Nutrient scavenging-fueled growth in pancreatic cancer depends on caveolae-mediated endocytosis under nutrient-deprived conditions

Abstract

Pancreatic ductal adenocarcinoma (PDAC) is characterized by its nutrient-scavenging ability, crucial for tumor progression. Here, we investigated the roles of caveolae-mediated endocytosis (CME) in PDAC progression. Analysis of patient data across diverse datasets revealed a strong association of high caveolin-1 (Cav-1) expression with higher histologic grade, the most aggressive PDAC molecular subtypes, and worse clinical outcomes. Cav-1 loss markedly promoted longer overall and tumor-free survival in a genetically engineered mouse model. Cav-1-deficient tumor cell lines exhibited significantly reduced proliferation, particularly under low nutrient conditions. Supplementing cells with albumin rescued the growth of Cav-1-proficient PDAC cells, but not in Cav-1-deficient PDAC cells under low glutamine conditions. In addition, Cav-1 depletion led to significant metabolic defects, including decreased glycolytic and mitochondrial metabolism, and downstream protein translation signaling pathways. These findings highlight the crucial role of Cav-1 and CME in fueling pancreatic tumorigenesis, sustaining tumor growth, and promoting survival through nutrient scavenging.

Fig. 1.. CAV1 expression is associated with aggressive molecular subtypes and shorter survival in patients with resectable and metastatic PDAC.

(A) Boxplots comparing CAV1 expression levels between molecular subtype groupings for the Moffitt, Karasinska, Collisson, and Bailey subtyping methods (left to right) in each of the five patient cohorts (top to bottom). Box plots indicate the median (central line), 25 to 75% interquartile range (IQR; bounds of box), and whiskers extend from box bounds to the largest value no further than 1.5 times the IQR. Two-tailed Wilcoxon mean rank sum P values are shown. (B) Kaplan-Meier plots showing overall survival (OS) between groups stratified by CAV1 expression levels. Hazard ratio (HR), 95% confidence interval (CI), and log-rank P value are shown and represent the comparison between low and high CAV1 groups. Tables showing the number at risk are included below.

Fig. 2.. Cav-1 expression in Human PDAC TMA.

(A) Representative immunohistochemistry (IHC) images of Cav-1 protein expression in normal pancreatic epithelium, pancreatic intraepithelial neoplasia (PanIN), moderately differentiated (Mod Diff), and poorly differentiated (Poorly Diff) pancreatic cancer tissue (original magnification, ×200) from samples in the OSU TMA. (B) Mean Cav-1 H-score correlated with tumor grade. (C) The BioMax TMA correlated with Cav-1 expression via IHC. (D and E) OS and distant metastasis–free survival (DMFS) in the patients with resected PDAC. The numbers below figure represent the number at risk for each time point. (F) Circulating Cav-1 in pancreatic cancer patients with metastasis (MP) compared to healthy volunteers (HV). (G) Cav-1 expression (H-score) compared between primary resected pancreatic tumors (RP) compared to sites of metastasis (MP) in those patients with developed metastatic disease and had metastatic tumors that were biopsied. *P < 0.05.

Fig. 3.. CAV1 deletion reduces tumorigenesis in the KPC mouse model.

(A) The generation of the KrasLSL-G12D;p53LSL-R270H/þ;Pdx1-cre; CAV1flox/flox (KPC-CAV1^fl/fl) mice (top). Representative IHC for Cav-1 protein in the KPC and KPC-CAV1^fl/fl mice (bottom). (B) Southern blotting CAV1neo allele in tail DNA of F1 offspring (top left); Southern blotting for CAV1^flox allele in tail DNA of mice after deletion of neo cassette (top right); genotyping of tail DNA from KPC, KPC-CAV1^fl/+, and KPC-CAV1^fl/fl mice (bottom left); genotyping of tail DNA from KPC and KPC-CAV1^fl/fl mice or genotyping of pancreatic tumor DNA from KPC-CAV1^fl/+ or KPC-CAV1^fl/fl mice (bottom right). (C) Kaplan-Meier analysis of OS of KPC, KPC-CAV1^fl/+, KPC-CAV1^fl/fl, and CAV1^fl/fl;Pdx1-cre mice. Numbers of animals per group are indicated. (D) Kaplan-Meier analysis of tumor-free survival of KPC, KPC-CAV1^fl/+, KPC-CAV1^fl/fl, and CAV1^fl/fl; Pdx1-cre mice. (E) The tumor-to-body weight ratio (%) was calculated for each of the four groups of mice. *P < 0.0001. (F) Tumor incidence in the pancreata of mice in the KPC mice compared to either the KPC-CAV1^fl/+ or KPC-CAV1^fl/fl mice. *P < 0.05; ns, not significant.

Fig. 4.. Loss of Cav-1 renders cells with decreased proliferative capabilities in serum-deprived conditions.

(A) Western blot analysis of Cav-1 expression in cell lysates from TDCLs from the KPC and KPC-CAV1^fl/fl tumors. Numbers refer to individual cell lines from different tumors. (B and C) Western blot analysis of (B) MiaPaca-2 (MP2) and (C) PANC-1 cells transfected with scrambled short hairpin RNA (shCtrl) or shRNA targeting CAV1 (shCAV1). (D to F) Proliferation assays measuring the change in confluence over time in regular media supplemented with 10% fetal bovine serum (FBS) in (D) KPC versus KPC-CAV1^fl/fl, (E) MP2 shCtrl versus shCAV1, and (F) PANC-1 shCtrl versus shCAV1 cells. (G to I) Proliferation assays measuring the change in confluence over time in regular media supplemented with 1% FBS in (G) KPC versus KPC-CAV1^fl/fl, (H) MP2 shCtrl versus shCAV1, and (I) PANC-1 shCtrl versus shCAV1 cells. (J to L) Cells were plated as single cells and incubated in either 10 or 1% FBS-supplemented media and colonies were assessed (>50 cells) after 2 weeks in (J) KPC versus KPC-CAV1^fl/fl, (K) MP2 shCtrl versus shCAV1, and (L) PANC-1 shCtrl versus shCAV1 cells. *P < 0.05.

