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Abstract

BACKGROUND:

Pancreatic ductal adenocarcinoma (PDA) is one of the major human health challenges with minimal therapeutic benefits due to its late detection, and de novo – and acquired chemotherapy resistance.

OBJECTIVE:

In this work we unravel the potential pro-survival role of RAB25 in pancreatic cancer chemotherapy resistance and aim to identify if RAB25 is a prognostic marker of patients’ survival in PDA.

METHODS:

We used RNA sequencing, shRNA mediated gene knockdown, BioGRID open repository of CRISPR screens (ORCS), GEPIA, kmplot.com, and cBioPortal.org databases to identify the role of RAB25 in PDA cell proliferation, chemotherapy response, expression in tumour versus normal tissues, and overall patients’ survival.

RESULTS:

RNA sequencing show Rab25 to be one of the top upregulated genes in gemcitabine resistance mouse PDA cells. Knockdown of Rab25 in these cells enhanced gemcitabine toxicity. In addition, re-analysis of previously published CRISPR/Cas9 data confirm RAB25 to be responsible for chemotherapy resistance in KRAS ${}^{\textit{G12D}}$ mutant human pancreatic cancer cell line. Finally, we used publicly available TCGA datasets and identify the upregulation of RAB25 in tumour tissues compared to the adjacent normal tissue, co-occurrence of KRAS ${}^{\text{G12}}$ mutations with RAB25 amplifications, and poor patients’ survival in cohorts with higher mRNA expression of RAB25.

CONCLUSION:

RAB25 expression is a prognostic marker for patient’s survival and gemcitabine resistance in PDA.

Keywords

RAB25 chemotherapy resistance pancreatic cancer KRAS TCGA

1. Introduction

Pancreatic cancer is a major human health challenge and the third leading cause of cancer-related deaths in the USA [1]. The death rate of pancreatic cancer varies among different populations and is reported to be $\sim$ 2.5% of all cancer related deaths worldwide [2]. Pancreatic cancer is a group of malignancies of pancreatic origin that is largely classified into exocrine and endocrine pancreatic cancer. The exocrine ductal adenocarcinoma or pancreatic ductal adenocarcinoma (PDA) accounts for more than 90% of pancreatic cancer patients worldwide. Due to its late diagnosis, widespread metastases, and resistance to chemotherapy, the overall survival rate of PDA is very low [3]. Although the molecular and genetic characteristics of PDA are extensively studied, its therapeutic targeting remains a challenge. Surgical resection is barely suitable for 10% patients while standard of care chemotherapy gemcitabine alone or in combination with oxaliplatin, and/or 5-fluorouracil, hardly produce any meaningful clinical response. Additionally, the combinatorial therapy often compromises patients’ life quality due to undesirable chemotherapy side effects [4, 5].

Despite being the standard of care for pancreatic cancer for decades gemcitabine treatment produces minimal benefits in PDA [6, 7, 8], largely due to de novo or rapid development of resistance [9, 10, 11]. Several factors contribute to gemcitabine resistance including tumor microenvironment, deregulation of nucleoside transporters/enzymes, epithelial to mesenchymal transition, and gene expression regulation by the miRNAs [11]. Despite the persistent resistance of PDA against gemcitabine, it is still widely used for its treatment largely due to lack of alternate effective treatment options. Thus, there is an urgent need to identify novel drug targets or biomarkers that could be effective against pancreatic cancer or predict the chemotherapy response accurately.

RAB25 (Ras-related protein Rab-25), is a small GTPase from the Rab protein family involved in vesicle transport, cell proliferation, signal transduction, cell motility and protein transport [12, 13]. In addition, RAB25 mediates tumorigenesis, cellular migration, metastasis, and chemoresistance [14, 15]. Depending on the context RAB25 may be an oncogene [14, 15, 16] or a tumour suppressor [17, 18, 19]. For example, in pancreatic cancer, RAB25 acts as an oncogene or tumour suppressor depending on the presence and absence of chloride intra cellular channel protein 3 (CLIC3) respectively [20]. Collectively, CLIC3 and RAB25 are responsible for recycling of integrin $\alpha$ 5 $\beta$ 1 to the plasma membrane which is responsible for enhanced cancer metastasis and poor patients’ survival [20]. Thus, the precise role of RAB25 in cancer biology and chemotherapy resistance in pancreatic cancer needs further evaluation.

In this study we generated gemcitabine resistant mPDA cells and performed whole transcriptomics analysis with a goal to identify novel proteins and pathways that could dictate gemcitabine resistant (GemR) in PDA cells. We used multiple approaches to narrow down on 10 most upregulated genes in GemR cells. Strikingly, we find that RAB25 protein which was previously not reported to be involved in gemcitabine resistance, is not only required for gemcitabine resistance, but it is also a prognostic marker for poor patients survival in PDA. Our results highlight the importance of targeting RAB25 to overcome gemcitabine resistance and use of RAB25 expression as a biomarker for stratifying gemcitabine responders from non-responders in PDA.

