Collagen-derived proline promotes pancreatic ductal adenocarcinoma cell survival under nutrient limited conditions

Abstract

Tissue architecture contributes to pancreatic ductal adenocarcinoma (PDAC) phenotypes. Cancer cells within PDAC form gland-like structures embedded in a collagen-rich meshwork where nutrients and oxygen are scarce. Altered metabolism is needed for tumour cells to survive in this environment, but the metabolic modifications that allow PDAC cells to endure these conditions are incompletely understood. Here we demonstrate that collagen serves as a proline reservoir for PDAC cells to use as a nutrient source when other fuels are limited. We show PDAC cells are able to take up collagen fragments, which can promote PDAC cell survival under nutrient limited conditions, and that collagen-derived proline contributes to PDAC cell metabolism. Finally, we show that proline oxidase (PRODH1) is required for PDAC cell proliferation in vitro and in vivo. Collectively, our results indicate that PDAC extracellular matrix represents a nutrient reservoir for tumour cells highlighting the metabolic flexibility of this cancer.

Figure 1. Collagen types I and IV and their associated collagenases are abundant in the stromal compartment of PDAC.

(a) Collagen by Masson’s trichrome staining and (b) immunohistochemistry (IHC) of collagen proteins type I and IV in human and mouse tumour sections. Representative images and their relative insets from n=4 patients and n=3 Pdx1-Cre;KrasG12D;Ink4a−/− (PKI) mice are illustrated. The percentage of total collagen and collagen types I and IV staining intensity calculated relative to total tumour area, expressed as mean±s.e.m., is indicated. Scale bar, 100 μm. (c) mRNA levels of the collagen specific MMP 13, 9 and 2 and prolidase (Pepd gene) measured by quantitative RT–PCR in PDAC from PKI mice (n=3) versus control pancreases from KrasG12D;Ink4a−/− (KI) mice (n=3). Data are mean±s.e.m. *P<0.05, **P<0.01, ***P<0.001; two-tailed unpaired Student’s t-test. (d) Prolidase localization in human and PKI mice tumours revealed by IHC (left) or immunofluorescence (IF, right), respectively. Representative images from n=4 patients and from n= 3 PKI mice. Scale bar, 100 μm. (e) Prolidase levels in supernatant from PK4A cells cultured during 24 or 48 h with coated collagen I in complete media. Total loaded protein was determined by Ponceau red staining (Std: standard). Marker sizes are indicated in kDa. Photos are representative of n=3 independent experiments. Uncropped images of blots are shown in Supplementary Fig. 10. MMP, matrix metalloproteases; RT–PCR, PCR with reverse transcription.

Figure 2. PDAC expresses proline metabolic enzymes.

(a) Free proline is metabolized in the mitochondrial matrix, first by proline oxidase (PRODH1), to generate 1-pyrroline-5-carboxylic acid (P5C) before conversion into glutamic-γ-semialdehyde (GSA). P5C dehydrogenase (P5CDH) metabolizes GSA into glutamate and NADH. Free proline is synthesized from P5C via pyrroline carboxylases 1 and 2 (PYCR1, 2) in the mitochondrion and PYCR3 in the cytosol. Proline incorporated into collagen (proline-Col) can be modified into hydroxyproline (OH-proline) by prolyl-4-Hydroxylases (P4H). (b,c) Localization of PRODH1 and P4HA3 in human and PKI mice PDAC by IHC (b) and in epithelial compartment by IF co-staining with the wide-spectrum cytokeratin (Ws KRT) epithelial marker in PKI mice (c). Representative images from n=4 patients (b) and n=3 PKI mice (b,c). Tumour glands and disseminated cells are indicated by asterisks and arrows, respectively. Scale bar, 100 μm. (d) Quantification of PRODH1 and P4HA3 protein levels in PKI mouse PDAC and in KI mouse control pancreas revealed by western blot analysis (upper panel, with marker size indicated in kDa). Protein levels in each tissue samples (histograms, lower panel, from n=3 control KI mice and n=3 PDAC PKI mice) are normalized to total loaded-proteins (Amido black staining). Data expressed as mean±s.e.m. (***P<0.001; two-tailed unpaired Student’s t-test). Uncropped images of blots are shown in Supplementary Fig. 10. (e) Quantification of PRODH1 protein levels in normal human pancreas (n=11) and human PDAC samples (n=10). PRODH1 levels are normalized to one normal pancreas used as a reference. Data are illustrated as mean±95% CI, *** P<0.001; Mann–Whitney test. Images of human PDAC and normal pancreas macroarray are displayed in Supplementary Fig. 1. CI, confidence interval.

Figure 3. Nutrient stress induces PDAC cells to take up collagen through macropinocytosis-dependent and independent mechanisms.

