RCC1 regulation of subcellular protein localization via Ran GTPase drives pancreatic ductal adenocarcinoma growth

Abstract

Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal malignancy, with limited therapeutic options. Here, we evaluated the role of regulator of chromosome condensation 1 (RCC1) in PDAC. RCC1 functions as a guanine exchange factor for GTP-binding nuclear protein Ran (Ran) GTPase and is involved in nucleocytoplasmic transport. RCC1 RNA expression is elevated in PDAC tissues compared to normal pancreatic tissues and correlates with poor prognosis. RCC1 silencing by RNAi and CRISPR-Cas9 knockout (KO) results in reduced proliferation in 2-D and 3-D cell cultures. RCC1 knockdown (KD) reduced migration and clonogenicity, enhanced apoptosis, and altered cell cycle progression in human PDAC and murine cells from LSL-Kras^G12D/+; LSL-Trp53^R172H/+; Pdx1-Cre (KPC) tumors. Mechanistically, RCC1 KO shows widespread transcriptomic alterations including regulation of PTK7, a co-receptor of the Wnt signaling pathway. RCC1 KD disrupted subcellular Ran localization and the Ran gradient. Nuclear and cytosolic proteomics revealed altered subcellular proteome localization in Rcc1 KD KPC-tumor-derived cells and several altered metabolic biosynthesis pathways. In vivo, RCC1 KO cells show reduced tumor growth potential when injected as sub-cutaneous xenografts. Finally, RCC1 KD sensitized PDAC cells to gemcitabine chemotherapy treatment. This study reveals the role of RCC1 in pancreatic cancer as a novel molecular vulnerability that could be exploited to enhance therapeutic response.

Keywords: Nuclear export; PDAC; Pancreatic cancer; RCC1; Ran GTPase.

Figure 1. RCC1 expression is elevated and is associated with poorer prognosis in PDAC.

(A) Box plot shows RCC1 RNA expression in normal pancreatic (n=167) and tumor tissues (n=178), Welch’s t-test p = 1.009e-9 (t= −6.357). Reads were realigned and reanalyzed using the same pipeline on Xena platform to eliminate any bias or batch effect. (B) Box plot shows RCC1 protein expression in normal pancreatic (n=74) and PDAC tumor tissues (n=137) from CPTAC database. (C) Kaplan-Meier plot depicting overall survival in TCGA patients according to RCC1 expression (n=183). Samples were obtained from patients who underwent pancreatectomy or Whipple procedure prior to therapy. Stage distribution of the patients: stage I: 10%; stage II: 83%; stage III: 3%; stage IV: 4%. (D) Dot plot of RCC1 IHC H-scores according to tumor grades from PDAC TMA shown in (S1C). RCC1 staining is higher in grade 3 cases (p = 0.017). TMA includes 44 cases of grades 1 and 2, 27 cases of grade 3 PDAC, 5 cases of normal pancreas tissue. Each case is represented by one core. (E) Dot plot of RCC1 IHC scores according to tumor stage from TMA in (D). (F) Western blot of RCC1 in a panel of PDAC cell lines. L3.6pl, HPAC, PANC-1, AsPC-1, COLO 357, HPAF-II have a KRAS G12D mutation, MIA PaCa-2 cells have a KRAS G12C mutation, and BxPC-3 cells have WT KRAS.

Figure 2. RCC1 silencing inhibits the proliferation of PDAC cells.

(A) Western blot showing efficiency of siRNA mediated RCC1 silencing at 72 hrs. (B) Bar graph showing reduced cell viability in RCC1 KD PDAC cells (72 hrs), n=6, experiments were repeated twice with similar results. (C) Scratch wound healing assay showing reduced cellular migration in MIA PaCa-2 upon RCC1 KD. Experiments were repeated twice. (D) Colony formation assay showing reduced colony number and size in RCC1 KD MIA PaCa-2 cells. Experiments were repeated three times. (E) KPC derived KCI-313 cells were transfected with mouse specific RCC1 siRNA or a non-targeting control. Western blot showing knockdown efficiency, and bar graphs showing reduced cell viability 72 hrs after knockdown (left, p = 0.0043), and reduced spheroid size in knockdown cells (right, p=0.0001), n=6, experiments were repeated twice with similar results.