2. Methods and materials

2.1 Mouse husbandry

Mice carrying conditional alleles for Kras ${}^{\text{G12D}}$ , and Tp53 ${}^{\text{Flox}}$ were previously described [21, 22]. Similarly, Pdx1-Cre mice were described before [23]. All mouse experiments were carried out in accordance with UK Home Office guidelines, overseen by the Animal Welfare and Ethical Review Body at the Francis Crick Institute.

2.2 Generation of mouse KPC cell lines

Small to medium sized palpable tumours from KPC (KRas ${}^{\text{G12D}}$ ; Tp53 ${}^{\Delta/+}$ ; Pdx-1-Cre, an autochthonous mouse model of PDA which faithfully recapitulates different stages of human PDA) mice were harvested under aesthetic conditions as described [24]. The tumour tissues were cut into small pieces using a scalpel blade and homogenized by using gentleMACS tumour isolation kit. Cells recovered in suspension were washed and resuspended in Matrigel/DMEM in a ratio of 1:1. Organoids were grown on 50% matrigel (Basal membrane matrx, Corning) in complete DMEM with 10% FBS, 1% glutamine and 1% penicillin/streptomycin as before [25]. Low passage tumour organoids were used to generate 2D mPDA cell lines which were subsequently used to generate gemcitabine resistant clones.

2.3 Generation of stable mPDA cell lines

2 to 3 shRNA targeting each shortlisted candidate genes were ordered from Sigma-Aldrich on an pLKO.1 background including a control non-targeting scrambled shRNA plasmid (shpLKO.1). The lentivirus preparation and stable cell line generation was done as before [26]. For Rab25, two independent shRNA, shRab25#1 and shRab25#2 were used to generate KPC-GemR stable cell lines.

2.4 Cell viability assays

Cell proliferation data in Fig. 3A was performed via quantification of cell density by taking live cell images at regular intervals of 3 hour up to 76 hours, by Incucyte FLR device equipped with 20x objective (Essen Biosciences). Cell viability was determined by either counting the viable cells in a 500 $\mu$ l of viable cell suspension on Beckman’s Vi-CELL XR 2.03 cell counter or by cell-titer blue viability assay (G8081, Promega) as before [27]. Briefly, cells were plated at a density of 300,000/well on a 6-well plate overnight. The next morning varying doses of gemcitabine was added and 72 hours after gemcitabine treatment cells were trypsinized, collected in PBS and counted on Vi-Cell counter. For celltiter blue viability assay, 10,000/well on a 96-well plate in triplicates were allowed to adhere overnight and next morning treated with increasing doses of gemcitabine. 72 hours after gemcitabine treatment, cell-titer blue viability reagent was added as per kit’s protocol and the plate was read on a TECAN’s plate reader as per kit’s protocol.

2.5 RNA sequencing analysis

RNA sequencing was performed as before [24]. Total RNA was isolated using Qiagen’s RNAesy kit and following the kit’s protocol. RNA was converted into strand-specific cDNA libraries using the KAPA mRNA HyperPrep kit (Roche) according to the manufacturer’s instructions. Sequencing was performed on Illumina HiSeq 4000 platform with single ended 75 bp reads with and 25–30 million reads per sample. Trimmomatic/0.36-Java-1.7.0_80 was used for adaptor trimming, with parameters “LEADING:3 TRAILING:3 SLIDINGWINDOW:4:20 MIN-LEN:36”. The RSEM package (v.1.2.31)54 in conjunction with the STAR alignment algorithm (v.2.5.2a) 55 was used for the read mapping and gene-level quantification with respect to mouse Ensembl GRCm38 – release 89. The parameters used were: – star-output-genome-bam – forward-prob 0. Differential expression analysis was performed with the DESeq2 package (v.1.20.0) 56 within the R programming environment (https://www.r-project.org/; v.3.5.1), where the significance threshold for the identification of differentially expressed transcripts was set to an adjusted $P$ value $\leqslant$ 0.05.

2.6 Pathway analysis of significantly deregulated genes in mPDA GemR cells

To cluster differentially regulated genes based on their common functionality, a set of genes was analyzed for pathway analysis using Ingenuity Pathway Analysis (IPA). The differentially expressed genes (DEG) that are adjusted for false discovery rate (FDR) with a corrected $p$ -value $<$ 0.05 were added to the software to investigate the molecular and cellular functional pathways enriched. We selected pathways with a Benjamini & Hochberg (B-H) $p$ -value of $<$ 0.01 and the data is presented as a -log B-H $p$ -value.

2.7 Meta analysis of RAB25 expression, genetic alterations, and patients’ survival datasets

The online database Gene Expression Profiling Interactive Analysis (GEPIA) (http://gepia.cancer-pku.cn/index.html) was used to visualize the expression of RAB25 in various tumors versus their matched normal tissues. GEPIA is an interactive web tool that analyses RNA sequencing expression of 9,736 tumors and 8,587 normal samples from TCGA and the GTEx projects [28]. Analysis was conducted using the ANOVA statistical method with a log2FC cutoff of 2 and a $q$ -value cutoff of 0.01 in TCGA tumors compared to TCGA normal and GTEx normal for matched normal data. In addition, log2 (TPM $+$ 1) transformed expression data was used for plotting.