Fig. 5.. Cav-1 regulates metabolic capacity in PDAC cells.

(A) The glycolysis stress test was performed in the Seahorse XFe96 Analyzer on KPC #1 and #4 as well as KPC-CAV1^fl/fl #2 and #3 TDCLs. The extracellular acidification rate (ECAR) over time was plotted after treatment at set time points of glucose (10 mM), oligomycin (1 μg/ml), and 2-DG (at 50 mM, inhibition of glycolysis). The maximal glycolytic capacity was significantly higher in KPC versus KPC-CAV1^fl/fl cells. (B) The glycolysis stress test was performed in MP2 shCtrl and shCAV1 cells as described above. The maximal glycolytic capacity was significantly higher in MP2 shCtrl versus shCAV1 cells. (C) The mitochondrial stress test was performed in the Seahorse XFe96 Analyzer KPC #1 and #4 TDCL and KPC-CAV1^fl/fl #2 and #3 TDCL following either overnight seeding in 10% FBS (left) or 1% FBS (right). The oxygen consumption rate (OCR) over time was plotted after injection of oligomycin (0.5 μM), carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) (1 μM), and rotenone/antimycin A (AA/Rot) (1 μM). Each data point represents an OCR measurement. (D) The mitochondrial stress test in MP2 shCtrl and shCAV1 cells following either overnight seeding in 10% FBS (left) or 1% FBS (right). (E) Basal consumption, adenosine 5′-triphosphate (ATP) production, and maximal respiration in 10% and 1% FBS media in the KPC cells compared to KPC-CAV1^fl/fl cells. (F) Basal consumption, ATP production, and maximal respiration in both 10 and 1% FBS conditions in MP2 shCtrl and shCAV1 cells. (G) Liquid chromatography–tandem mass spectrometry chromatograms of TCA cycle intermediates extracted from KPC and KPC-CAV1^fl/fl TDLC TDCLs after overnight incubation in 1% FBS. Data are expressed as means ± SEM, n = 2 independent experiments. (H) Diagram of the TCA cycle displaying the conversion of glutamine into alpha-ketoglutarate. *P < 0.05.

Fig. 6.. Cav-1 regulates growth under low glutamine environments and albumin uptake.

(A to D) KPC #4 TDCL, KPC-CAV1^fl/fl #2 TDCL, MP2 shCtrl, and MP2 shCAV1 cells were grown in glutamine dilutions (0 to 4 mM) and proliferation was assessed by IncuCyte confluence measurements every 4 hours. (E to H) Cells were grown in the glutamine concentration just below the glutamine threshold of each individual cell line (KPC #4 = 0.2 mM, KPC-CAV1^fl/fl = 0.8 mM, MP2 shCtrl = 0.4 mM, and MP2 shCAV1 = 1.0 mM) with or without supplementation with either mouse serum albumin (KPC) or human serum albumin (MP2) at 3% per volume and proliferation capacity measured by the IncuCyte. (I) Representative immunofluorescence images of 10 pancreatic cell lines after treatment with albumin for 30 min showing expression of Cav-1 (red), albumin (green), and 4′,6-diamidino-2-phenylindole (DAPI) (blue). Experiments were performed in triplicate and repeated three times with similar results. (J) The Cav-1 index was plotted against the albumin indexes to determine correlations.

Fig. 7.. Cav-1 regulates the activation of ribosomal S6 under nutrient stress.

(A) Mean comparisons of RPPA data for KPC versus KPC-CAV1^fl/fl cells. Differential protein abundance was used using RPPA measures through limma to compare the two cell lines. P values are from a two-sided, unpaired t test comparing the mean of KPC cells versus KPC-CAV1^fl/fl cells. Error bars denote SEM. (B) Representative IHC images of S6 (p235/236) protein expression KPC and KPC-CAV1^fl/fl tumors (n = 3 each). (C) Immunoblots showing levels of phosphorylated S6 (Ser^235/236), total S6, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in KPC/ KPC-CAV1^fl/fl, MP2, and PANC-1 shCtrl/shCAV1 cells. (D) Immunoblots displaying levels of phosphorylated S6 (234/235), total S6, Cav-1, and GAPDH in KPC and PANC-1shCtrl cells in both 10 and 1% FBS ± LY-2584702 (5 μM, 2 hours). (E) Proliferation assays in the KPC #4 or PANC-1 shCtrl cells in 10 or 1% FBS ± 5 μM of LY-2584702 (**P < 0.01). (F) PANC-1 shCtrl and shCAV1 cells transfected with S6K mutant plasmids (S6K1 Mut or S6K2 Mut) for 48 hours followed by media exchange with 1% FBS overnight. Levels of phospho-S6 (S235/236), total S6, Cav-1, and GAPDH are shown. (G) Proliferation assays in the PANC-1 shCAV1 cells transfected with the S6K plasmids (S6K1 WT, S6K1 Mut, or S6K2 Mut) following 48-hour transfection with the S6K plasmids in either 10 or 1% FBS (*P < 0.05). (H) Immunoblots showing higher albumin uptake in PANC-1 shCtrl cells compared to shCAV1 cells, with induction of phospho-S6 activation at 1 hour. (I) Immunoblots indicating S6 activation in KPC cells compared to KPC-CAV1^fl/fl cells. *P < 0.05 and **P < 0.01.