Inhibition of endoplasmic-reticulum-stress-mediated autophagy enhances the effectiveness of chemotherapeutics on pancreatic cancer

The human PDAC cell lines Panc02.03, Panc3.27, Miapaca-2, and the murine PDAC cell lines, Panc02, and KPCP1 were originally procured from ATCC (Manassas, VA). Miapaca-2 was cultured in DMEM medium, and the rest others were cultured in ATCC-recommended RPMI-1640 supplemented with 10% fetal bovine serum and maintained at 5% CO₂ at 37 °C. For long-term storage, the cells were frozen in a 5% DMSO containing the respective tissue culture medium in liquid nitrogen. Cell viability assays were carried out using Trypan-blue exclusion method using Beckman Coulter Vi-CELL™ cell viability analyzer and Image analysis [25].

The following drugs were used in this study: Tunicamycin (Sigma-Aldrich) was prepared fresh in DMSO media for 5 mM stock solution. STF-083010 (Sigma-Aldrich) was prepared fresh in dark room with DMSO for 25 mM stock solution. 4-Phenylbutyric acid, sodium salt (Sigma-Aldrich) was dissolved in water at 100 mM stock solution. Chloroquine (Sigma-Aldrich) was prepared fresh in water at 50 mM stock solution. Gemcitabine and taxol solutions were freshly prepared in aliquots of 5 mM for one-time usage. Sunitinib maleate salt (Sigma-Aldrich) was dissolved in DMSO in dark room at 5 mM stock solution.

About 10,000 cells were seeded onto 12-well microtiter plates and allowed to attach overnight. Drug treatments typically started at about 50% confluence for 72 h incubation and dosing. After the drug treatment, cells were washed 2 × with fresh culture media and trypsinized (0.15% Trypsin, Invitrogen) for cell viability assays. For lysosome staining, 50 nM of lysotracker dye (LysoTracker™ Red DNN-99, Invitrogen) was added to the wells and the live cells were incubated for 45 min followed by 3 × washes with tissue culture media and imaged by fluorescent microscopy (Zeiss Axiovert) and quantified using ImageJ [26]. For TUNEL assays, cells were seeded onto sterile 8-chamber borosilicate cover glass (Tissue-Tek) and after treatment, cells were fixed with 4% PFA for 2 h, followed by the TUNEL protocol recommended by the Cell death detection kit, Fluorescein (Roche) [27]. In brief, the fixed cells were washed with PBS, permeabilized with freshly prepared 0.1% Triton X-100 and 0.1% sodium citrate for 2 min on ice, followed by washing with PBS. The cells in Tissue-Tek glass chambers were then overlaid with 100 μl TUNEL reaction mix, according to manufacturer’s instructions and incubated at 37 °C for 1 h, washed again with PBS and mounted with anti-fading reagent with DAPI (Molecular Probes) and imaged using Zeiss Axiovert with 488 nm filter. The TUNEL positive cells were quantified using ImageJ. Statistical analysis was performed by using t-test, and a p-value < 0.05 was considered significant.

Total mRNA was extracted from cell pellets using Exiquon™ cell RNA kit, and the concentration was measured using spectrophotometer. A consistent amount of template mRNA was used for RT-PCR assay using Ambion™ RT-PCR kit, following manufacturer’s protocol. The PCR products were subjected to 2% Agarose electrophoresis in TBE buffer and the gel was imaged using Bio-Rad gel documentation unit.