The cBioPortal for Cancer genomics (http://www.cbioportal.org/) was used to analyse the genetic alterations associated with in different cancer types in the TCGA pipeline. The keyword “RAB25” was searched within the cBioPortal database to obtain a cross-cancer summary and identify the genetic alterations (amplification, missense mutations, deep deletion and CNV) of RAB25 associated with pancreatic cancer.

Patient prognoses were evaluated by Kaplan-Meier survival curves of Pancreatic ductal adenocarcinoma patients with low or high expression of RAB25 using the Kaplan-Meier plotter (http://kmplot.com) and the GEPIA platforms. The database includes RNA-seq information based on TCGA and GEO datasets

2.8 Re-analysis of CRISPR/Cas9 screen data

The CRISPR/Cas9 screen data were collected from BioGRID Open Repository of CRISPR Screens (ORCS) (https://orcs.thebiogrid.org/), which is a publicly available scientific online resource. The keyword “RAB25” was searched within the database and 22 hits were identified out of 1099 screens. We selected screens with the phenotype “response to chemicals” and a $p$ -value of $<$ 0.05 in both KRAS wild type and muted pancreatic cancer cell lines. The data was presented as Log 2FC.

Figure 1.

Generation of gemcitabine resistant mouse PDA cell lines. A, Scheme of functional genomics screen to identify mediators of gemcitabine resistance. B, mPDA cell viability as judged by counting of viable cells after 72 hours $\pm$ indicated gemcitabine doses. C, mPDA cell viability as judged by cell titer blue cell viability assay.

Figure 2.

RNA-sequencing of KPC-Par versus KPC-GemR cells. A, Heat map showing changes at mRNA levels of 79 upregulated and 279 downregulated genes in gemcitabine resistant mPDA cells. B, Molecular and cellular functional pathway analysis generated by IPA. Ca’–Ch’, Immunohistochemistry of pancreatic adenocarcinoma tissues obtained from The Human Protein Atlas (https://www.proteinatlas.org/).

3. Results

3.1 Establishment of mPDA GemR cell lines

To understand the mechanisms of gemcitabine resistance in pancreatic cancer, we first harvested small-to-medium-sized tumours from the pancreas of KPC mice. To prevent the confounding factors such as immune and stromal cells affecting our studies, we derived 3 independent primary organoid tumour cell lines (Fig. 1A) (KPC-Par). To model gemcitabine resistance in PDA we treated these low passage primary cell lines with gradually increasing doses of gemcitabine and derived 3 gemcitabine resistant mouse PDA (KPC-GemR) cell lines (Fig. 1A). Indeed, mPDA tumour cells develop resistance to gemcitabine and provide a model to study mechanisms of acquired gemcitabine resistance (Fig. 1B and C).

3.2 RNA-sequencing revealed diverse set of genes deregulated in KPC-GemR cells

Next, we performed a whole transcriptome comparison of low passage KPC-Par and KPC-GemR cells and found 79 genes significantly up-regulated while 279 genes downregulated in resistant cells (Fig. 2A). Interestingly, we found downregulation of Ent4 – one of the four members of the equilibrative nucleotide transporter (ENT) family involved in gemcitabine uptake, suggesting that gemcitabine uptake might be affected in those cells [29]. In addition, S100 Calcium binding protein 4 (S100a4) – a proposed prognostic marker for gemcitabine resistance in PDA [30] – is also upregulated in resistant mPDA cell lines. Thus, our data suggest that pro-survival mechanisms of chemotherapy resistance are conserved between murine and human PDA. We performed ingenuity pathway analysis to uncover the molecular and cellular pathways deregulated in KPC-GemR cells (Fig. 2A). The top 10 most deregulated pathways in KPC-GemR cells include cell movement, growth and proliferation, and drug metabolism (Fig. 2B). Importantly we made a shortlist of 10 druggable genes (proteins with enzymatic activity) (Table 1), that were significantly upregulated in KPC-GemR cells, and reason that one or more of those candidate proteins might be involved in gemcitabine resistance in those cells. We first confirmed the pancreatic adenocarcinoma tissue expression of our shortlisted targets by human protein atlas (Fig. 2C). We find various degrees of expression of our target proteins in human PDA ranging from tumour cell specific expression to stromal or no expression at all (Fig. 2C). Four proteins showed tumour specific expression in the order of strongest to weakest including RAB25, S100a4, MOAa, and AK5 (Fig. 2Ca’–Cd’). Two proteins CXCR2 and TACSTD2, showed strong stromal expression although weak to moderate expression was detected in tumour cells, (Fig. 2Ce’ and Cf’), while two proteins, PPBP and RAB33b were undetected (Fig. 2C-g’ and C-h’). Tumour expression data for two of our shortlisted proteins (ILR5a and GLT1D1) was not available with the human protein atlas.