FACS analysis of collagen I or collagen type IV uptake by PK4A cells (a and b (upper) respectively). Cells were cultured 48 h in normoxia (21% oxygen (O₂)) in complete (25 mM glucose, 4 mM glutamine), glucose-free or glutamine-free media with or without DQ-fluorescein-conjugated collagen I or IV (Col I or IV DQ). For each culture condition, either vehicle (DMSO) or 30 μM EIPA was added. Light grey peaks represent PKA cells not exposed to collagen I or IV DQ and dark grey peaks shifted to the right side reveal cells taking up collagen I or IV DQ. The percentage of positive cells in the dark grey shifted distributions, delimited by vertical bar, was indicated as mean±s.e.m. and represented as bar graph in charts below (lower a and b panels respectively). (c) Col I or (d) Col IV DQ MFI relative to each culture conditions during 48 h in normoxia. Data represent mean±s.e.m. (n=6 independent experiments). P values, indicated by asterisks, show statistical significance relative to respective culture media without EIPA, while letters indicate statistical significance relative to complete media without EIPA. (^cP<0.05, ^bP<0.01, ^aP<0.001; two-tailed unpaired Student's t-test). (e,f) Comparison of the percentage (left panels) and MFI (right panels) between normoxic and hypoxic (1% oxygen (O₂)) PK4A cells taking up Col I (e) or Col IV (f) DQ in each 48 h culture conditions. Data represent mean±s.e.m. (normoxia n=6 independent experiments; hypoxia n=3 independent experiments). *P<0.05, **P<0.01, two-tailed unpaired Student’s t-test. (g,h) Representative co-staining of Col IV DQ and Lamp1 lysosomal marker in PK4A cells cultured in glucose-free (g) or glutamine-free (h) media under normoxic conditions with or without EIPA. Insets show magnification of Col IV, Lamp1 or Col IV/Lamp1 (merge) PKA4 cells in each condition. Images are representative of n=3 independent experiments. Scale bar: 50 μm. FACS, fluorescence-activated cell sorting. MFI, mean fluorescence intensity.

Figure 4. Collagen uptake promotes survival and proline promotes both survival and proliferation.

(a) PK4A cell survival in indicated media supplemented with vehicle or water-soluble collagen IV alone or in combination with EIPA treatment (upper). After treatments, cells were submitted to crystal violet assay. (upper) Representative crystal violet images (n=3 independent experiments). (middle) Crystal violet intensity, expressed as mean±s.e.m. (n=3 independent experiments) is presented as fold-induction relative to intensity in glucose-free condition without EIPA and without col IV. ND: Value below detection level. **P<0.01, two-tailed unpaired Student’s t-test. (lower) Glutamine concentration in media of PK4A cells cultured in glucose-free medium supplemented or not with water-soluble collagen IV over 72 h. Data represent mean±s.e.m. (n=3 independent experiments). Two-way ANOVA followed by Bonferroni post-hoc test revealed no significant difference in PK4A glutamine uptake between the two culture conditions. (b) ERK1/2 and p70S6K phosphorylation status in PK4A cells cultured with or without water-soluble collagen IV in indicated media during 72 h. (c) Prolidase levels in supernatant from PK4A cells cultured with coated collagen I in indicated media. Total protein loading was determined by Ponceau red staining. (b,c) marker sizes are indicated in kDa. Photos are representative of n=2 (b) and n=3 (c) independent experiments. Uncropped images of blots are shown in Supplementary Fig. 10. (d) Survival of PK4A cells cultured under normoxic (21% O₂) or hypoxic (1% O₂) conditions (upper and lower charts, respectively) in indicated nutrient conditions, in media alone or supplemented with either DHP (1 mM) or with DHP and L-proline (500 μM). Living cells, determined 24 h after treatment, are expressed as percentage of untreated cells in complete media (mean±s.e.m., n=4 independent experiments). *P<0.05, **P<0.01, ***P<0.001; two-tailed unpaired Student's t-test. (e) Proliferation of PK4A cells cultured for 24–96 h under normoxia as in d. Data are mean±s.e.m., n=4 independent experiments. P value, is relative to corresponding untreated value at each time point.*P<0.05, **P<0.01, ***P<0.001; two-way ANOVA followed by Bonferroni post-hoc test. ANOVA, analysis of variance.

Figure 5. Proline catabolism diminishes glycolytic flux under glutamine deprivation.

(a) Glutamine and (b) glucose (upper) and lactate (lower) concentration in supernatant of subconfluent PK4A cells cultured for 8–24 h under gradual decrease of glutamine concentration (4, 2, 0.5 and 0 mM). Data are mean±s.e.m., n=3 independent experiments. P value, indicated by green and dark asterisks in b, is relative to glucose or lactate value in 4 mM glutamine at each time point. **P<0.01, ***P<0.001; two-way ANOVA followed by Bonferroni post-hoc test. (c) Fractional labelling (M3) of lactate from U-¹³C-glucose at 1, 3, 6 and 24 h under various glutamine concentrations (4 mM, 2 mM, 0.5 mM). All data were presented as mean±s.e.m. (n=3 independent experiments). (d) Glucose consumption and lactate production by PK4A cells cultured during 24 h in 0.5 mM glutamine media alone, or supplemented with DHP (1 mM) or with DHP and L-proline (500 μM). Data are presented as fold induction (mean±s.e.m., n=3 independent experiments) relative to untreated media values. *P<0.05, ***P<0.001; two-tailed unpaired Student’s t-test. ANOVA, analysis of variance.