Forward, 5′-CCAAGAGAGGGTTCTTGAATCTCG-3′

Reverse, 5′-ATGGGCCAGCCTGGATATACAACA-3′

Forward, 5′-GGAGTTAAGACAGCGCTTGGGGA-3′

Reverse, 5′-TGTTCTGGAGGGGTGACAACTGGG-3′

Female C57BL/6 (B6, H-2^b) mice, 8–10 weeks old, were purchased from Taconic (Germantown, NY). Animals were maintained in a specific-pathogen-free facility at the University of Pittsburgh Cancer Institute in accordance with the Institutional IACUC and NIH guidelines. Cell suspension of KPCP1 or Panc02 were prepared to a concentration of 1 million cells in 20 µl PBS solution. Pre-weighted mice were anesthetized, and a small incision of 1-cm was created at the left abdominal flank medial to the splenic silhouette. The pancreas was gently exposed and the cell suspension was injected under direct visualization into the pancreatic tail. The abdominal muscle and skin layer were subsequently sutured. Mice were monitored until 2 weeks for palpable tumor growth and general signs of morbidity. After 2 weeks of tumor growth, the drug treatment was initiated using 10 mice for each treatment group. At first the chloroquine (50 mg/Kg) was administered daily by intraperitoneal injection, sunitinib (25 mg/Kg) was administered daily by oral gavage, and Chemotherapy (Chemo) containing gemcitabine (25 mg/Kg) plus paclitaxel (10 mg/Kg) was intraperitoneally injected once weekly. Treatment was continued until the mice were alive for the survival analysis. For the tissue corollary study, 3 mice from each treatment group were sacrificed after 4-weeks of treatment, and pancreatic tissues were surgically removed and fixed in 4% PFA/PBS solution. All animal experiments were conducted in strict adherence with the Institutional IACUC and NIH guidelines. Statistical analysis was performed by using t-test, and a p-value < 0.05 was considered significant. The Kaplan–Meier method and log-rank test were used to evaluate the survival analysis.

Human PDAC tissue sections from different tumor grades, pancreatitis tissue and normal healthy pancreatic tissue sections were obtained from the Department of Pathology, University of Pittsburgh Medical Center. PDAC Tissue microarrays with normal adjacent tissues were purchased from US Biomax (Catalog # PA241d). Mouse tissue samples were removed for corollary studies and fixed in 4% PFA in PBS, processed and embedded in paraffin. All paraffin-embedded tissues were sectioned at 5 µM sections. Sequential sections were stained with Hematoxylin and Eosin (H&E) or left unstained for IHC studies. For the quantitative analysis of apoptosis, paraffin-embedded tissue sections were assayed using TUNEL method standardized at the UPCI Tissue and Research Pathology resources. Anti-active Caspase-3 (Abcam) IHC was performed to further analyze apoptosis in murine tissues. Statistical analysis was performed by using t-test, and a p-value < 0.05 was considered significant. For cell proliferation assays, anti-Ki67 (Abcam) IHC was performed on alternate sections and imaged using Zeiss Axiovert microscope. TUNEL-positive cells, active Casp-3 positive cells and Ki67-positive cells were counted in viable regions of the ductal carcinoma in the pancreas in 5 random 20 × fields and expressed as percentage of the total cells counted in the foci. For each corollary study, representative tissue sections from 3 mice were used. Statistical analysis was performed by using t-test, and a p-value < 0.05 was considered significant. GRP78 expression was analyzed by IHC using respective anti-GRP78 antibodies (Abcam) specific to mouse or human for the respective tissue sections and the intensity of immunostaining was analyzed in both ductal carcinoma and adjacent acini and surrounding inflammatory regions. Intensity of anti-GRP78 staining was ranked as weak (scores: 1–2) or strong (scores: 3–4), and the Mann–Whitney U-test was used to compare GRP78 expression levels in the PDAC tissues and the non-tumor adjacent tissues (NAT) and a p-value < 0.05 was considered significant.

To assess the ER stress and UPR activity in the tumor microenvironment, we analyzed 24 PDAC core tissues obtained in tissue microarray, representing six different tumor cases. We examined the expression of the proximal ER stress sensor GRP78 by anti-GRP78 immunohistochemistry (IHC) on the tumor tissues and the non-tumor adjacent tissues (NAT). These studies revealed elevated expression of GRP78 in pancreatic ductal tissues of PDAC compared to normal healthy tissues (p < 0.05, Fig. 1a, b). We further examined tissues exhibiting different grades of PDAC (2 cases prior to neo-adjuvant therapies), pancreatitis (1 case) and apparently healthy pancreatic tissue (2 cases) obtained from routine human tissue biopsies in our clinic. GRP78 staining was significantly enriched in the invasive PDAC cells as well as the surrounding PDAC stromal cells in both the cases of PDAC (Fig. 1c). GRP78 was also elevated in the acinar and inflammatory cells of the tissues exhibiting pancreatitis pathology (Fig. 1c).

We used several human PDAC cell lines carrying KRAS mutations, namely, Panc02.03, Miapaca-2 and Panc03.27 to examine the effect of dysregulated ER stress on autophagy and cell viability. Tunicamycin inhibits protein glycosylation and causes accumulation of unfolded proteins in ER lumen, thus triggering ER stress and constitutive upregulation of UPR pathways [28]. STF-083010, on the other hand, targets the IRE1alpha arm of UPR by inhibiting XBP1 splicing, resulting in unresolved ER stress [20]. As shown in Fig. 2a, both GRP78 expression and XBP1 splicing activity are increased in human PDAC cells (Panc02.03) treated with 2.5 µM tunicamycin. XBP1 splicing could be reduced to normal level when also treated with STF-083010 at 25 µM or above, suggesting that PDAC cells are responsive to chemical activation and inhibition of the ER stress-UPR pathway. STF-083010 treatment did not alter the tunicamycin-induced elevated expression of GRP78, suggesting its specificity to IRE1α arm of UPR (Fig. 2a).