Figure 3. RCC1 knockout using CRISPR-Cas9 attenuates cellular proliferation and alters cell cycle.

(A) Western blot showing efficiency of RCC1 KO in HPAF-II and MIA PaCa-2 PDAC cell lines. (B) Bar graph showing reduced growth of RCC1 KO cells, n=6. (C) Western blot showing RCC1 and Ran levels in MIA PaCa-2 WT and two isolated RCC1 KO clones termed KO1 and KO4. (D) Bar graph of colony number and size quantification of clonogenic assay demonstrating reduced colony size in HPAF-II RCC1 KO cells. Experiments were done in three biological replicates. (E) Bar graph summarizing MIA PaCa-2 clonogenic assay revealing decreased number and size of RCC1 KO colonies, four biological replicates. (F) Representative pictures of colonies quantified in (D) and (E). (G) Bar graph depicting smaller spheroids in MIA PaCa-2 RCC1 KO cells, n=6. (H) Bar graph depicting alteration in cell cycle progression in RCC1 KO1 and KO4 MIA PaCa-2 cells, data from three biological replicates.

Figure 4. RCC1 silencing alters the transcriptome of PDAC cells.

(A) Volcano plot of differentially expressed genes (DEG) in MIA PaCa-2 RCC1 KO cells, three independent biological replicates. Downregulated genes = 292. Upregulated genes = 192. (B) KEGG pathway and gene ontology enrichment analysis of DEGs in RCC1 KO MIA PaCa-2 cells. (C) Relative gene expression fold change normalized to GAPDH by RT-qPCR of selected up or downregulated DEGs in MIA PaCa-2 RCC1 KO clones 1 and 4, and HPAF-II RCC1 KO cells (n=3). (D) Western blot showing PTK7 protein downregulation in HPAF-II and MIA PaCa-2 RCC1 KO cells. (E) PTK7 siRNA KD. Western blot showing KD efficiency, and bar graph of cell viability after 72 hrs.

Figure 5. RCC1 regulates Ran downstream in PDAC.

(A) Kaplan-Meier plots of overall and progression free survival of PDAC patients in the TCGA in the high quartile vs the low quartile of RAN expression. (B) Scatter plot correlation analysis of RNA expression of RCC1 and RAN in TCGA PDAC patients and CCLE PDAC cells lines. (C) Nuclear cytoplasmic fractionation Western blot showing depleted nuclear Ran in MIA PaCa-2 and HPAF-II RCC1 KO cells. (D) Ran-GTP assay showing reduced Ran GTP loading in RCC1 KO cells. (E) Volcano plots of differentially expressed proteins in nuclear and cytoplasmic fractions of RCC1 silenced KPC derived cells. Four independent biological replicates were used for analysis. (F) Selected significantly enriched pathways in the cytoplasmic proteomics dataset. (G) Significantly enriched pathways in nuclear proteomics. (H) Nuclear cytoplasmic fractionation showing elevated nuclear HDAC3 in RCC1 KO cells. (I) Immunofluorescence staining of HDAC3 in MIA PaCa-2 RCC1 WT and KO cells. (J) Kaplan-Meier plot demonstrating difference in overall survival of PDAC patients in the TCGA based on HDAC3 expression.

Figure 6. RCC1 KO restricts PDAC tumor growth in vivo.

(A) H&Es and immunohistochemical staining of Rcc1 and Ran in a c-myc overexpressing PDAC mouse model tissues. (B) An equal number of HPAF-II WT and HPAF-II RCC1 CRISPR/Cas9 knockout cells (1×10⁶) were bilaterally injected subcutaneously in ICR-SCID mice. Growth was monitored over 1 month (** p<0.01). (C) same as (B) for MIA PaCa-2 followed over 3 months. (D) Photograph showing excised HPAF-II derived tumors from (B), and bar graph showing the difference in tumor weights. Picture of harvested tumors is shown. The experiment was repeated with n=2 (E) Pictures of MIA PaCa-2 tumors in mice after euthanasia is shown. (F) Immunohistochemical staining of FFPE sections from excised HPAF-II derived xenografts showing Ran, PTK7, and ELOVL2 in residual tumor tissues.