Translational study reveals a two-faced role of RBM3 in pancreatic cancer and suggests its potential value as a biomarker for improved patient stratification

Emelie Karnevi, Liv Ben Dror, Adil Mardinoglu, Jacob Elebro, Margareta Heby, Sven-Erik Olofsson, Björn Nodin, Jakob Eberhard, William Gallagher, Mathias Uhlén, Karin Jirström, Emelie Karnevi, Liv Ben Dror, Adil Mardinoglu, Jacob Elebro, Margareta Heby, Sven-Erik Olofsson, Björn Nodin, Jakob Eberhard, William Gallagher, Mathias Uhlén, Karin Jirström

Abstract

Periampullary adenocarcinoma, including pancreatic cancer, is a heterogeneous group of tumors with dismal prognosis, partially due to lack of reliable targetable and predictive biomarkers. RNA-binding motif protein 3 (RBM3) has previously been shown to be an independent prognostic and predictive biomarker in several types of cancer. Herein, we examined the prognostic value of RBM3 in periampullary adenocarcinoma, as well as the effects following RBM3 suppression in pancreatic cancer cells in vitro. RBM3 mRNA levels were examined in 176 pancreatic cancer patients from The Cancer Genome Atlas. Immunohistochemical expression of RBM3 was analyzed in tissue microarrays with primary tumors and paired lymph node metastases from 175 consecutive patients with resected periampullary adenocarcinoma. Pancreatic cancer cells were transfected with anti-RBM3 siRNA in vitro and the influence on cell viability following chemotherapy, transwell migration and invasion was assessed. The results demonstrated that high mRNA-levels of RBM3 were significantly associated with a reduced overall survival (p = 0.026). RBM3 protein expression was significantly higher in lymph node metastases than in primary tumors (p = 0.005). High RBM3 protein expression was an independent predictive factor for the effect of adjuvant chemotherapy and an independent negative prognostic factor in untreated patients (p for interaction = 0.003). After siRNA suppression of RBM3 in vitro, pancreatic cancer cells displayed reduced migration and invasion compared to control, as well as a significantly increased resistance to chemotherapy. In conclusion, the strong indication of a positive response predictive effect of RBM3 expression in pancreatic cancer may be highly relevant in the clinical setting and merits further validation.

Keywords: RBM3; pancreatic cancer; periampullary cancer; prediction; prognosis.

Conflict of interest statement

CONFLICTS OF INTEREST KJ and JEB are inventors on a patent related to RBM3 as a predictor of chemotherapy response: ”Prediction of response to platinum-based therapy: European Patent no. (EP)2396660 (validated in Denmark, France, Ireland, The Netherlands, Switzerland, Great Britain, Sweden and Germany); Australia Patent no. (AU)2010212772; US Patent no. (US)8747910; Chinese Patent no. ZL201080007960.0; Hong Kong Patent no. (HK)1166371; Japanese Patent no. (JP)5767116 and Brazilian Patent Application no. PI1008875–0.

Figures

Figure 1. RBM3 expression in primary tumors…
Figure 1. RBM3 expression in primary tumors and lymph node metastases
Immunohistochemical images of (A) negative RBM3 expression, (B) intermediate RBM3 expression and (C) strong RBM3 expression. A) Negative staining in normal pancreatic tissue and paired intestinal type primary tumour and metastasis. B-C) Pictures represent three paired primary tumors and metastasis from intestinal type tumors (B) and pancreatobiliary-type tumors (C). Box plots visualizing the distribution of RBM3 expression in (D) the entire cohort, (E) intestinal type tumors and (F) pancreatobiliary-type tumors. The images were taken at 20X magnification using cellSens dimension software. Scale bar represents 20 μm.
Figure 2. Prognostic value of RBM3 expression
Figure 2. Prognostic value of RBM3 expression
Kaplan-Meier analysis of 5-year overall survival in relation to (A) RBM3 mRNA expression in pancreatic cancer patients from the TCGA, (B) RBM3 protein expression in the periampullary cohort using the median cutoff value of the nuclear score, and (C) overall survival in strata according to RBM3 expression and any adjuvant chemotherapy, with low RBM3 expression/no adjuvant treatment as reference. Log rank p-value for high RBM3 expression /adjuvant treatment compared to high RBM3 expression/ no adjuvant treatment p = 0.035, and log rank p-value for high RBM3 expression /adjuvant treatment compared to low RBM3 expression /adjuvant treatment p = 0.070.
Figure 3. RBM3 mRNA and protein expression…
Figure 3. RBM3 mRNA and protein expression after transfection
(A) Representative images of RBM3 protein expression in pancreatic cancer cell lines BxPC-3, PANC-1 and MIAPaCa-2 after transfection with siRNA against RBM3 or negative control (CTRL). (B) RBM3 relative mRNA expression levels in cancer cells after transfection with siRNA against RBM3 or siRNA negative control. (C) Representative images of COX-2 protein expression following transfection, as well as COX-2 and IL-8 relative mRNA expression levels in cancer cells after RBM3 suppression. Images and graphs represent one of at least three independent experiments. Representative images have been taken at 20X magnification with cellSens dimension software. Scale bar represents 20 μm. N.D. = not detected. ***p < 0.001
Figure 4. Influence by RBM3 suppression on…
Figure 4. Influence by RBM3 suppression on cancer cell behavior
(A) Representative images of transwell migration with BxPC-3 or PANC-1 cells after transfection with siRNA against RBM3 or control. (B) Representative images of organotypic gel sections stained with hematoxylin and eosin after 7 days incubation. All images were taken at 10X (A) or 20X (B) magnification using cellSens dimension software. Scale bar represents 20 (B) or 50 (A) μm. (C) Graphs represent cell viability after incubation with gemcitabine, oxaliplatin and 5-FU of MIAPaCa-2 cells with or without suppressed RBM3, relative to control (no treatment). Grey lines represent cells transfected with negative control siRNA, and black lines the RBM3 suppressed cells. Significant differences between control and siRNA were analyzed with non-linear regression.

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Source: PubMed

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