RABL6A Promotes Oxaliplatin Resistance in Tumor Cells and Is a New Marker of Survival for Resected Pancreatic Ductal Adenocarcinoma Patients

Abstract

Pancreatic ductal adenocarcinoma (PDAC) is characterized by early recurrence following pancreatectomy, rapid progression, and chemoresistance. Novel prognostic and predictive biomarkers are urgently needed to both stratify patients for clinical trials and select patients for adjuvant therapy regimens. This study sought to determine the biological significance of RABL6A (RAB, member RAS oncogene family-like protein 6 isoform A), a novel pancreatic protein, in PDAC. Analyses of RABL6A protein expression in PDAC specimens from 73 patients who underwent pancreatic resection showed that RABL6A levels are altered in 74% of tumors relative to adjacent benign ductal epithelium. Undetectable RABL6A expression, found in 7% (5/73) of patients, correlated with improved overall survival (range 41 to 118 months with 3/5 patients still living), while patients with RABL6A expression had a worse outcome (range 3.3 to 100 months, median survival 20.3 months) (P = 0.0134). In agreement with those findings, RABL6A expression was increased in pancreatic cancer cell lines compared to normal pancreatic epithelial cells, and its knockdown inhibited pancreatic cancer cell proliferation and induced apoptosis. Moreover, RABL6A depletion selectively sensitized cells to oxaliplatin-induced arrest and death. This work reveals that RABL6A promotes the proliferation, survival, and oxaliplatin resistance of PDAC cells, whereas its loss is associated with extended survival in patients with resected PDAC. Such data suggest RABL6A is a novel biomarker of PDAC and potential target for anticancer therapy.

Keywords: ARF; RABL6A; chemoresistance; oxaliplatin; pancreatic ductal adenocarcinoma.

Figure 1.

RABL6A is a pancreatic protein with elevated levels in PDAC cell lines. (A) Western blot of C57Bl/6 mouse tissues (100 µg protein per lane) show high RABL6A expression in the normal pancreas. Relative expression of RABL6A in each tissue was quantified and normalized to γ-tubulin levels. (B) Western blots of RABL6A and γ-tubulin (loading control) show elevated RABL6A levels in PDAC cells (Panc-1, MIA PaCa-2 [MP2], BxPC-3, and CAPAN-1) compared to immortalized human pancreatic epithelial (HPNE) cells. RABL6A levels in cells relative to HPNE cells were quantified and denoted for each lane. (C) qRT-PCR analysis of RABL6 isoforms A-D in Panc-1 cells, normalized to GAPDH levels, show RABL6A mRNA is the most abundant form.

Figure 2.

RABL6A expression is altered in PDAC tumors and its loss is associated with increased survival in resected PDAC patients. (A) Representative images of IHC stains for RABL6A expression in human resected PDAC tumor specimens. RABL6A staining was scored relative to matched (adjacent) non-neoplastic ducts present in the same specimen. RABL6A levels were scored as −2 (undetectable), −1 (modestly decreased), 0 (equivalent), +1 (modestly increased), and +2 (markedly increased) relative to surrounding benign ducts. Patient specimen stains representative of −2 and +2 RALB6A expression are shown. T = tumor (red arrow), N = normal (black arrow) tissue. (B) Graphical quantification of data presented in A from stains of 73 patient specimens. Seventy-four percent of tumors displayed altered RABL6A expression. (C) Kaplan–Meier plot of overall survival in patients who underwent resective surgery. Survival analysis shows prolonged survival in the group where RABL6A tumor expression is absent ((−) RABL6A) compared to patients who express RABL6A in their tumors ((+) RABL6A). P = 0.0134; hazard ratio = 6.16; 95% confidence interval = 1.46-26.01.

Figure 3.

RABL6A loss promotes cell death through apoptosis. (A) Top, schematic of the 4 known RABL6 isoforms A-D and regions recognized by KD1 and KD2 shRNAs. Bottom, qRT-PCR analysis of RABL6 mRNA levels (A-D) in Panc-1 cells expressing control (CON), KD1, or KD2 shRNAs. KD1 silences all RABL6 isoforms while KD2 is specific for RABL6A. *, P < 0.05 for comparison of each isoform in KD cells to CON cells. (B) Western blot showing effective RABL6A depletion, increased cleaved caspase-3 (CC3) and reduced ERK1/2 phosphorylation (p-ERK1/2) in Panc-1 cells expressing KD1 and KD2 shRNAs compared to CON cells. GAPDH levels served as loading control. (C) Decreased viability of RABL6A depleted Panc-1 cells compared to CON cells. The mean and standard deviation of data from 3 independent experiments is shown (*, P < 0.05 for comparison to CON). (D) Representative cell cycle distributions of Panc-1 cells expressing CON, KD1, or KD2 shRNAs 4 days after transduction.

Figure 4.

Effects of sustained RABL6A depletion on PDAC cell proliferation. (Left panel) Panc-1 cells; (Middle panel) MiaPaCa-2 [MP-2] cells; (Right panel) BxPC-3 cells. The indicated PDAC cells expressing CON, KD1, or KD2 shRNAs were cultured for 8 weeks in puromycin to maintain shRNA expression and then examined by western blotting (top) and growth curve analyses (bottom). Western blots show effective maintenance of RABL6A depletion. Levels of γ-tubulin or GAPDH were used as loading control. Growth curves show KD1 cells proliferate more slowly than KD2 and CON cells in all lines assayed while only MP-2 KD2 cells proliferate more slowly than controls (*P < 0.0001). Representative data from 3 or more independently derived sets of stable lines for each cell type are shown.

Figure 5.

Sustained RABL6A depletion sensitizes Panc-1 cells to oxaliplatin-induced cell cycle arrest and apoptosis. Established Panc-1 CON, KD1, and KD2 cells were exposed to the indicated concentrations of oxaliplatin for 4 days and examined for cell number (A) and viability (B). Graphs are representative of several experiments with data normalized to values for untreated cells within each group. *, P < 0.001 for KD1 and KD2; **, P < 0.001 for KD1 only. (C) Western blot detection of RABL6A, cleaved caspase-3, phosphorylated ERK1/2 (p-ERK1/2), total ERK1/2, and GAPDH (loading control) in Panc-1 CON, KD1, and KD2 cells exposed for 4 days to 3 µg/mL oxaliplatin or left untreated (−). (D) Representative cell cycle distributions of Panc-1 CON, KD1, and KD2 cells treated with 3 µg/mL oxaliplatin for 4 days. The 4N/2N ratio of cells (averaged from 3 independent experiments) for each population is displayed, revealing heightened mitotic arrest in oxaliplatin-treated RABL6A knockdown cells.