Ran Is a Potential Therapeutic Target for Cancer Cells with Molecular Changes Associated with Activation of the PI3K/Akt/mTORC1 and Ras/MEK/ERK Pathways

This article is featured in Highlights of This Issue, p. 321

Normal cells acquire mutations to activate/suppress various pathways to fulfill certain functions to become cancerous (1). Targeting these cancer hallmarks may exert a selective killing effect on cancer cells. Cancer cells are addicted to oncogenes or oncogenic pathways (2), which are usually uniquely present or hyperactivated, and are essential for tumor cell growth and survival. The 2 most frequently dysregulated signaling pathways in cancers are the phosphoinositide 3-kinase (PI3K)/Akt/mTORC1 and Ras/MEK/ERK [mitogen-activated protein/extracellular signal-regulated kinase (ERK; MEK)] pathways (3). Due to the presence of parallel pathways in cancer cells, targeting these pathways in cancer therapy leads to limited success (3).

These growth signaling pathways usually exert their ultimate effect by regulating the translocation of transcription factors into or out of the nucleus, thereby altering the transcriptome and consequently, the expressome (4). The activity of several oncogenic or tumor suppressive transcription factors, such as NF-κB, FOXO1, p53, and β-catenin (4), and nontranscription factors, such as survivin (5), are regulated by their subcellular localization. Therefore, the uncoupling of nucleocytoplasmic transport from growth and survival signaling pathways has been suggested to be a potential target for cancer therapy (6).

Ran GTPase (Ran), a Ras-related G protein, is involved in several important cellular processes, the best known being nucleocytoplasmic transportation (7). Ran guanine nucleotide exchange factor, RCC1, is retained in the nucleus and Ran GTPase activating protein, RanGAP, is retained in the cytoplasm. Their cellular compartmentalization creates a RanGTP gradient, which regulates the transport of macromolecules between nucleus and cytoplasm (8). Ran also plays an important role in cancer development and progression. It is overexpressed in various cancers with prognostic significance (9, 10), and its overexpression is correlated with increased aggressiveness of the cancer cells in vitro and in vivo (11–13). Ran has been shown to be a promising cancer therapeutic target; silencing Ran expression induces more apoptosis in cancer cells than normal cells (14) as well as in activated K-Ras–mutant cells than their isogenic K-Ras wild-type counterparts (15). However, the reasons for these selective killing effects are far from clear.

There are reports showing that Ran may be a mediator between growth signaling and nucleocytoplasmic transport. Ran is upregulated by the PI3K/Akt pathway in H₂O₂-induced mitosis (16) and is also activated by growth factors (13). Ran binding protein 3 (RanBP3) is phosphorylated in response to Ras/MEK/ERK and PI3K/Akt activation, whereas the phosphorylation of RanBP3 modulates Ran-dependent nucleocytoplasmic transport (17, 18). Taken together, these findings suggest that the expression level and the activity of Ran, and thereby the capacity of nucleocytoplasmic transportation, are regulated by growth and survival signaling pathways. Here, we show that Ran silencing results in a selective killing effect on cancer cells with stronger activation of the PI3K/Akt/mTORC1 and MEK/ERK pathways probably through dysregulation of nucleocytoplasmic transportation and downregulation of Mcl-1.

See details in Supplementary Materials and Methods.

pLKO.1-shRan1, -2, -3, -4, and -5 (clone IDs were NM_006325.2-697s1c1, NM_006325.2-198s1c1, NM_006325.2-484s1c1, NM_006325.2-625s1c1, and NM_006325.2-142s1c1, respectively) were obtained from Sigma-Aldrich. pLKO.1-shScramble (Scr, #1864), pCMV-dR8.2 dvpr (#8455), and pCMV-VSV-G (#8454) were obtained from Addgene.

