate days. Once the cells reached 50%–60% confluence, they were collected or passaged (via trypsinization) for the subsequent experiments.

Potential contamination by mycoplasma was monitored by PCR for conserved 16S rRNA coding sequence in the newly plated cells, and follow-up routine examination was done by DAPI staining.

Antibodies

Antibodies for Western blot analyses and the final working dilutions were as follows: mouse mAbs against VHL (1:1,000; BD Biosciences), BiP (1:1,000; BioLegend), p-JNK (1:1,000; Santa Cruz Biotechnology), FLAG (1:1,000; Cell Signaling Technology), and β-actin (1:500, Santa Cruz Biotechnology); rabbit polyclonal antibodies against IRE1α (1:1,000; Cell Signaling Technology), p-IRE1α (1:1,000; Abcam), PERK (1:500; Santa Cruz Biotechnology), p-PERK (1:500; Santa Cruz Biotechnology), eIF2α (1:1,000; Cell Signaling Technology), p-eIF2α (1:1,000; Cell Signaling Technology), ATF4 (1:500; Santa Cruz Biotechnology), p65 (1:1,000; GeneTex), fibrillarin (1:1,000, Santa Cruz Biotechnology), TNF-α (1:1,000, GeneTex), and IL1β (1:1,000, GeneTex); and goat polyclonal antibody against albumin (1:250; Santa Cruz Biotechnology). All horseradish peroxidase–conjugated secondary antibodies were purchased from Jackson ImmunoResearch Inc. (Jackson ImmunoResearch).

Protein synthesis rate analysis

The Protein Synthesis Assay Kit (Cayman Chemical) was used according to manufacturer's protocol. Briefly, cells were cultured with or without the protein synthesis inhibitor cycloheximide (at a final concentration of 50 μg/mL) at 37°C for 30 minutes. After incubation, cells were trypsinized and collected by centrifugation at 400 × g for 5 minutes. Cells were resuspended in 0.5 mL of 20 mmol/L O-propargyl-puromycin (OPP) and incubated at 37°C for 2 hours. OPP is an alkyne analogue of puromycin that is incorporated into the nascent peptide chain and causes premature chain termination (41). After incubation, cells were collected by centrifugation as above. Cells were resuspended in 0.5 mL cell-based assay fixative solution and incubated at room temperature for 5 minutes. Cells were then collected, resuspended in 1 mL cell-based assay wash buffer, and incubated at room temperature for 5 minutes. Cells were collected by centrifugation as above and resuspended in 1 mL FAM-Azide [conjugated with the fluorescein isothiocyanate (FITC) dye] staining solution and incubated in the dark at room temperature for 30 minutes. After incubation, cells were collected as above and resuspended in 1 mL cell-based assay wash buffer and incubated at room temperature for 5 minutes. Cells were collected and resuspended in 1 mL assay buffer and FITC-stained cells quantified by NovoCyte Flow Cytometer (ACEA Biosciences).

Measurement of intracellular ROS generation

Cells were washed with PBS and incubated with 10 μmol/L dihydroethidium (DHE; Santa Cruz Biotechnology) in Hank's Balanced Salt Solution (HBSS) at 37°C for 30 minutes in the dark. In the presence of ROS, DHE is oxidized and becomes fluorescent (42). After incubation, cells were trypsinized and washed with ice-cold PBS three times. The cellular ROS level was quantified by NovoCyte Flow Cytometer (ACEA Biosciences).

Collagen detection

Histologic staining of paraffin sections was conducted using previously described methods (19). Collagen deposition was visualized using Picro-Sirius Red Stain Kit (Abcam) following the manufacturer's instruction. The slides were cleared and mounted using Cytoseal Mounting Media-Cytoseal 60 (Thermo Fisher). The sections were evaluated under light microscope Olympus IX81 and the images were analyzed with ImageJ software (NIH, Bethesda, MD).

RNA interference

For VHL knockdown, cells were transfected with plasmids expressing short hairpin RNAs (shRNA) as described (43, 44). The target sequences shVHL1, gagaactgggacgaggccg, and shVHL3, gagcctagtcaagcctgag, were used in this study.

For IRE1α knockdown, two independent siRNA was used: (i) siIRE1α1 is a mixture of four duplex RNAs (from Santa Cruz Biotechnology), consisting of the following target sequences: caaccucucuucuguaucu, ggaaggugaugcacaucaa, cuggaggagacgaaugaua, cuguacucuuggaguaaca; and (ii) siIRE1α2 targets the sequence cugcuuaaugucagucuac, as described previously (45).

Culture of primary renal tubular epithelial cells

Mouse primary renal tubule cells were isolated using the previously described methods (46, 47).

Transfection

HK-2 cells were transfected with scrambled siRNA or siRNA specific for IRE1α, or vectors expressing scrambled shRNA, shRNA specific for VHL (shVHL), FLAG-IRE1αwt, or FLAG-IRE1αK599A (provided by Dr. K. Mori in Kyoto University, Japan; ref. 27) using the BTX Gemini X2 Electroporation System (BTX Harvard Apparatus). Briefly, 5 × 105 cells were electroporated with 10 pmol of siRNA or 4 μg of plasmid in 1 mL serum-free media with one 100 V pulse for 10 ms and plated in 6-well plates.