Table 1
List of selected DEG in gemcitabine resistant mPDA cells

Gene name	Gene symbol	Biological significance	Inhibitors	Mutations in Pancreatic cancer
Adenylate Kinase 5	AK5	Involved in the conversion of di/tri-phosphates, as well as in the regulation of ERK	–	–
C-X-C Motif Chemokine Receptor 2	CXCR2	Recognize CxC chemokines and act as a receptor of IL-8, which results in activation of neutrophiles	Acetylcysteine Clotrimazole Ibuprofen Reparixin	Amplification mutation
Pro-Platelet Basic Protein	PPBP	Activates neutrophils and stimulates DNA synthesis, glycolysis, intracellular cAMP accumulation and prostaglandin E2 secretion. In addition, involved in the formation and secretion of plasminogen activators by synovial cells	Copper	Amplification mutation
Interleukin 5 Receptor Subunit Alpha	IL5RA	Interacts with syntenin and results in IL5 mediated activation of the transcription factor SOX4. Mediators of the immune response as they control proliferation, differentiation and cell survival/apoptosis	Benralizuma	–
Ras-Related Protein Rab-25	RAB25	Involved in membrane trafficking, cell survival and promotes invasive migration of cells	–	Missense Mutation (A88T ) Amplification mutation
Ras-Related Protein Rab-33B	RAB33B	Involved in autophagy and vesicle transport during protein secretion and endocytosis.	–	–
Tumor Associated Calcium Signal Transducer 2	TACSTD2	May function as a growth factor receptor And as a cell surface receptor that transduces calcium signals.	Sacituzumab Govitecan	–
Glycosyltransferase 1 Domain Containing 1	GLT1D1	Predicted to enable glycosyltransferase activity	–	–
S100 Calcium Binding Protein A4	s100a4	Involved in the regulation of cell cycle progression, differentiation, motility, angiogenesis, apoptosis, and autophagy.	Trifluoperazine Niclosamide	Amplification mutation
Monoamine Oxidase A	MAOA	Involved in the oxidative deamination of biogenic and xenobiotic amines. it is involved in the metabolism of neuroactive and vasoactive amines in the central nervous system and peripheral tissues.	Isocarboxazid Linezolid Phenelzine	Deletion mutation

Figure 3.

RAB25 mediates cell survival in response to gemcitabine. A, Cell viability shown as percent confluency for stable mPDA cells with indicated shRNA expression, shpLKO.1 $=$ non-target control. B, Percent cell viability as judged by cell titer blue assay in stable mPDA cells targeted by a control or two different shRNA against Rab25 $\pm$ indicated doses of gemcitabine. C, Cell viability for indicated shRNAs as judged by crystal violet staining of fixed viable cells. D, Schematic representation of CRISPR/Cas9 screening data analyses from BioGRID-ORCS (https://orcs.thebiogrid.org/). E, Log2 Fold change data for RAB25 sgRNA enrichment for indicated conditions. For each condition, the highest scoring sgRNA targeted gene is shown as a positive control in both KRAS ${}^{\text{G12D}}$ and KRAS ${}^{\text{WT}}$ pancreatic cell lines.

Figure 4.

Genetic aberrations of Rab25 in different cancer types and survival rate. A, Alteration frequency of RAB25 gene in different cancers, including pancreatic cancer obtained from cBioPortal (http://www.cbioportal.org/). Alteration frequency included mutations (green), amplification (red) and deep deletion (blue). B and C, RAB25 expression levels across TCGA tumors compared to TCGA normal and Genotype-Tissue Expression (GTEx) data through the use of GEPIA (http://gepia.cancer-pku.cn/index.html) and the TNMplot,com.

3.3 RAB25 inhibition sensitizes pancreatic cancer cells to chemotherapy

Nevertheless, we generated stable KPC-GemR cell lines expressing at least two different lentivirus mediated shRNA targeting each of our shortlisted genes and treated those cells with gemcitabine to monitor the viability of those cells in response to gemcitabine (data not shown). Interestingly, knockdown of Rab25 despite having minimal effect on KPC-GemR cell proliferation, strongly sensitized KPC-GemR cells to gemcitabine (Fig. 3A–C). To understand if RAB25 plays a similar pro-survival role in human PDA, we used publicly available datasets from pooled CRISPR/Cas9 viability screens performed in PDA cell lines (Fig. 3D). As indicated by the results from CRISPR/Cas9 viability screen (CERES Score $<$ 1.0 and $>$ $-$ 0.2), the sgRAB25 used for deletion of RAB25 is neither depleted not enriched in the total cell population suggesting that RAB25 deletion does not affect overall viability in PDA KRAS ${}^{\text{wt}}$ as well as KRAS ${}^{\text{G12D}}$ mutant cell lines consistent with our results in mPDA cells (Fig. 3A and E). Then we analysed CRISPR/Cas9 pooled synergistic activation module (SAM) screen data. In this experiment, a genome wide sgRNA library was used to see which genes upon activation will result in conferring resistance to PDA cells against gemcitabine and oxaliplatin [31]. Interestingly, KRAS ${}^{\text{G12D}}$ mutant but not wildtype cell line showed strong enrichment of sgRNA RAB25 in response to two independent chemotherapy drugs gemcitabine and oxaliplatin (Fig. 3E–G). Thus, loss of function and gain of function experiments confirmed the requirement of RAB25 for survival of PDA cells in response to chemotherapy. These results suggest a possibility that RAB25 mediates pro-survival activities of PDA cells specifically in KRAS ${}^{\text{G12D}}$ background.