As it is hypothesized that ER stress regulates autophagy and lysosomal activity via arms of UPR pathway, we assayed autophagy in the tunicamycin and STF-083010 treated human PDAC cells (Panc02.03) by TEM and lysotracker staining. As shown by TEM analysis, both tunicamycin and STF-083010 significantly increased the number of matured lysosomes in the PDAC cells (p < 0.01, Fig. 2b). ER dilation, a hallmark feature of ER stress, was also frequently noticed in these treated cells (Fig. 2b). As evident in Fig. 2c, tunicamycin or STF-083010 increased the acidophilic lysotracker staining, suggesting increased lysosomal activities.

We used 4-PBA, a chemical chaperone that alleviates ER stress by augmenting protein folding and thereby reduces GRP78 expression, to examine its effect on lysosomal activity. Interestingly, 4-PBA could suppress both tunicamycin and STF-083010 induced upregulation of autophagy (Fig. 2c). This indicates that 4-PBA may be alleviating the accumulation of unfolded proteins thereby reducing ER stress and autophagy. This suggests that ER stress activates autophagy in PDAC cells, and that ER stress-UPR can be targeted to control cancer cell viability.

Autophagy is one of the downstream UPR effect by which cells degrade/recycle excess unfolded proteins and defective organelles as a pro-survival adaptive mechanism. We examined the effect of inhibiting autophagy by chloroquine on the growth and survival of PDAC cells. Chloroquine, a well-known autophagy inhibitor, functions by preventing endosomal acidification, which inhibits both fusion of the autophagosome and lysosome, and lysosomal enzyme mediated degradation during the late autophagy process [29]. Lysotracker staining was diminished in chloroquine treated cells consistent with autophagy inhibition (Fig. 2c).

We then proceeded to evaluate the effect of combining STF-083010 and chloroquine with gemcitabine in several PDAC cell lines. The IC50 concentration for each of these drugs were derived and an optimized drug combination regimen was developed below the respective IC50 range, prior to proceeding with combination therapy. For most of our assays, cells were inoculated and allowed to reach 50% confluence and then treated for 72 h to evaluate their yield and viability (Fig. 2d).

Administration of STF-083010 at 25 µM caused cytotoxicity as evidenced by significantly reduced cell viability compared to control (p < 0.05). Gemcitabine alone at 250 nM could also significantly reduce cell viability (p < 0.05). Co-administration of STF-083010 and chloroquine could further reduce the cell survival (p < 0.01), showing an additive effect (Fig. 2d). Similarly, additive effects were evident when STF-083010 and gemcitabine were co-administered (p < 0.01). Co-administering chloroquine, STF-083010 and gemcitabine together did not show significant reduction of cell viability, compared to dual treatment of gemcitabine and STF-083010 (p = 0.61). Although, STF-083010 can reduce PDAC cell viability in vitro, it is physiologically unstable and hence is not suitable for clinical development.

Chemotherapeutics are often limited in their efficacy as tumors progressively acquire chemoresistance. ER stress-induced autophagy is believed to be an important mechanism through which tumors develop chemoresistance. Hence, we sought to compare the utility of clinically approved drugs that may affect the autophagy/lysosomal processes. Sunitinib is a multi-tyrosine kinase inhibitor that also targets VEGF receptors, PDGF receptors and KIT, thus disrupting angiogenesis [30]. Sunitinib has been approved for the treatment of metastatic renal carcinoma, gastrointestinal stromal tumor (GIST) and pancreatic neuroendocrine tumor [31, 32]. In addition, it was hypothesized that sunitinib may alter the autophosphorylation and activity of IRE1α, as well as lysosomal enzymatic activity [21, 22, 33, 34]. Hence, we chose to use sunitinib to assess its effects in altering the ER stress, autophagy and tumor response.