Nuclear extraction

The cells were collected, resuspended in hypotonic buffer (10 mmol/L HEPES, pH 7.9; 10 mmol/L KCl; 1.5 mmol/L MgCl2; 0.2 mmol/L PMSF; 20 μg/mL aprotinin; 0.5 mmol/L DTT; and 0.5% NP-40), and incubated on ice for 15 minutes. After centrifugation at 6,000 × g for 15 minutes at 4°C, the supernatant was collected (the cytosolic fraction). The pellet was then washed once with basal buffer (hypotonic buffer without 0.5% NP-40). After centrifugation again at 6,000 × g for 15 minutes at 4°C, the pellet was collected and resuspended in hypertonic buffer (20 mmol/L HEPES, pH 7.9; 400 mmol/L KCl; 1.5 mmol/L MgCl2; 0.2 mmol/L PMSF; 20 μg/mL aprotinin; 0.5 mmol/L DTT; 0.2 mmol/L Na-EDTA; 10% glycerol) and incubated at room temperature for 30 minutes. After centrifugation at 10,000 × g for 30 minutes at 4°C, the nuclear fraction contained in the supernatant was collected.

Chemotaxis assay

Transwell plates (24-well; 8-μm pore size, Corning) were used to measure chemotaxis of the RAW264.7 cells. HK-2 cells were transfected with siRNA or vector-expressing shRNA by electroporation (see above) and seeded into the bottom well. After incubation for 48 hours, cells were treated with solvent (DMSO:PBS = 1:3; or DMSO), tunicamycin, APY29, or 4-PBA as described above, for 24 hours in serum-free media supplemented with 5% BSA (BIONOVAS). RAW264.7 cells (1 × 105) were seeded in the top well and then placed over the bottom well containing the treated or untreated HK-2 cells. In a control experiment, RAW264.7 cells in the top well were pretreated with tunicamycin (at 1 μg/mL) before being placed over HK-2 cells, to assess whether tunicamycin can directly influence the migratory capability of the macrophages. In a separate experiment, the HK-2 cells were pretreated with chemical chaperon 4-PBA at a final concentration of 1 mmol/L for 24 hours. The media containing 4-PBA were replaced with the serum-free media described above, and the top well containing RAW264.7 cells inserted for the chemotaxis assay.

The chemotaxis was allowed to proceed for 24 hours, after which the remaining cells on the upperside of the membrane was wiped off with a cotton swap and the migrated cells on the underside of the membrane were stained with 0.1% crystal violet for 5 minutes and washed with H2O. The excess crystal violet was destained with methanol for 15 minutes. The stained membrane was imaged with the EPSON V750 PRO scanner. Quantification was performed by colorimetric measurement at OD570 using Synergy HT (BioTek).

Statistical analysis

Data are expressed as means ± SEM. Groups were compared via a one-way or two-way ANOVA followed by Bonferroni post hoc analysis. P < 0.05 was considered statistically significant. The sample size is designed for 80% power and statistical significance set at P < 0.05. On the basis of previous experiences, the normal mean and deviation should give an effective size of 3. We therefore estimate the sample size for our two-way ANOVA to be 3.

Loss of VHL function causes ER stress and UPR

In our previous study, inactivation of the mouse VHL gene (designated as Vhlh) in a subpopulation of kidney distal tubule cells, using the HOXB7-Cre-GFP driver, resulted in hyperplastic clear-cell lesions, inflammation (i.e., infiltration of immune cells) and severe fibrosis (Supplementary Fig. S1; ref. 19). As mentioned above, a potential link between VHL mutation and inflammatory response is the metabolic abnormalities in the VHL mutant. We first examined whether metabolic abnormalities exist in noncancerous kidney epithelial cells upon VHL knockdown. As we are primarily interested in cellular conditions before the onset of cancer, in this study, commonly used VHL-mutant cancer cell lines will not be appropriate. We therefore first examined noncancerous human kidney cell lines HK-2 or HEK293 (containing wild-type VHL) and their VHL knockdown counterparts.

As shown in Fig. 1 and Supplementary Fig. S2A and S2B, in human kidney cells, VHL knockdown using either of the shVHL constructs resulted in increased protein synthesis and elevated levels of ROS, consistent with the previous notion (20, 25). This was confirmed using a different kidney cell line HEK293 (Supplementary Fig. S2C and S2D). We therefore hypothesize that the metabolic abnormalities may lead to ER stress and UPR, which in turn can induce inflammatory response.