3.4 RAB25 is amplified across multiple human cancers

We next determine the mutational spectrum of RAB25 across multiple human cancers through cBioPortal.org platform using publicly available TCGA datasets. Strikingly, RAB25 was amplified in multiple human cancers including up to 5% PDA (Fig. 4A), suggesting an oncogenic role for RAB25. Consistent with that, the RAB25 gene expression was increased $\sim$ 2–3 folds in tumour tissues compared to adjacent normal tissues as judged by two independent publicly available datasets (Fig. 4B and C). These results are in line with the possible role of RAB25 in PDA progression, survival, and response to chemotherapy.

Figure 5.

RAB25 genomic alterations in pancreatic cancer. A, Data showing alteration frequency of RAB25 gene in pancreatic cancer as obtained from cBioPortal (http://www.cbioportal.org/). B, Dot plot showing the % altered frequency of indicated genes in RAB25 altered versus unaltered PDA patients. C, Data from B, shown as bar graph. D, and E, Fraction genome altered and Total mutational burden analysis in human PDA cohort from A.

Figure 6.

Higher expression of RAB25 is associated with poor patients’ survival in PDA cohorts. A, Overall survival of PDA patients with RAB25 high versus low expression cohorts as evaluated by kmplot.com survival analysis. B, Overall survival of PDA patients in RAB25 high versus low expression cohorts from GEPIA platform. C, Schematic showing possible mechanisms of RAB25 pro-survival activities in KRAS ${}^{\text{G12D}}$ mutant PDA cells.

3.5 RAB25 amplifications co-occur with KRAS mutations in PDA and are responsible for poor survival in PDA patients

Next, we aim to detect the genetic alteration frequency of RAB25 specifically in pancreatic cancer. We use cBioPortal platform to visualize the TCGA pancreatic cancer dataset of $\sim$ 184 patients and use this data to search for RAB25 mutations and/or gene expression pattern. RAB25 is deregulated in 16% of the total cases (Fig. 5A). Interestingly, RAB25 amplifications and mRNA upregulation consisted more than 90% of RAB25 alterations in this cohort (Fig. 5A and B). To understand potential role of RAB25 in the pathology of PDA, we search for genetic alterations and clinical parameters of patients with RAB25 amplifications and upregulation. Strikingly, oncogenic KRAS mutations were significantly enriched in patients with RAB25 amplifications (Fig. 5B and C). Indicating a potential correlation between RAB25 and KRAS in PDA. Patients with RAB25 alterations showed higher load of genomic alterations, and total mutational burden compared to unaltered controls (Fig. 5D and E). These results suggest a possibility that RAB25 amplifications preferentially occur in KRAS ${}^{\text{G12D}}$ positive PDA patients and may confer these patients with more aggressive and chemotherapy resistant phenotypes.

Finally, we used two independent publicly available datasets to check whether RAB25 expression correlates with patients’ survival in PDA. As expected, higher expression of RAB25 is associated with significantly reduced survival in patients cohorts from both the datasets (Fig. 6A and B).

4. Discussion

Pancreatic cancer continues to be one of the most difficult to treat cancers which is in sharp contrast to other cancers including breast, colon, and lung cancer, and myeloid malignancies where the overall patient survival has significantly improved over the last few decades. The frontline chemotherapy gemcitabine shows minimal benefits against PDA [32]. Thus, there is an urgent need to identify novel drug targets and prognostic markers of clinical relevance that can help tackle the menace of pancreatic cancer more efficiently. In this study we identify RAB25 to be associated with chemotherapy resistance in pancreatic cancer cell lines and with genomic instability and poor patients’ survival.

Interestingly we show that mPDA cells rapidly develop resistance to increasing doses of gemcitabine in a long-term culture. This resistance is associated with dramatic upregulation of genes from multiple cellular signalling pathways including cell survival, cell death, and drug metabolism. One of the highest upregulated gene RAB25, is very strongly expressed in human PDA, leading us to believe that RAB25 must be involved in PDA pathophysiology. However, RAB25 is not essential for survival of neither mPDA nor human PDA cell lines but rather its inhibition sensitized those cells to gemcitabine. These results highlight the possibility that RAB25 is specifically required for survival of pancreatic cancer cells in response to chemotherapy.