Transfection was carried out by GeneJuice according to the manufacturer's instructions. Viral particles were harvested 48 hours posttransfection and were applied to the target cells with 6 μg/mL polyprene supplement. The target cells were allowed to be infected for 4 hours and medium was refreshed. The same amount and same batch of viral particles were used for any comparison made in this study.

Cells were cultured in their normal medium until 24 hours postinfection. At 24 hours postinfection, the medium was changed to the desired medium with different drugs (the concentrations of the drugs used are listed in Supplementary Table S1, unless otherwise specified). Cells were harvested at 72 hours postinfection for protein and apoptosis analyses, unless otherwise specified.

Western blotting was carried out as previously described (19). Nuclear/cytoplasmic fractionation was carried out by N-Per kit from Pierce. The details of the antibodies used in this study are listed in Supplementary Table S2.

The extent of apoptosis was estimated by the percentage of sub-G₁ phase cells by propidium iodide (PI) staining. Annexin V staining was done with the Annexin V–Fluos staining Kit (Roche) according to the manufacturer's instructions. Stained cells were analyzed by a BD LSRII Flow cytometer.

Breast cancer patient specimens were obtained from University of Liverpool, United Kingdom (20), whereas lung cancers patient specimens were obtained from St. James Hospital, Dublin, Ireland. See details of patient information in Supplementary Materials and Methods.

Immunohistochemical staining and evaluation were carried out as previously described (20). See details in Supplementary Materials and Methods.

A total of 4 breast cancer data sets (GSE1378, GSE1379, GSE2034, and GSE3143), consisting of 564 patients and a total of 6 lung cancer data sets (GSE6253, GSE3141, GSE8894, GSE4573, GSE5123, and GSE13213) consisting of 601 patients with their corresponding microarray and survival data available in Gene Expression Omnibus (GEO) were included in this study. The data sets were preprocessed as previously described using R and Bioconductor for normalization (21). See details in Supplementary Materials and Methods.

Because infection of shRan1 and shRan2 led to the lowest expression of Ran protein and highest apoptotic responses among the 5 shRan sequences (Supplementary Fig. S1), these 2 shRNAs were used in this study. Ran silencing induced significantly fewer cell deaths and lower activation of apoptotic pathways in the immortalized human embryonic kidney 293 (HEK 293) cell line than cancer cell lines from breast (MDA-MB-231), lung (A549), prostate (DU145), esophageal (EC109), and colon (HCT116 and DLD-1; P < 0.01; Supplementary Fig. S2A and S2B). Ran silencing also resulted in a significantly higher apoptotic response in breast cancer MDA-MB-231 than immortalized MCF10a cells (Supplementary Fig. S3), in line with a previous study (14).

Growth signaling pathways are usually aberrantly activated in cancers. Therefore, we investigated whether uncoupling of the activated growth signal and Ran expression underpins the differential response to Ran silencing in immortalized and cancer cells. Significantly more apoptotic sub-G₁ phase and Annexin V–positive cells were detected upon Ran silencing when MDA-MB-231 breast and A549 lung cancer cells were grown in the presence of FBS, which stimulates the growth signaling pathways, compared with serum-free conditions (P < 0.01; Fig. 1A and B). The lowest apoptosis induced by Ran silencing was detected when cells were grown in Hank's buffered salt solution (HBSS), in which the growth signal is inactive because of the absence of growth factors and amino acids (P < 0.01; Fig. 1A and B). PI3K/Akt/mTORC1 and Ras/MEK/ERK are 2 of the most frequently hyperactivated pathways in cancer cells, we therefore investigated whether inhibiting these 2 pathways by mTORC1 inhibitor, rapamycin, class I PI3K/mTORC1 dual inhibitor, PI103 and MEK1/2 inhibitor, PD184352 may affect the sensitivity of cancer cells to Ran silencing–induced apoptosis. As expected, treatment of the MDA-MB-231 and A549 cells with these drugs inhibited phosphorylation of their corresponding downstream targets (Supplementary Fig. S4) and reduced cell proliferation (Supplementary Fig. S5). These inhibitors of the PI3K/Akt/mTORC1 or MEK/ERK pathways significantly reduced Ran silencing–induced apoptosis in MDA-MB-231 and A549 cells (P < 0.05; Fig. 1C and D). Similar results were obtained with MTT cell survival assay (Supplementary Fig. S6). These results suggest that Ran may be preferentially required for survival in cancer cells with higher activities of the PI3K/Akt/mTORC1 and MEK/ERK pathways.