We next examined ER stress and UPR at the cellular level. In HK-2 cells, VHL knockdown induced ER stress and UPR, as indicated by the increased levels of BiP, phosphorylated IRE1α (p-IRE1α), and IRE1α target XBP1 (Fig. 2A; Supplementary Fig. S3A–S3C). The level of ER stress induction via VHL knockdown is similar to that induced by protein glycosylation inhibitor (and thus ER stress inducer) tunicamycin (Fig. 2A; Supplementary Fig. S3A–S3C). The other UPR pathway, the PERK–eIF2α–ATF4 axis, was also induced by VHL knockdown and tunicamycin treatment (Fig. 2A; Supplementary Fig. S3D–S3F), although the ATF4 induction is not as prominent as BiP, p-IRE1α, and XBP1. This indicates that the eIF2α pathway may be under more stringent feedback control such as that mediated by phosphatase GADD34 (48). In step with IRE1α activation, JNK was phosphorylated (activated) without changes in the total JNK protein levels (Fig. 2A; Supplementary Fig. S3G). Also, the level of NFκB activation was significantly increased in VHL knockdown cells, as shown by the increase of NFκB subunit p65 nuclear localization with concomitant decrease in cytosolic localization (Fig. 2B; Supplementary Fig. S4A and S4B), as well as by the increase in the expression of NFκB target gene products TNFα and IL1β (Fig. 2C; Supplementary Fig. S4C and S4D). The effects of VHL knockdown and tunicamycin treatment are additive in the nuclear localization of NFκB, but the additive effects are modest in ER stress response and in the activation of JNK, suggesting a feedback control in these UPR signaling pathways, but the exact mechanism in this context is unknown. Note that these effects were sustained as long as VHL is reduced or knocked out. We have observed effective knockdown as long as 7 days after transfection (data not shown), but the cells were usually analyzed within 3 days after transfection.

We sought to verify the above results by examining primary kidney tubule cells isolated from wild-type and Vhlh conditional knockout mice (Hoxb7-Cre-GFP-driven; ref. 46). The primary cells were confirmed for their respective VHL protein expression level (Fig. 2D, top). The increased ER stress response was observed in the increased expression of BiP, p-IRE1α, XBP1, p-PERK, p-eIF2α, and ATF4 in Vhlh-mutant cells and after treatment with tunicamycin (Fig. 2D; Supplementary Fig. S5A–S5F). JNK and NFκB were also activated in step with IRE1α activation (Fig. 2D and E; Supplementary Fig. S5G–S5I).

JNK and NFκB activation is dependent on the IRE1α kinase activity

As mentioned earlier, IRE1α is a critical link between ER stress and inflammatory response, possibly via its noncanonical function of JNK and NFκB activation (see Introduction). To test whether VHL inactivation (knockdown or knockout) indeed induces inflammatory response via IRE1α, we used a kinase inhibitor APY29 that showed preference for IRE1α (49). APY29 is a type 1 inhibitor that occupies the IRE1α kinase–active site (50). While it blocks the access of kinase substrates, it also keeps the active site in an open conformation. Because the RNase activity of IRE1α depends on the open conformation induced by autophosphorylation, APY29 can block IRE1α kinase activity yet allosterically activates the IRE1α RNase domain (49). Thus, APY29 will not inhibit, or may actually enhance, the canonical UPR. Specifically inhibiting the IRE1α kinase activity without blocking UPR is advantageous for our assays as the strategy will avoid complicating side effects from further increasing ER stress.

As shown in Fig. 3, treatment with APY29 decreased the level of p-IRE1α while increasing or maintaining the levels of XBP1 in VHL wild-type and knockdown HK-2 cells (Fig. 3A; Supplementary Fig. S6A and S6B). The BiP levels in APY29-treated VHL knockdown cells were reduced compared with untreated VHL knockdown cells (Fig. 3A; Supplementary Fig. S6C), possibly the result of increased XBP1 levels. More importantly, APY29 also significantly decreased the levels of p-JNK in VHL knockdown cells to near the wild-type level without significantly affecting the total IRE1α and JNK protein levels (Fig. 3A; Supplementary Fig. S6D). The same response was reproduced in the mouse primary renal tubule cells comparing wild-type and Vhlh mutant (Fig. 3B; Supplementary Fig. S5J–S5M). Nuclear localization of NFκB and TNFα and IL1β expression in VHL knockdown HK-2 cells were also reduced to the background level upon APY29 treatment (Fig. 3C and D; Supplementary Fig. S6E–S6H).

VHL loss of function in kidney epithelial cells enhances recruitment of macrophages

One key event in tissue inflammation is the infiltration of macrophages, and we have previously observed increased macrophage infiltration into the Vhlh conditional knockout kidney (19). We therefore devised an in vitro system to test whether VHL loss of function in epithelial cells possesses increased capability to induce macrophage chemotaxis. In this assay, HK-2 cells (with or without VHL knockdown) were plated in the bottom well of a Boyden chamber, whereas the mouse monocyte/macrophage cell line RAW264.7 was plated on the porous membrane above the HK-2 cells. The number of RAW264.7 cells migrated toward the HK-2 cells to the underside of the membrane could then be quantified as an index of the chemotactic response. The efficacy of VHL knockdown in HK-2 cells (comparing triplicates each of the scrambled and the two shVHL constructs) was monitored by Western blot analysis for VHL and HIF-1α protein expression (Supplementary Fig. S7A and S7B). In the transwell assay, knockdown of VHL induced a 3- to 6-fold increase in macrophage chemotaxis compared with wild-type cells (Fig. 4A). Treatment of HK-2 cells with tunicamycin increased the macrophage chemotaxis toward wild-type cells to a level comparable with the VHL knockdown cells (3- to 4-fold), and further increased the chemotaxis toward VHL knockdown cells by about 2-fold. Tunicamycin did not directly stimulate macrophage migration as treatment of RAW264.7 cells with tunicamycin did not increase chemotaxis (Supplementary Fig. S7C). Taken together, the results suggest that ER stress in VHL loss-of-function cells can induce macrophage chemotaxis, which is further increased by exacerbating the stress level with tunicamycin.