Strikingly, by reanalysing the published data from the CRISPR/Cas9 viability screens in response to chemotherapy drugs gemcitabine and oxaliplatin in PDA cell lines, we find that RAB25 sgRNA was significantly enriched only in KRAS ${}^{\text{G12D}}$ mutant human PDA cell line PANC-1 while not in KRAS ${}^{\text{wt}}$ BXPC3 cells. Suggesting that the gain of RAB25 gene provided resistance to KRAS ${}^{\text{G12D}}$ mutant cells against chemotherapy. Consistent with that we find $>$ 90% PDA patients with RAB25 alterations to have KRAS ${}^{\text{G12D}}$ mutations in a cohort from TCGA. Additionally, patients with RAB25/KRAS alterations showed enhanced genomic instability and increased mutational burden – both of which might be responsible for aggressive phenotypes and chemotherapy resistance in pancreatic cancer. Indeed, using two different publicly available datasets of PDA patients, we find higher expression of RAB25 to be strongly associated with poor clinical outcome. These results suggest a possible synthetic lethality between RAB25 and KRAS genes in pancreatic cancer and warrants further investigation of the role of RAB25 in mediating chemotherapy resistance in KRAS ${}^{\text{G12D}}$ mutant PDA.

Whereas our study shed some important light on the role of RAB25 in pancreatic cancer, the exact molecular mechanism of how RAB25 helps PDA cells evade gemcitabine induced cell death is merely speculative. There could be several possibilities that may explain RAB25’s anti-chemotherapy activities in PDA (Fig. 6C). For example, RAB25 overexpression is associated with aggressive phenotypes of ovarian, breast, and pancreatic cancer [33]. Some of those aggressive phenotypes can be explained by CLIC3/RAB25 mediated recycling and activation of integrin/EGFR signalling [20]. Additionally, RAB25 is associated with chemotherapy and radiotherapy resistance in multiple cancers [33], most likely due to the inhibition of mitochondrial apoptotic pathway [34]. Noticeably, RAB25 helps cancer cells encounter bioenergetic stress by mTOR activation and production of ATP [35]. Tumours in PDA are largely hypoxic and under continuous nutritional and hypoxic stress due to poor vascularization [36]. Thus, RAB25 upregulation may help those tumours by providing prosurvival signalling and maintaining the ATP levels for cellular growth and signalling (Fig. 6C). This is further highlighted by strong upregulation of RAB25 in tumour versus normal pancreatic cancer tissues as shown in our analyses.

Recent advances in the field of stapled peptide technology for targeting difficult to target small molecules like RAB25 has generated a lot of interest for clinical manifestation. Particularly, targeting RAB25 in multiple cancer cell lines using a RAB25 specific peptide showed context dependent anti-tumour effects [37]. Our study highlights the importance of targeting RAB25 in combination with chemotherapy in human PDA cell lines and in autochthonous mice models of PDA and provides evidence that RAB25 might be an interesting therapeutic target in addressing chemotherapy resistance in highly aggressive otherwise difficult to treat PDA. Finally, our results indicate that high RAB25 expression in human PDA is strongly associated with poor clinical outcome and stratification of PDA patients based on RAB25 expression can identify gemcitabine responders and help identify alternate treatment plans for non-responders.

Footnotes

Acknowledgments

We would like to thank Advanced Sequencing, and Bioinformatics and Biostatistics Core facilities of the Francis Crick Institute for RNA sequencing and data analysis. This work was supported by the Francis Crick Institute (U.K) and Hamad bin Khalifa University (Qatar). O.M.K. was funded by an EMBO long-term postdoctoral fellowship award (ALT-549-213), a Swedish International postdoctoral fellowship award (VR-537-2013-359), and by an intramural grant from Hamad Bin Khalifa University (Qatar Foundation, Qatar).

Author contributions

Conception: OK designed the study. OK performed mouse experiments and generated the RNA sequencing data.

Interpretation or analysis of data: AK analysed ORCS, GEPIA, and TCGA datasets. AK performed IPA analysis on the RNA sequencing data. OK analysed survival curves from the TCGA datasets.

Preparation of the manuscript: OK, AK, and SA assembled figures and wrote manuscripts. AB provided important feedback and final approval for submission.

Revision for important intellectual content: N/A.

Supervision: AK and SA are graduate students under supervision of OK.

References

Siegel

R.L.

Miller

K.D.

Fuchs

H.E.

and Jemal

, Cancer statistics, 2022, CA: a cancer journal for clinicians, 2022.

Bray

Ferlay

Soerjomataram

Siegel

R.L.

Torre

L.A.

and Jemal

, Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries, CA: A Cancer Journal for Clinicians 68 (2018), 394–424.

Zhang

Sanagapalli

and Stoita

, Challenges in diagnosis of pancreatic cancer, World Journal of Gastroenterology 24 (2018), 2047.

Dumont

Puleo

Collignon

Meurisse

Chavez

Seidel

Gast

Polus

Loly

and Delvenne

, A single center experience in resectable pancreatic ductal adenocarcinoma: The limitations of the surgery-first approach. Critical review of the literature and proposals for practice update, Acta Gastro-Enterologica Belgica 80 (2017), 451–461.

Labori

K.J.

Katz

M.H.

Tzeng

C.W.

Bjørnbeth

B.A.

Cvancarova

Edwin

Kure

E.H.

Eide

T.J.

Dueland

and Buanes

, Impact of early disease progression and surgical complications on adjuvant chemotherapy completion rates and survival in patients undergoing the surgery first approach for resectable pancreatic ductal adenocarcinoma – a population-based cohort study, Acta Oncologica 55 (2016), 265–277.