Ran silencing resulted in a significantly higher apoptotic response in the mutant K-Ras transformed cells compared with the wild-type (WT) K-Ras counterparts in the DLD-1/DKO-3 colon cancer isogenic pair (P < 0.01; Fig. 2A–C). This result suggests that cancer cells with Ras activation are more susceptible to Ran silencing–induced apoptosis. Mutant K-Ras activates the MEK/ERK and PI3K/Akt/mTORC1 pathways to increase cell proliferation and cell survival (22). Inhibitors of PI3K or MEK, but not mTORC1, reduced the mutant K-Ras-mediated sensitization of the cells to Ran silencing–induced apoptosis (Fig. 2D). The results were confirmed by Western blot of cleaved PARP, caspase 3 and 9 (Fig. 2E–G). Similar results were obtained by the HCT116 and Hkh-2 colon cancer isogenic pair, in which inhibitors of PI3K/Akt or MEK/ERK pathways abolished mutant K-Ras–mediated sensitization to Ran silencing–induced apoptosis (Supplementary Fig. S7). The results were further confirmed by MTT cell survival assay showing that cancer cells with mutant K-Ras were more susceptible to Ran silencing–induced apoptosis than their wild-type counterparts (Supplementary Fig. S8A and S8B). Our results show that isogenic cancer cells with activated K-Ras are more susceptible to Ran silencing–induced apoptosis, and this may be due to the higher activities of the PI3K/Akt and MEK/ERK pathways.

c-Met amplification has been shown to promote resistance to the EGFR inhibitor, gefitinib, in lung cancer cells through persistent activation of the PI3K/Akt and MEK/ERK pathways (23). The effect of Ran knockdown on apoptotic induction in a pair of lung cancer cell lines, HCC827 parental cells and HCC827 GR5 c-Met amplified cells was also tested. HCC827 parental cells have a significant level of c-Met phosphorylation, whereas HCC827 GR5 cells, generated by prolonged exposure of gefitinib, have increased levels of total c-Met and phosphorylated c-Met (23). Upon treatment of gefitinib, the pAkt and pERK levels were greatly decreased in HCC827 parental cells, whereas their levels are persistent in GR5 cells (23). Ran silencing resulted in a significantly higher apoptotic response in GR5 than in HCC827 cells (P < 0.01; Fig. 3A and Supplementary Fig. S8C), suggesting that cells with c-Met amplification are more susceptible to Ran silencing–induced apoptosis. Similar to mutant K-Ras, inhibitors of the PI3K/Akt and Ras/MEK/ERK pathways significantly reduced c-Met amplification–mediated sensitization to Ran silencing–induced apoptosis (P < 0.01; Fig. 3B). Interestingly, Ran silencing induced a significant apoptotic response in GR5 cells in the presence of gefitinib but not in HCC827 parental cells (P < 0.01; Fig. 3B), suggesting that cells that are resistant to gefitinib may maintain their sensitivity to Ran silencing–induced apoptosis. Our results show that isogenic cancer cells with c-Met amplification are more susceptible to Ran silencing–induced apoptosis and this effect is probably dependent on the PI3K/Akt and MEK/ERK pathway.

In another colon cancer isogenic cell pair, the HCT116 WT and Pten-null cell lines, the activation of the PI3K/Akt pathway through deletion in Pten sensitizes the cancer cells to Ran silencing–induced apoptosis also potentially in a PI3K/Akt/mTORC1 pathway-dependent manner (Fig. 3C–D).