Macrophage recruitment by VHL loss-of-function cells is dependent on the IRE1α kinase activity

To further ascertain that the increased chemotaxis of macrophage RAW264.7 cells toward VHL loss-of-function cells is mediated by the IRE1α signaling pathway, we performed the in vitro chemotaxis experiment in the presence of APY29. In this experiment, the primary renal tubule cells were included, in addition to the HK-2 cells. In both cell systems, loss of VHL function significantly increased the chemotactic migration of the RAW264.7 cells (Fig. 4B and C). In the presence of APY29, RAW264.7 cell migration toward VHL loss-of-function HK-2 (Fig. 4B) or primary (Fig. 4C) kidney tubule cells was reduced to the wild-type levels. The specificity of APY29 and the involvement of IRE1α were further verified by knocking down IRE1α in the VHL knockdown background. VHL-IRE1α double knockdown ameliorated the capacity of VHL knockdown HK-2 cells to recruit macrophages (Fig. 4D).

To verify whether ER stress is necessary for inducing macrophage chemotaxis, HK-2 cells were pretreated with a chemical chaperon and ER stress inhibitor 4-PBA that improves protein folding (51). We confirmed that 4-PBA treatment reduced ER stress marker expression including BiP, XBP1, p-IRE1α, and p-PERK, as well as p-JNK (data not shown). Importantly, 4-PBA could also inhibit the chemotaxis of RAW264.7 cells toward VHL knockdown cells (Fig. 4E). Therefore, we conclude that the enhanced ability of VHL loss-of-function cells to attract macrophage is the result of ER stress signaling and is dependent on the IRE1α kinase activity.

IRE1α kinase function is required for JNK activation in VHL knockdown kidney cells

We have shown so far that the IRE1α kinase inhibitor APY29 can effectively block the inflammatory response in the VHL knockdown or mutant kidney cells. To further confirm the direct involvement of IRE1α kinase activity, we used an IRE1α kinase–active site mutation K599A (27, 52). As shown in Fig. 5 and Supplementary Fig. S8, the increase of p-JNK level in VHL knockdown HK-2 cells (compare lanes 2 with 1) was ameliorated by the knockdown of IRE1α (compare lanes 3 with 2). Interestingly, the reduction of JNK activation in IRE1α knockdown cells could be restored by expressing a wild-type IRE1α exogenously, but not by reexpressing the kinase-active site mutant K599A (compare lane 3 with lanes 4 with 5). The expression of the exogenous wild-type or mutant IRE1α was monitored by the fusion marker peptide FLAG (top). As controls, without VHL knockdown (lanes 6–10), JNK activation (p-JNK) was very low regardless of the IRE1α status. Note that in these conditions, IRE1α showed low levels of activation (p-IRE1α). This level of activation is likely a constitutive background activity of IRE1α that is under a separate control, as it is not affected by exogenously expressed IRE1α (wild-type or kinase-site mutant). Note that this background level is very low when the extent of IRE1α knockdown is enhanced using a different siRNA construct (Supplementary Fig. S8). We conclude that JNK activation in VHL loss-of-function kidney cells is dependent on the IRE1α kinase function.

APY29 reverses the VHL knockout kidney phenotype in vivo

The VHL-mutant kidney phenotypes including fibrosis and proteinaceous blockage of tubules are developed at approximately 3–4 months of age in HOXB7-Cre-GFP; Vhlhfl/fl mice (Supplementary Fig. S1; ref. 19). To verify whether these phenotypes are dependent on the IRE1α kinase activity, we injected 1.3 μg of APY29 (in 50 μL canola oil) intraperitoneally into 2.5-month-old HOXB7-Cre; Vhlhfl/fl mice (canola oil alone as control), three times a week for 6 weeks. The injection schedule and dosage were determined empirically, taking into account the sublethal effective dosage needed and the age of mouse when the phenotypes are fully developed. As shown in Fig. 6A, mutant kidneys showed extensive fibrosis compared with wild-type (top). With APY29 treatment, the pathologic fibrosis was undetectable (bottom). Quantification of the Sirius red staining showed that APY29 could reduce the fibrosis to the wild-type level (right). It is interesting to point out that, because our injection regimen begins at 2.5 months of age, when the phenotypes are barely detectable (19), the effect of APY29 is likely a preventive one. Concomitant to the reduction of tissue fibrosis, the loss of kidney function observed in knockout mice, as measured by the increased albumin content in equal volume urine (Fig. 6B, compare lanes 1 with 2), was also rescued by APY29 treatment (lanes 2, 3, 4).

Inflammation has been implicated in the initiation of various cancers (10, 11). This is an important finding because if the link can be verified, such cancers are potentially preventable by anti-inflammatory therapeutics. This is particularly relevant for high-risk patients such as family members predisposed to hereditary cancers, and patients of inflammatory diseases such as hepatitis, colitis, and chronic kidney disease. We have previously demonstrated a link between inflammation and development of ccRCC in a knockout mouse model. Genetic and epigenetic inactivation of the VHL tumor suppressor gene has been shown to be the driver in up to 70%–80% of the ccRCC cases (2). We therefore generated conditional knockout of Vhlh in a subset of kidney tubule cells using the HOXB7-Cre-GFP driver, which resulted in hyperplastic lesions containing abnormal clear cells typical of the ccRCC (19). Surprisingly, these hyperplastic lesions were accompanied by severe inflammation and fibrosis. It thus provides support for the notion that inflammatory kidney disease is a risk factor for RCC development (12–15).