After a dose response study, an IC50 and optimal dosing regimen was derived for sunitinib in Panc3.27 and Miapaca-2 cells. As shown in Fig. 3a, sunitinib-treated cells showed reduced growth. Co-administration of sunitinib significantly enhanced the cytocidal effect of gemcitabine in both Panc3.27 and Miapaca-2 cell lines (p < 0.01, Fig. 3b). Sunitinib and chloroquine combination exhibited an additive effect without gemcitabine (p < 0.01, Fig. 3b). Interestingly, Miapaca-2 cells showed a greater sensitivity to chloroquine and sunitinib compared to Panc3.27 cells (Fig. 3b).

We analyzed XBP1 splicing and lysosomal function in sunitinib and chloroquine treated cells. Sunitinib did not affect the XBP1 splicing (Additional file 2: Figure S2A), indicating it does not directly alter the IRE1alpha-mediated splicing activity. However, lysotracker staining was significantly diminished in sunitinib or chloroquine treated cells, suggesting that both are affecting the lysosomal degradation process.

To examine further the effect of Sunitinib on cellular organelles, ER stress and autophagy, we performed ultrastructural analyses of PDAC cells by transmission electron micrography (TEM). In the control groups of both Panc3.27 and Miapaca-2 cell lines, normal organelles, normal ER structures, normal lysosomes and mitochondria were apparent (Fig. 4a). While matured lysosomes can be frequently seen in control cells, large incomplete multivesicular auto-lysosome bodies were abundantly seen in sunitinib-treated Panc3.27 and Miapaca-2 cells (p < 0.001, Fig. 4b). These multivesicular autolysosome bodies contained largely undigested materials, suggesting incomplete autophagy. Expanded ER lumen and macroautophagic vesicles were also observed in sunitinib-treated cells. These data suggest that sunitinib may inhibit late-stage autophagy and lysosomal degradation leading to apoptotic cell death.

TUNEL staining, which demonstrates DNA fragmentation seen in apoptosis revealed only moderately increased apoptosis in gemcitabine alone treatment compared to the DMSO treatment (p = 0.062, Fig. 5), whereas all other drug treatment groups showed significantly increased apoptosis (p < 0.05). Within the treatment groups, sunitinib treatment alone caused similar level of apoptosis as seen in the gemcitabine + chloroquine treatment groups. Significantly increased (p < 0.05) apoptosis was noticed in sunitinib + chloroquine, as well as in gemcitabine + sunitinib treated cells (p < 0.01, Fig. 5). The highest rate of apoptosis though was noticed in the tripslet combinations of gemcitabine + sunitinib + chloroquine (eightfold increase, p < 0.01).

To examine the efficacy of sunitinib co-administration with conventional chemotherapeutics, we analyzed its effects in relation to tumor progression and survival when administered in orthotopically transplanted Panc02 or KPCP1 murine tumor models. Panc02 cell lines carry an induced mutation in K-ras alone, while KPCP1 cell lines carry putative mutations in both K-ras and P53.

Panc02 or KPCP1 cells were directly injected into the pancreatic tail of female C57BL/6 mice via mini laparotomy incision and were allowed to grow in vivo. These orthotopic mice developed pancreatic cancer with 100% penetrance by 2–3 weeks post-transplantation. Mice with advanced tumors after 2-weeks post-transplantation were treated by daily oral administration of chloroquine, intra-peritoneal injection with sunitinib and/or gemcitabine + paclitaxel (Chemo) and control vehicle. For survival analysis, mice were followed until mortality by predetermined guidelines in accordance with the institutional animal protocol. For tissue corollary assays, mice were sacrificed at 4-week post-treatment and the pancreatic tissues were dissected to analyze histological features, proliferation, apoptosis and UPR marker expression.

The combinatorial drug treatments reduced ductal carcinoma formation. Large focal necrotic areas were evident in tissues from the Chemo + sunitinib + chloroquine treatment cohort (Additional file 2: Figure S2). The expression of the UPR sensor Grp78 was significantly upregulated in the PDAC tissues of mice treated with gemcitabine with or without sunitinib (p < 0.05, Fig. 6), whereas it was to the basal level of expression in the sunitinib + chloroquine and the Chemo + sunitinib + chloroquine cohorts (Fig. 6). To further examine tumorigenesis, we performed anti-Ki67 IHC on the pancreatic tissues of the treated and control mice and analyzed the cell proliferation. As expected, the proportion of Ki67 expressing cells were significantly reduced in all the combination treatment groups compared to the control (Fig. 7). There was also a statistically significant reduction in cell proliferation in sunitinib alone treatment (p = 0.044), although the gemcitabine alone treatment did not have significant effect (p = 0.16). There was no significant difference in cell proliferation between sunitinib + chloroquine and Chemo + sunitinib and the Chemo + chloroquine treatments (p = 0.113). However, the triplet combination of Chemo + sunitinib + chloroquine showed the highest reduction of cell proliferation compared to any other single or double treatment groups (p < 0.01).