We investigated whether a high level of immunohistochemical detectable Ran is associated with aggressive breast and lung cancers. Breast cancer patients with a higher percentage of malignant cell nuclei stained for Ran in their primary tumors had a shorter median survival time than those with less than 1% of malignant cell nuclei stained for Ran in their primary tumors (Fig. 4A and B, P < 0.001). When analyzed with the other prognostic of tumor size, grade, and lymph node involvement in the Cox-regression analysis, staining for nuclear Ran was independently associated with patient survival time [relative risk (RR) = 12.53, 95% CI: 3.95–39.758, P < 0.001; Supplementary Table S3A]. Similar results were obtained for cytoplasmic staining for Ran. Thus, patients with a higher cytoplasmic staining for Ran had a significantly shorter survival time than those with less than 1% of cytoplasmic staining for Ran in their primary tumors (P < 0.001; Fig. 4A and C). When analyzed with the other prognostic factors, staining for cytoplasmic Ran was independently associated with patient survival time (RR = 2.50, 95% CI: 1.59–3.93, P = 0.001; Supplementary Table S3B). Higher levels of staining for Ran were also significantly correlated with a shorter patient survival time in lung cancer patients (P = 0.001; Supplementary Fig. S9A and S9B). When analyzed with other prognostic factors of clinical stage, lymph node involvement, and histology, this overall staining for Ran was independently associated with patient survival times in the Cox-regression analysis (RR = 1.63, 95% CI: 1.08–2.44, P = 0.019; Supplementary Table S3C). Results were substantiated with patient data available in GEO database. A high level of Ran mRNA expression was associated with poorer prognosis in both breast (RR = 1.57, 95% CI: 1.06–2.32, P = 0.022; Fig. 4D) and lung (RR = 1.591, 95% CI: 1.139–2.222, P = 0.006; Supplementary Fig. S9C) cancer patients.

We have also analyzed the GEO breast cancer data set with more than 200 patients (GSE2034; Fig. 4E), in which we have sufficient information to identify patients with PIK3CA activation mutations that lead to an activation of the PI3K/Akt/mTORC1 pathway based on the gene signature described previously (24). In this data set, a high level of Ran was significantly correlated with shorter survival time in patients with PIK3CA mutation gene signature (Fig. 4F, P = 0.018), but not in those with PIK3CA wild-type gene signature (Fig. 4G, P = 0.186).

There was a much greater difference in patient survival time between high and low level of Ran in c-Met–positive tumors (median survival 58.1 months, final proportion surviving 18% vs. >216 months, 81%, respectively; Wilcoxon–Gehan χ² = 16.5, P < 0.001; Fig. 4H) than in c-Met–negative tumors (>216 months, 78% vs. >216 months, 100%, respectively; Wilcoxon–Gehan χ² = 9, P = 0.003; Fig. 4I). Similar results were obtained when using a metastasis-promoting protein, osteopontin (OPN), which has been shown to activate the PI3K/Akt and MEK/ERK pathways and is available in the same cohort (20, 25, 26), to stratify the patients. The difference in patient survival time was much greater between high and low Ran in OPN-positive tumors (57.2 months, 16% vs. >216 months, 79%, respectively; Wilcoxon–Gehan χ² = 16.7, P < 0.001; Fig. 4J) than in OPN-negative tumors (>216 months, 86% vs. >228 months, 100%, respectively; Wilcoxon–Gehan χ² = 6.58, P = 0.01; Fig. 4K).