One critical question to address is how a single tumor suppressor gene mutation in a subpopulation of tubule epithelial cells can induce a systemic inflammatory response, as the answer to this question may provide an avenue for cancer prevention. Two of the critical inflammation-inducing master switches, NFκB and JNK, have been known to be activated in cells under ER stress via the IRE1α pathway (37, 38). In VHL loss-of-function cells, because of the imbalance in proteostasis induced by metabolic abnormalities (Fig. 1), chronic ER stress is a likely pathophysiologic condition. If the above reasoning is valid, such condition could lead to activation of an inflammatory response beyond the epithelia that exhibit reduced VHL function. This study provides the proof for such a model.

In VHL knockdown or mutant cells, ER stress–responsive pathway emanating from IRE1α is activated, which is accompanied by increased levels of p-JNK and activation of NFκB (Fig. 2). Significantly, these responses can be blocked by an IRE1α kinase inhibitor APY29 (Fig. 3). Functionally, ER stress and VHL loss of function exert additive chemotactic attraction for macrophages, which can also be ameliorated by IRE1α knockdown and treatment with APY29 (Fig. 4). The involvement of IRE1α kinase activity was further confirmed using a kinase-active site mutant of IRE1α, which could not rescue the loss of JNK activation after the endogenous IRE1α was knocked down (Fig. 5). In vivo, APY29 can prevent the fibrotic phenotype and improve the kidney function of the knockout mice (Fig. 6).

We also observed certain degrees of additive effects of VHL inactivation and tunicamycin treatment, which is more prominent in NFκB activation and macrophage recruitment but less so in ER stress marker expression. There are three possibilities for the additive effect: (i) the extent of ER stress has different gradations, ranging from maintenance of proteostasis to induction of apoptosis (28, 29). It is possible that VHL inactivation does not induce ER stress to a maximum extent; therefore addition of tunicamycin can exacerbate the stress. (ii) VHL inactivation can elicit inflammatory response via an ER stress-independent pathway. Indeed VHL loss of function has been shown to result in NFκB and JNK activation via stabilizing a scaffold complex containing Card9 (53, 54), although these were shown in malignant ccRCC cell lines, not in premalignant kidney cells (3). The IRE1α pathway can be activated by an ER stress–independent mechanism, such as membrane lipid saturation (55). We favor the first possibility as our result indicates that knocking down IRE1α or reducing ER stress could completely ameliorate the inflammatory response after VHL inactivation.

APY29 has been known to target the IRE1α-TRAF2 scaffold activity without inhibiting XBP1 activation, thus reducing the UPR (49, 50). As such, APY29 was chosen as an IRE1α kinase inhibitor because we could avoid the complex side effects associated with further increasing ER stress. It is also important to note that in our in vivo experiment, the mice treated with APY29 did not show drug-related lethality during and after the treatment, indicating that the compound may have relatively low cytotoxicity at our chosen dosage. We have previously shown that ruxolitinib, the inhibitor of JAK1/2 that is a main mediator of the inflammatory cytokine signaling pathway, could reduce fibrosis and hyperplasia in the HOXB7-Cre-GFP–driven Vhlh knockout kidney (19). Here we demonstrate that rescue of inflammatory and fibrotic phenotypes can be achieved by intervening at further upstream of the pathologic chain of events—that is, before the inflammatory pathway is activated in the epithelial cells.

Two key events in early cancer progression are hypoxic response (the Warburg effect) and tumor-associated inflammation (“tumors are wounds that do not heal”), but the potential link between these two events has not been elucidated. This study provides a mechanistic view of the origin of inflammatory response induced by hypoxia-induced metabolic imbalance in epithelial cells. The finding can suggest a strategy for early cancer detection and prevention in high-risk cohorts. One high-risk group that may benefit from the knowledge is familial VHL disease patients. Up to 60% of the carriers of germline VHL mutations develop ccRCC in their life time (2). These high-risk patients should be considered for preventive monitoring or treatment for inflammatory kidney conditions. It is also worth noting that the IRE1α-TRAF2–mediated inflammatory pathway identified in the VHL loss-of-function cells is induced by the ER stress, but this IRE1α–TRAF2 pathway does not contribute to the modulation of ER stress, as APY29 treatment could block the inflammatory response but did not reduce the UPR. This provides a therapeutic strategy that can avoid altering the status of ER stress and the associated UPR. Therapeutics that aim to reduce ER stress in noncancerous tissues may decrease inflammatory response but may also cause unwanted side effects, as ER stress and UPR are an important protective mechanism in normal cellular physiology. In this sense, the type of IRE1α kinase inhibitors, such as APY29, that specifically block the inflammatory function of IRE1α offers a good avenue for cancer prevention. Future studies should further delineate the exact roles of ER stress and inflammation at different stages of cancer development.

No potential conflicts of interest were disclosed.