Burris

H.r.

Moore

M.J.

Andersen

Green

M.R.

Rothenberg

M.L.

Modiano

M.R.

Christine Cripps

Portenoy

R.K.

Storniolo

A.M.

and Tarassoff

, Improvements in survival and clinical benefit with gemcitabine as first-line therapy for patients with advanced pancreas cancer: A randomized trial, Journal of Clinical Oncology 15 (1997), 2403–2413.

Steinfeld

Scott

Vilander

Marx

Quirk

Lindberg

and Koerner

, The role of lean process improvement in implementation of evidence-based practices in behavioral health care, The Journal of Behavioral Health Services & Research 42 (2015), 504–518.

Zhu

Zhao

and Gou

, Gemcitabine plus S-1 for metastatic pancreatic cancer, Medicine 97 (2018).

Samulitis

B.K.

Pond

K.W.

Pond

Cress

A.E.

Patel

Wisner

Patel

Dorr

R.T.

and Landowski

T.H.

, Gemcitabine resistant pancreatic cancer cell lines acquire an invasive phenotype with collateral hypersensitivity to histone deacetylase inhibitors, Cancer Biology & Therapy 16 (2015), 43–51.

10.

H.-Q.

Gocho

Aguilar

Zhuang

Z.-N.

Yanaga

Huang

and Chiao

P.J.

, Mechanisms of overcoming intrinsic resistance to gemcitabine in pancreatic ductal adenocarcinoma through the redox modulation, Molecular Cancer Therapeutics 14 (2015), 788–798.

11.

Zeng

Pöttler

Lan

Grützmann

Pilarsky

and Yang

, Chemoresistance in pancreatic cancer, International Journal of Molecular Sciences 20 (2019), 4504.

12.

Bhuinand

and Roy

J.K.

, Rab proteins: The key regulators of intracellular vesicle transport, Experimental Cell Research 328 (2014), 1–19.

13.

Chavrierand

and Goud

, The role of ARF and Rab GTPases in membrane transport, Current Opinion in Cell Biology 11 (1999), 466–475.

14.

Wang

and He

, Rab25 GTPase: Functional roles in cancer, Oncotarget 8 (2017), 64591.

15.

Cheng

K.W.

Lahad

J.P.

Kuo

W.-l.

Lapuk

Yamada

Auersperg

Liu

Smith-McCune

K.H.

and Fishman

, The RAB25 small GTPase determines aggressiveness of ovarian and breast cancers, Nature Medicine 10 (2004), 1251–1256.

16.

Dai

She

Zhao

Jiang

Chen

and Zhao

, Identification and characterization of nine novel human small GTPases showing variable expressions in liver cancer tissues, Gene Expression The Journal of Liver Research 10 (2002), 231–242.

17.

Goldenringand

and Nam

, Rab25 as a tumour suppressor in colon carcinogenesis, British Journal of Cancer 104 (2011), 33–36.

18.

Tong

Chan

K.W.

Bao

J.Y.

Wong

K.Y.

Chen

J.-N.

Kwan

P.S.

Tang

K.H.

Qin

Y.-R.

and Lok

, Rab25 is a tumor suppressor gene with antiangiogenic and anti-invasive activities in esophageal squamous cell carcinoma, Cancer Research 72 (2012), 6024–6035.

19.

Clausen

Melchers

Mastik

Slagter-Menkema

Groen

Laan

B.v.d.

Van Criekinge

De Meyer

Denil

and Van Der Vegt

, RAB25 expression is epigenetically downregulated in oral and oropharyngeal squamous cell carcinoma with lymph node metastasis, Epigenetics 11 (2016), 653–663.

20.

Dozynkiewicz

M.A.

Jamieson

N.B.

MacPherson

Grindlay

van den Berghe

P.V.

von Thun

Morton

J.P.

Gourley

Timpson

and Nixon

, Rab25 and CLIC3 collaborate to promote integrin recycling from late endosomes/lysosomes and drive cancer progression, Developmental Cell 22 (2012), 131–145.

21.

Jackson

E.L.

Willis

Mercer

Bronson

R.T.

Crowley

Montoya

Jacks

and Tuveson

D.A.

, Analysis of lung tumor initiation and progression using conditional expression of oncogenic K-ras, Genes & Development 15 (2001), 3243–3248.

22.

Marino

Vooijs

van Der Gulden

Jonkers

and Berns

, Induction of medulloblastomas in p53-null mutant mice by somatic inactivation of Rb in the external granular layer cells of the cerebellum, Genes & Development 14 (2000), 994–1004.

23.

Hingorani

S.R.

Petricoin

E.F.

Maitra

Rajapakse

King

Jacobetz

M.A.

Ross

Conrads

T.P.

Veenstra

T.D.

Hitt

B.A.

Kawaguchi

Johann

Liotta

L.A.

Crawford

H.C.

Putt

M.E.

Jacks

Wright

C.V.

Hruban

R.H.

Lowy

A.M.

and Tuveson

D.A.

, Preinvasive and invasive ductal pancreatic cancer and its early detection in the mouse, Cancer Cell 4 (2003), 437–450.