A colorectal cancer data set (GSE16125) that records K-Ras mutation, survival data and Ran mRNA expression was also analyzed. In patients whose tumors did not contain mutated K-Ras, 71% and 73% patients died with high and low levels of Ran, respectively (Fisher Exact test, P = 1.000). In contrast, in patients with K-Ras–activating mutation and a high level of Ran, the median survival time was 10 months (7 out of 7 patients died), whereas for those with K-Ras–activating mutation and a low level of Ran, the median survival time was 62 months (6 out of 12 patients died; Fisher Exact test, P = 0.044). Taken together, these results suggest that a high level of Ran is specifically required for human cancers with mutations that correlate with activation of the PI3K/Akt/mTORC1 or Ras/MEK/ERK pathways.

We also investigated the role of nucleocytoplasmic transport on Ran silencing–induced apoptosis. Rapamycin significantly inhibited Ran silencing–induced apoptosis in MDA-MB-231 breast cancer cells (P < 0.001) and this was reversed by Leptomycin B (LMB; P < 0.001; Fig. 5A and B), which impairs nuclear export by covalently binding to CRM1 (27). LMB treatment also suppressed the inhibitory effect of PD184352 on Ran silencing–induced apoptosis in MDA-MB-231 cells (P < 0.01; Fig. 5C and D). LMB also reversed the rapamycin and PI103-mediated suppression on Ran silencing–induced apoptosis in A549 lung cancer cells (Supplementary Fig. S10A–S10D). In addition, nuclear localization of β-catenin and NF-κB was decreased and that of p53 and p27 was increased in MDA-MB-231 cells (Fig. 5E); similarly, nuclear localization of β-catenin was decreased and that of p53, p27, and c-jun was increased in A549 lung cancer cells upon Ran silencing (Supplementary Fig. S10E). In MDA-MB-231 cells, the decrease in β-catenin and increase in p27, but not that of p53 and NF-κB, in the nucleus were diminished by treatment with rapamycin or PD184352 (Fig. 5F and G). Similarly, in A549 cells, the decrease in β-catenin and increases in p53 and p27 in the nucleus were diminished by treatment with rapamycin or PI103 (Supplementary Fig. S10F–S10G). These results suggest that the dysregulation of nuclear localization of transcription factors upon Ran silencing requires active PI3K/Akt/mTORC1 and MEK/ERK pathways and that nucleocytoplasmic transportation plays an important role in governing PI3K/Akt/mTORC1 and MEK/ERK pathway-mediated sensitization of cancer cells to Ran silencing–induced apoptosis.

In addition to the above changes in subcellular localization of transcription factors, the protein level of a prosurvival upstream regulator of caspase 9, Mcl-1, was decreased upon Ran silencing, but not that of other prosurvival and proapoptotic factors tested (data not shown). Indeed, Mcl-1 mRNA was significantly downregulated in both MDA-MB-231 breast (Fig. 5H) and A549 lung (Supplementary Fig. S10H) cancer cells upon Ran knockdown. This result suggests that the observed reduction in Mcl-1 occurs at the transcriptional level and is probably due to the dysregulation of nucleocytoplasmic transportation upon Ran silencing. Most importantly, Ran silencing–mediated Mcl-1 downregulation was reversed by inhibitors of the PI3K/Akt/mTORC1 and Ras/MEK/ERK pathways in both MDA-MB-231 (Fig. 5I–L) and A549 cells (Supplementary Fig. S10I–S10L).

Activation of the PI3K/Akt/mTORC1 and Ras/MEK/ERK pathways is common in different types of cancer. In addition to cell survival, these 2 pathways promote cell proliferation, angiogenesis, metastasis as well as other important cellular responses (28, 29). In this report, we show that cancer cells with K-Ras–activating mutation, c-Met amplification, or Pten deletion are more sensitive to Ran silencing–induced apoptosis. Because they can be inhibited by relatively specific inhibitors of the PI3K/Akt/mTORC1 and MEK/ERK pathways, our results, therefore, show that Ran silencing may be potentially dependent on these 2 pathways. Thus, Ran silencing–induced apoptosis is likely to be specific for cancers with mutations that correlate with activation of these 2 pathways. Activating mutations of PIK3CA and K-Ras, c-Met activation, and OPN overexpression have been previously shown to correlate with activation of the PI3K/Akt/mTORC1 and MEK/ERK pathways (3, 24–26, 30, 31). Because Ran expression is associated with reduced survival of patients with mutation of PIK3CA or K-Ras, or increased expression of c-Met or OPN, our results suggest that Ran may be a potential therapeutic target for cancers with hyperactivation of the PI3K/Akt/mTORC1 and MEK/ERK pathways induced by various mutations or overexpression of specific protooncogenes.