Conception and design: C.-Y. Kuo, T. Hsu

Development of methodology: C.-Y. Kuo, T. Hsu

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): C.-Y. Kuo, C.-H. Lin

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): C.-Y. Kuo, C.-H. Lin, T. Hsu

Writing, review, and/or revision of the manuscript: C.-Y. Kuo, T. Hsu

Study supervision: T. Hsu

We are indebted to Dr. Ann Chen, Chief of Pathology, Triservice General Hospital, Taiwan, for analysis of the mouse kidney sections. We also thank the Taiwan Bio-development Foundation (TBF) for support of the T. Hsu laboratory.

This work was supported by a grant from the NIH (#R01CA109860 to T. Hsu), a grant from the Ministry of Science and Technology, Taiwan (MOST103-2320-B-008-002-MY3 to T. Hsu), a grant from National Health Research Institute, Taiwan (EX105-0501BI to T. Hsu), and a grant from Cathay General Hospital (CGH-MR-A10304 to C.-H. Lin).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1.
Cancer Genome Atlas Research Network
. 
Comprehensive molecular characterization of clear cell renal cell carcinoma
.
Nature
2013
;
499
:
43
9
.
2.
Lonser
RR
,
Glenn
GM
,
Walther
M
,
Chew
EY
,
Libutti
SK
,
Linehan
WM
, et al
von Hippel-Lindau disease
.
Lancet
2003
;
361
:
2059
67
.
3.
Shen
C
,
Kaelin
WG
 Jr
. 
The VHL/HIF axis in clear cell renal carcinoma
.
Semin Cancer Biol
2013
;
23
:
18
25
.
4.
Kim
JW
,
Tchernyshyov
I
,
Semenza
GL
,
Dang
CV
. 
HIF-1-mediated expression of pyruvate dehydrogenase kinase: a metabolic switch required for cellular adaptation to hypoxia
.
Cell Metab
2006
;
3
:
177
85
.
5.
Forsythe
JA
,
Jiang
BH
,
Iyer
NV
,
Agani
F
,
Leung
SW
,
Koos
RD
, et al
Activation of vascular endothelial growth factor gene transcription by hypoxia-inducible factor 1
.
Mol Cell Biol
1996
;
16
:
4604
13
.
6.
Haase
VH
. 
The VHL/HIF oxygen-sensing pathway and its relevance to kidney disease
.
Kidney Int
2006
;
69
:
1302
7
.
7.
Ohh
M
,
Park
CW
,
Ivan
M
,
Hoffman
MA
,
Kim
TY
,
Huang
LE
, et al
Ubiquitination of hypoxia-inducible factor requires direct binding to the beta-domain of the von Hippel-Lindau protein
.
Nat Cell Biol
2000
;
2
:
423
7
.
8.
Ciccarese
C
,
Brunelli
M
,
Montironi
R
,
Fiorentino
M
,
Iacovelli
R
,
Heng
D
, et al
The prospect of precision therapy for renal cell carcinoma
.
Cancer Treat Rev
2016
;
49
:
37
44
.
9.
Dvorak
HF
. 
Tumors: wounds that do not heal. Similarities between tumor stroma generation and wound healing
.
N Engl J Med
1986
;
315
:
1650
9
.
10.
Elinav
E
,
Nowarski
R
,
Thaiss
CA
,
Hu
B
,
Jin
C
,
Flavell
RA
. 
Inflammation-induced cancer: crosstalk between tumours, immune cells and microorganisms
.
Nat Rev Cancer
2013
;
13
:
759
71
.
11.
Crusz
SM
,
Balkwill
FR
. 
Inflammation and cancer: advances and new agents
.
Nat Rev Clin Oncol
2015
;
12
:
584
96
.
12.
de Vivar Chevez
AR
,
Finke
J
,
Bukowski
R
. 
The role of inflammation in kidney cancer
.
Adv Exp Med Biol
2014
;
816
:
197
234
.
13.
Tan
W
,
Hildebrandt
MA
,
Pu
X
,
Huang
M
,
Lin
J
,
Matin
SF
, et al
Role of inflammatory related gene expression in clear cell renal cell carcinoma development and clinical outcomes
.
J Urol
2011
;
186
:
2071
7
.
14.
Butler
AM
,
Olshan
AF
,
Kshirsagar
AV
,
Edwards
JK
,
Nielsen
ME
,
Wheeler
SB
, et al
Cancer incidence among US Medicare ESRD patients receiving hemodialysis, 1996–2009
.
Am J Kidney Dis
2015
;
65
:
763
72
.
15.
Stengel
B
. 
Chronic kidney disease and cancer: a troubling connection
.
J Nephrol
2010
;
23
:
253
62
.
16.
Iliopoulos
D
,
Hirsch
HA
,
Struhl
K
. 
An epigenetic switch involving NF-kappaB, Lin28, Let-7 MicroRNA, and IL6 links inflammation to cell transformation
.
Cell
2009
;
139
:
693
706
.
17.
Shimizu
T
,
Marusawa
H
,
Endo
Y
,
Chiba
T
. 
Inflammation-mediated genomic instability: roles of activation-induced cytidine deaminase in carcinogenesis