We performed TUNEL assays on these tissue samples to quantify the apoptosis related cell death. Both the Chemo + sunitinib + chloroquine and sunitinib + chloroquine treatment groups had high proportion of TUNEL positive cells in the pancreatic tissue, especially in the ductal epithelium (p < 0.01, Fig. 8). Although, the Chemo alone and Chemo + sunitinib group also showed significant apoptosis (p < 0.05), there was no significant increase in apoptosis in the sunitinib, or Chemo + chloroquine groups in this assay (p = 0.18 and 0.22 respectively). We further validated the cellular apoptosis by examining the levels of active Casp3, a well-known early indicator of apoptosis. Anti-active Casp3 IHC showed increased Casp3 activity in Chemo + CQ, Sun + CQ and Chemo + Sun + CQ in significantly increasing order (p < 0.05, Additional file 3: Figure S3). The triplet combination of Chemo + Sun + CQ showed significantly more proportion of active Casp3 positive PDAC cells compared to the either of the doublet treatments Chemo + Sun (p < 0.01), Chemo + CQ (p < 0.01) or Sun + CQ (p < 0.05).

In the KPCP1 orthotopic cohort, treatment with Chemo alone, sunitinib alone, or sunitinib + chloroquine did not cause any significant increase in survival compared to the control group, as determined by Kaplan–Meier curve analyses (Fig. 9a). The survival of mice undergoing combination therapy of Chemo + sunitinib, Chemo + chloroquine and Chemo + sunitinib + chloroquine showed a significant increase of survival (p < 0.05, Fig. 9a). These treatment groups also showed significantly increased survival in the Panc02 orthotopic cohort (p < 0.01, Additional file 3: Figure S3). These three treatment groups were able to survive an average 2 weeks longer compared to the control groups in the KPCP1 cohort (63 ± 3 days vs 48 ± 4 days). The reduction of pancreatic tumor growth was also evident from a significant reduction of pancreatic weight after 4-weeks of drug treatments in the dual treatments of Chemo + sunitinib (p < 0.05), Chemo + chloroquine (p < 0.01) and the triplet combination of Chemo + sunitinib + chloroquine (p < 0.01) as shown in Fig. 9b. Either of monotherapy with Chemo or sunitinib and the dual therapy of sunitinib + chloroquine did not result in any significant reduction of pancreatic mass (Fig. 9b).

The mean survival rate was much higher in the Panc02 cohort, with the Chemo + sunitinib + chloroquine group showing an increase in maximum survival of more than 68 days (90% increase) as compared to the control group (115 ± 26 days vs 47 ± 11 days, Additional file 4: Figure S4). The Chemo + chloroquine (99 ± 22 days), and Chemo + sunitinib group (87 ± 19 days) also survived significantly longer compared to the control or single treatment groups in the Panc02 model (p < 0.05, Additional file 4: Figure S4). While gemcitabine treatment alone did not increase survival in the KPCP1 cohort, as compared to the control group (52 ± 2 vs 48 ± 4 days), it was significantly effective in the Panc02 cohort (80 ± 26 days vs 47 ± 11 days, Additional file 4: Figure S4). The sunitinib + chloroquine combination did not significantly increase survival compared to either sham groups or sunitinib treatment group in either KPCP1 or Panc02 orthotopic model (p = 0.32 and 0.43 respectively). Thus, the results from the survival analysis suggest that Chemo + sunitinib + chloroquine treatment significantly increases survival of the mice developing PDAC derived from either KPCP1 or Panc02 cells. Furthermore, dual treatments of both Chemo + chloroquine and Chemo + sunitinib also offers therapeutic opportunities in increasing survival rate.

Our data shows that sunitinib alone was sufficient to suppress cell growth, but does not increase cell death, and the sunitinib therapy alone was not sufficient to increase overall survival. However, sunitinib presents an effective therapeutic intervention if co-administered with gemcitabine and chloroquine in the murine PDAC models. Taken together, these results suggest that targeting multiple pathways of ER stress and autophagy is well tolerated by animals and these drug combinations increase the response of PDAC to chemotherapy and increases animal survival.