Ran is involved in regulating the rate of nucleocytoplasmic shuttling, both nuclear import as well as nuclear export (7, 32). Growth signaling pathways transmit their signals by regulating the subcellular location of transcription factors, which finally convey the signals to modulate the transcriptome for an appropriate response (4). mTORC1 has been shown to affect translation of different transcription factors in response to growth and stress signals (33, 34). Akt is regulated through its nuclear export signal (35), whereas PI3K/Akt pathway also regulates expression and localization of growth, apoptosis, and stress response–mediated transcription factors (36–39). Similarly, ERK is regulated by nucleocytoplasmic transport (40, 41) and itself also regulates localization of transcription factors in response to various stimuli (42, 43). Ran is an important mediator in nucleocytoplasmic transportation, Ran activity, which itself is regulated by growth signaling (13, 17, 18), may be required to fulfill nucleocytoplasmic transport requirements for proper response to various stimuli (4, 6).We suggest that Ran silencing may result in an impaired or decreased rate of nucleocytoplasmic transportation, leading to the uncoupling of the growth stimuli and the required translocation of transcription factors. Indeed, we have found that the nuclear localization of β-catenin, NF-κB, p53, p27, and c-jun is altered upon Ran silencing and the Mcl-1 transcript is downregulated. Interestingly, p53 has been shown to transcriptionally repress (44), whereas NF-κB, the PI3K/Akt, and Ras/MEK/ERK pathways have been shown to transcriptionally stimulate, Mcl-1 (45–47). Thus, the overall effect of the alteration of the nuclear levels of transcription factors upon Ran silencing may lead to altered transcription of the survival/apoptotic effectors thereby resulting in apoptosis. Further analysis, probably through DNA microarray and 2D gel electrophoresis of nuclear/cytoplasmic fractionated proteins, is required for a clearer picture of the overall effect of Ran silencing on nucleocytoplasmic transportation and gene expression.

Another possible explanation is that Ran silencing may induce apoptosis in cancer cells with a higher proliferation rate. As serum withdrawal, inhibitors of the PI3K/Akt/mTORC1 and Ras/MEK/ERK pathways and removal of the active K-Ras allele in the isogenic cells result in decreased cell proliferation, which also leads to a reduced sensitivity to Ran silencing–induced apoptosis.

The GR5 cells is different from its parental HCC827 cells, in which c-Met amplification in GR5 cells sustains the PI3K/Akt signaling in the presence of EGFR inhibitor (23). We found that knockdown of Ran leads to a significant increase in apoptotic response in GR5 cells, but not in the HCC827 parental cells, in the presence of gefitinib. The persistent activation of PI3K/Akt and MEK/ERK pathways in GR5 cells treated with gefitinib may contribute to the Ran silencing–induced apoptosis. Our results therefore suggest that targeting Ran can also be used in combination with other therapeutic agents for cancer treatment in the future.

In conclusion, this study shows that Ran silencing results in apoptosis preferentially in cancer cells with mutations that correlate with activation of the PI3K/Akt/mTORC1 and Ras/MEK/ERK pathways.

No potential conflicts of interest were disclosed.

The authors thank CRUK for the postdoctoral fellowship to H.-F. Yuen.

This work was supported by Cancer and Polio Research Fund for funding the immunohistochemical studies on breast cancer.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.