.
Cancer Sci
2012
;
103
:
1201
6
.
18.
Kiraly
O
,
Gong
G
,
Olipitz
W
,
Muthupalani
S
,
Engelward
BP
. 
Inflammation-induced cell proliferation potentiates DNA damage-induced mutations invivo
.
PLoS Genet
2015
;
11
:
e1004901
.
19.
Pritchett
TL
,
Bader
HL
,
Henderson
J
,
Hsu
T
. 
Conditional inactivation of the mouse von Hippel-Lindau tumor suppressor gene results in wide-spread hyperplastic, inflammatory and fibrotic lesions in the kidney
.
Oncogene
2015
;
34
:
2631
9
.
20.
Galban
S
,
Fan
J
,
Martindale
JL
,
Cheadle
C
,
Hoffman
B
,
Woods
MP
, et al
von Hippel-Lindau protein-mediated repression of tumor necrosis factor alpha translation revealed through use of cDNA arrays
.
Mol Cell Biol
2003
;
23
:
2316
28
.
21.
Robb
VA
,
Karbowniczek
M
,
Klein-Szanto
AJ
,
Henske
EP
. 
Activation of the mTOR signaling pathway in renal clear cell carcinoma
.
J Urol
2007
;
177
:
346
52
.
22.
Hervouet
E
,
Cizkova
A
,
Demont
J
,
Vojtiskova
A
,
Pecina
P
,
Franssen-van Hal
NL
, et al
HIF and reactive oxygen species regulate oxidative phosphorylation in cancer
.
Carcinogenesis
2008
;
29
:
1528
37
.
23.
Metallo
CM
,
Gameiro
PA
,
Bell
EL
,
Mattaini
KR
,
Yang
J
,
Hiller
K
, et al
Reductive glutamine metabolism by IDH1 mediates lipogenesis under hypoxia
.
Nature
2012
;
481
:
380
4
.
24.
Guzy
RD
,
Hoyos
B
,
Robin
E
,
Chen
H
,
Liu
L
,
Mansfield
KD
, et al
Mitochondrial complex III is required for hypoxia-induced ROS production and cellular oxygen sensing
.
Cell Metab
2005
;
1
:
401
8
.
25.
Sabharwal
SS
,
Schumacker
PT
. 
Mitochondrial ROS in cancer: initiators, amplifiers or an Achilles' heel?
Nat Rev Cancer
2014
;
14
:
709
21
.
26.
Inagi
R
,
Ishimoto
Y
,
Nangaku
M
. 
Proteostasis in endoplasmic reticulum–new mechanisms in kidney disease
.
Nat Rev Nephrol
2014
;
10
:
369
78
.
27.
Mori
T
,
Hayashi
T
,
Hayashi
E
,
Su
TP
. 
Sigma-1 receptor chaperone at the ER-mitochondrion interface mediates the mitochondrion-ER-nucleus signaling for cellular survival
.
PLoS One
2013
;
8
:
e76941
.
28.
Walter
P
,
Ron
D
. 
The unfolded protein response: from stress pathway to homeostatic regulation
.
Science
2011
;
334
:
1081
6
.
29.
Zhang
K
,
Kaufman
RJ
. 
From endoplasmic-reticulum stress to the inflammatory response
.
Nature
2008
;
454
:
455
62
.
30.
Fougeray
S
,
Bouvier
N
,
Beaune
P
,
Legendre
C
,
Anglicheau
D
,
Thervet
E
, et al
Metabolic stress promotes renal tubular inflammation by triggering the unfolded protein response
.
Cell Death Dis
2011
;
2
:
e143
.
31.
Chen
Y
,
Brandizzi
F
. 
IRE1: ER stress sensor and cell fate executor
.
Trends Cell Biol
2013
;
23
:
547
55
.
32.
Bertolotti
A
,
Wang
X
,
Novoa
I
,
Jungreis
R
,
Schlessinger
K
,
Cho
JH
, et al
Increased sensitivity to dextran sodium sulfate colitis in IRE1beta-deficient mice
.
J Clin Invest
2001
;
107
:
585
93
.
33.
Li
H
,
Korennykh
AV
,
Behrman
SL
,
Walter
P
. 
Mammalian endoplasmic reticulum stress sensor IRE1 signals by dynamic clustering
.
Proc Natl Acad Sci U S A
2010
;
107
:
16113
8
.
34.
Korennykh
AV
,
Egea
PF
,
Korostelev
AA
,
Finer-Moore
J
,
Zhang
C
,
Shokat
KM
, et al
The unfolded protein response signals through high-order assembly of Ire1
.
Nature
2009
;
457
:
687
93
.
35.
Tirasophon
W
,
Lee
K
,
Callaghan
B
,
Welihinda
A
,
Kaufman
RJ
. 
The endoribonuclease activity of mammalian IRE1 autoregulates its mRNA and is required for the unfolded protein response
.
Genes Dev
2000
;
14
:
2725
36
.
36.
Lee
KP
,
Dey
M
,
Neculai
D
,
Cao
C
,
Dever
TE
,
Sicheri
F
. 
Structure of the dual enzyme Ire1 reveals the basis for catalysis and regulation in nonconventional RNA splicing
.
Cell
2008
;
132
:
89
100
.
37.
Urano
F
,
Wang
X
,
Bertolotti
A
,
Zhang
Y
,
Chung
P
,
Harding
HP
, et al