Figure 6. Proline supports TCA metabolism under nutrient limited conditions.

(a) Schematic representation of the protocol used to create extracellular matrix (ECM) containing U-¹³C-proline. MEFs: mouse embryonic fibroblasts. (b) GCMS analysis of acid hydrolysis of de-cellularized ECM proteins from experiment outline in part a. Data is from n=1 experiment and is representative of n=2 experiments. (c) Fractional labelling of free U-¹³C-proline pools inside PK4A cells after 24 h on labelled ECM under indicated nutrient conditions. (d) U-¹³C-proline tracing by GCMS analysis into TCA intermediates in PK4A cells for 24 h under indicated nutrient conditions. M0 represents the unlabelled molecule with each increasing number representing another labelled carbon. Empty circles represent unlabelled ¹²C-species, while filled circles represent ¹³C-labelled carbons. For c and d data are mean±s.e.m. of n=3 biological replicates and representative of at least n=2 independent experiments. (e) Fractional labelling of proline from U-¹³C-proline after 24 h at the indicated conditions at normoxia (21% O₂) and hypoxia (1% O₂) (upper panels). All data are presented as mean±s.e.m. n=3 per condition. Complete mass isotopomer distribution (MID) of citrate from U-¹³C-proline after 24 h at the indicated conditions at normoxia (21% O₂) and hypoxia (1% O₂) (lower panel). All data are presented as mean±s.e.m. n=3 per condition. GCMS, gas chromatography-mass spectrometry.

Figure 7. DHP limits production of proline-derived glutamate and mitochondrial respiration.

(a) Fractional labelling of proline and glutamate from U-¹³C-proline after 24 h at 5 mM glucose (upper charts) or 0.5 mM glutamine (lower charts) media alone or supplemented with DHP (1 mM). Ratio of M5 glutamate (Glu) to M5 proline (Pro) at 5 mM glucose or 0.5 mM glutamine are presented next to labelled fractions. All data are presented as mean±s.e.m. n=3 per condition. ***P<0.001; two-tailed unpaired Student’s t-test. (b) Representative oxygen consumption rate (OCR) profile of PK4A cells cultured in 5 mM glucose media alone, or supplemented with DHP (1 mM) or with DHP and L-proline (500 μM) (upper panel). Sequential injections of 1 μM oligomycin, 1 μM FCCP and 0.5 μM rotenone/antimycin A mix are indicated. Maximal respiration was determined under indicated limited-nutrient conditions (lower panel). Data are mean±s.e.m., n=3 independent experiments. *P<0.05, ***P<0.001; two-tailed unpaired Student’s t-test.

Figure 8. PRODH1 inhibition limits pancreatic cancer cell resistance to nutrient limitation and impedes pancreatic tumour growth.

(a) Representative clonogenic assay and quantification of sg-Control and sg-PRODH1_clone9 and _clone3 PK4A cells colony-forming area in 5 and 1 mM glucose media. Data are mean±s.e.m. (n=3 independent experiments) and are expressed in percentage of 5 mM glucose sg-Control value. **P<0.01, ***P<0.001; two-tailed unpaired Student's t-test. Scale bar: 5 mm. (b) Tumour regression measured in sg-PRODH1_clone9 and _clone3 mice as compared to sg-Control one. Mice were subcutaneously implanted with sg-Control or sg-PRODH1_clone9 or _clone3 PK4A cells and caliper measurement of tumour size was performed over 15 post-implantation days. Data are mean±s.e.m. (n=10 mice per group). P value, indicated by green and blue asterisks, is relative to sg-Control tumour volume after 15 days post-implantation. **P<0.01, *** P< 0.001; two-way ANOVA followed by Bonferroni post-hoc test. (c) Ki67 IHC staining in sg-Control, sg-PRODH1_clone9 or _clone3 subcutaneous pancreatic tumour sections. Representative images from n=4 mice per group are illustrated. Scale bar: 100 μm. Chart represents quantification of Ki67 stained area/ quantified tumor area (n=7 fields quantified per tumour). *P<0.05, **P<0.01; two-tailed unpaired Student’s t-test. (d) Schematic summary. Upon nutrient limitation PDAC cells degrade collagen-rich PDAC extracellular matrix proteins and take up free proline through membrane transporters (SLC) as well as collagen fragments through macropinocytosis-dependent and independent processes. Internalized vesicles fuse with lysosome and collagen I and IV (ColI and ColIV) fragments undergo further degradation towards free amino acids. Collagen-derived proline (Pro) is further metabolized by the tricarboxylic acid (TCA) cycle to promote PDAC cell survival. MMPs: Matrix metalloproteases, Glu: Glutamate, α-KG: α-ketoglutarate, Asp: Aspartate. Mito: Mitochondria. ANOVA, analysis of variance.