24.

Nelson

J.K.

Thin

M.Z.

Evan

Howell

Almeida

Legrave

Koenis

D.S.

Koifman

and Sugimoto

, USP25 promotes pathological HIF-1-driven metabolic reprogramming and is a potential therapeutic target in pancreatic cancer, Nature Communications 13 (2022), 1–18.

25.

Wang

V.M.

Ferreira

R.M.M.

Almagro

Evan

Legrave

Zaw Thin

Frith

Carvalho

Barry

D.J.

Snijders

A.P.

Herbert

Nye

E.L.

MacRae

J.I.

and Behrens

, CD9 identifies pancreatic cancer stem cells and modulates glutamine metabolism to fuel tumour growth, Nat Cell Biol 21 (2019), 1425–1435.

26.

Aldaalis

Bengoechea-Alonso

M.T.

and Ericsson

, The SREBP-dependent regulation of cyclin D1 coordinates cell proliferation and lipid synthesis, Front Oncol 12 (2022), 942386.

27.

Khan

O.M.

Almagro

Nelson

J.K.

Horswell

Encheva

Keyan

K.S.

Clurman

B.E.

Snijders

A.P.

and Behrens

, Proteasomal degradation of the tumour suppressor FBW7 requires branched ubiquitylation by TRIP12, Nature Communications 12 (2021), 2043.

28.

Tang

Kang

Gao

and Zhang

, GEPIA: A web server for cancer and normal gene expression profiling and interactive analyses, Nucleic Acids Research 45 (2017), W98–W102.

29.

Fryer

R.A.

Barlett

Galustian

and Dalgleish

A.G.

, Mechanisms underlying gemcitabine resistance in pancreatic cancer and sensitisation by the iMiDTM lenalidomide, Anticancer Research 31 (2011), 3747–3756.

30.

Mahon

P.C.

Baril

Bhakta

Chelala

Caulee

Harada

and Lemoine

N.R.

, S100A4 contributes to the suppression of BNIP3 expression, chemoresistance, and inhibition of apoptosis in pancreatic cancer, Cancer Research 67 (2007), 6786–6795.

31.

Ramaker

R.C.

Hardigan

A.A.

Gordon

E.R.

Wright

C.A.

Myers

R.M.

and Cooper

S.J.

, Pooled CRISPR screening in pancreatic cancer cells implicates co-repressor complexes as a cause of multiple drug resistance via regulation of epithelial-to-mesenchymal transition, BMC Cancer 21 (2021), 632–632.

32.

Binenbaum

Na’ara

and Gil

, Gemcitabine resistance in pancreatic ductal adenocarcinoma, Drug Resist Updat 23 (2015), 55–68.

33.

Mitra

Cheng

K.W.

and Mills

G.B.

, Rab25 in cancer: A brief update, Biochem Soc Trans 40 (2012), 1404–1408.

34.

Temel

S.G.

Giray

Karakas

Gul

Kozanoglu

Celik

Basaga

Acikbas

Sucularli

Oztop

Aka

and Kutuk

, RAB25 confers resistance to chemotherapy by altering mitochondrial apoptosis signaling in ovarian cancer cells, Apoptosis 25 (2020), 799–816.

35.

Cheng

K.W.

Agarwal

Mitra

Lee

J.S.

Carey

Gray

J.W.

and Mills

G.B.

, Rab25 increases cellular ATP and glycogen stores protecting cancer cells from bioenergetic stress, EMBO Mol Med 4 (2012), 125–141.

36.

Yamasaki

Yanai

and Onishi

, Hypoxia and pancreatic ductal adenocarcinoma, Cancer Lett 484 (2020), 9–15.

37.

Mitra

Montgomery

J.E.

Kolar

M.J.

Jeong

K.J.

Peng

Verdine

G.L.

Mills

G.B.

and Moellering

R.E.

, Stapled peptide inhibitors of RAB25 target context-specific phenotypes in cancer, Nat Commun 8 (2017), 660.

A small Rho GTPase RAB25 with a potential role in chemotherapy resistance in pancreatic cancer

Abstract

BACKGROUND:

OBJECTIVE:

METHODS:

RESULTS:

CONCLUSION:

Keywords

1. Introduction

2. Methods and materials

2.1 Mouse husbandry

2.2 Generation of mouse KPC cell lines

2.3 Generation of stable mPDA cell lines

2.4 Cell viability assays

2.5 RNA sequencing analysis

2.6 Pathway analysis of significantly deregulated genes in mPDA GemR cells

2.7 Meta analysis of RAB25 expression, genetic alterations, and patients’ survival datasets

2.8 Re-analysis of CRISPR/Cas9 screen data

3.1 Establishment of mPDA GemR cell lines

3.2 RNA-sequencing revealed diverse set of genes deregulated in KPC-GemR cells

Table 1 List of selected DEG in gemcitabine resistant mPDA cells

3.4 RAB25 is amplified across multiple human cancers

4. Discussion

Footnotes

Acknowledgments

Author contributions

References

Table 1
List of selected DEG in gemcitabine resistant mPDA cells