Coupling of stress in the ER to activation of JNK protein kinases by transmembrane protein kinase IRE1
.
Science
2000
;
287
:
664
6
.
38.
Hu
P
,
Han
Z
,
Couvillon
AD
,
Kaufman
RJ
,
Exton
JH
. 
Autocrine tumor necrosis factor alpha links endoplasmic reticulum stress to the membrane death receptor pathway through IRE1alpha-mediated NF-kappaB activation and down-regulation of TRAF2 expression
.
Mol Cell Biol
2006
;
26
:
3071
84
.
39.
Oeckinghaus
A
,
Ghosh
S
. 
The NF-kappaB family of transcription factors and its regulation
.
Cold Spring Harb Perspect Biol
2009
;
1
:
a000034
.
40.
Schonthaler
HB
,
Guinea-Viniegra
J
,
Wagner
EF
. 
Targeting inflammation by modulating the Jun/AP-1 pathway
.
Ann Rheum Dis
2011
;
70
Suppl 1
:
i109
12
.
41.
Liu
J
,
Xu
Y
,
Stoleru
D
,
Salic
A
. 
Imaging protein synthesis in cells and tissues with an alkyne analog of puromycin
.
Proc Natl Acad Sci U S A
2012
;
109
:
413
8
.
42.
Rothe
G
,
Valet
G
. 
Flow cytometric analysis of respiratory burst activity in phagocytes with hydroethidine and 2′,7′-dichlorofluorescin
.
J Leukoc Biol
1990
;
47
:
440
8
.
43.
Champion
KJ
,
Guinea
M
,
Dammai
V
,
Hsu
T
. 
Endothelial function of von Hippel-Lindau tumor suppressor gene: control of fibroblast growth factor receptor signaling
.
Cancer Res
2008
;
68
:
4649
57
.
44.
Hsu
T
,
Adereth
Y
,
Kose
N
,
Dammai
V
. 
Endocytic function of von Hippel-Lindau tumor suppressor protein regulates surface localization of fibroblast growth factor receptor 1 and cell motility
.
J Biol Chem
2006
;
281
:
12069
80
.
45.
Son
SM
,
Byun
J
,
Roh
SE
,
Kim
SJ
,
Mook-Jung
I
. 
Reduced IRE1alpha mediates apoptotic cell death by disrupting calcium homeostasis via the InsP3 receptor
.
Cell Death Dis
2014
;
5
:
e1188
.
46.
Hsouna
A
,
Nallamothu
G
,
Kose
N
,
Guinea
M
,
Dammai
V
,
Hsu
T
. 
Drosophila von Hippel-Lindau tumor suppressor gene function in epithelial tubule morphogenesis
.
Mol Cell Biol
2010
;
30
:
3779
94
.
47.
Nowak
G
,
Price
PM
,
Schnellmann
RG
. 
Lack of a functional p21WAF1/CIP1 gene accelerates caspase-independent apoptosis induced by cisplatin in renal cells
.
Am J Physiol Renal Physiol
2003
;
285
:
F440
50
.
48.
Novoa
I
,
Zhang
Y
,
Zeng
H
,
Jungreis
R
,
Harding
HP
,
Ron
D
. 
Stress-induced gene expression requires programmed recovery from translational repression
.
EMBO J
2003
;
22
:
1180
7
.
49.
Wang
L
,
Perera
BG
,
Hari
SB
,
Bhhatarai
B
,
Backes
BJ
,
Seeliger
MA
, et al
Divergent allosteric control of the IRE1alpha endoribonuclease using kinase inhibitors
.
Nat Chem Biol
2012
;
8
:
982
9
.
50.
Ghosh
R
,
Wang
L
,
Wang
ES
,
Perera
BG
,
Igbaria
A
,
Morita
S
, et al
Allosteric inhibition of the IRE1alpha RNase preserves cell viability and function during endoplasmic reticulum stress
.
Cell
2014
;
158
:
534
48
.
51.
Rubenstein
RC
,
Zeitlin
PL
. 
Sodium 4-phenylbutyrate downregulates Hsc70: implications for intracellular trafficking of DeltaF508-CFTR
.
Am J Physiol Cell Physiol
2000
;
278
:
C259
67
.
52.
Tirasophon
W
,
Welihinda
AA
,
Kaufman
RJ
. 
A stress response pathway from the endoplasmic reticulum to the nucleus requires a novel bifunctional protein kinase/endoribonuclease (Ire1p) in mammalian cells
.
Genes Dev
1998
;
12
:
1812
24
.
53.
Yang
H
,
Minamishima
YA
,
Yan
Q
,
Schlisio
S
,
Ebert
BL
,
Zhang
X
, et al
pVHL acts as an adaptor to promote the inhibitory phosphorylation of the NF-kappaB agonist Card9 by CK2
.
Mol Cell
2007
;
28
:
15
27
.
54.
An
J
,
Liu
H
,
Magyar
CE
,
Guo
Y
,
Veena
MS
,
Srivatsan
ES
, et al
Hyperactivated JNK is a therapeutic target in pVHL-deficient renal cell carcinoma
.
Cancer Res
2013
;
73
:
1374
85
.
55.
Kitai
Y
,
Ariyama
H
,
Kono
N
,
Oikawa
D
,
Iwawaki
T
,
Arai
H
. 
Membrane lipid saturation activates IRE1alpha without inducing clustering
.
Genes Cells
2013
;
18
:
798
809
.