Phospholipase D1 Acts through Akt/TopBP1 and RB1 to Regulate the E2F1-Dependent Apoptotic Program in Cancer Cells

Deregulation of the retinoblastoma tumor suppressor, RB1, and E2F pathway is a common feature of many cancers (1). RB1 mainly exerts its tumor-suppressive effects by restraining the activity of E2F transcription factor, which subsequently regulates cellular proliferation and apoptosis depending on the cellular contexts (2). Although RB1 is inactivated in a wide variety of human cancers, loss or mutations of the RB1 gene are rare, and the RB1 gene is frequently overexpressed in colorectal cancer (3), which is linked to the antiapoptotic function of the protein. However, the mechanisms by which RB1 regulates apoptosis are yet to be fully elucidated. Although recent studies suggest that there is a cross-talk of the RB1/E2F1 pathway with the Wnt signaling pathways in colorectal cancer (4, 5), the mechanism underlying their functional interaction remains elusive. Most human colorectal cancers involve somatic mutations in the adenomatous polyposis coli (APC) tumor suppressor gene, which leads to the activation of Wnt signaling via stabilization and nuclear accumulation of β-catenin and its interaction with TCF transcription factor and subsequent transcriptional activation of Wnt target genes (6). E2F1 has been identified as a negative regulator of β-catenin/TCF-dependent transcription (4). Although a recent study attributed the retention of RB1 function in colorectal cancer cells to its ability to repress E2F-1–dependent inactivation of β-catenin signaling (7), little is known about the molecular mechanism linking these entangled pathways in colorectal cancer. The expression of RB1 can promote cell survival through repression of E2F-driven apoptosis (8). Thus, the interplay between RB1/E2F1 and Wnt/β-catenin signaling pathways might be even more complex than originally thought. Accordingly, identification of critical effectors regulating the multifaceted cross-talk between signaling pathways will lead to the nomination of new candidates for creation of targeted therapies useful to colorectal cancer management. We recently demonstrated that phospholipase D1 (PLD1) as a new transcriptional target of β-catenin/TCF4 promotes Wnt signaling (9, 10), revealing bidirectional cross-talk between the PLD1 and Wnt/β-catenin pathways. In addition, targeting of PLD1 attenuates intestinal tumorigenesis by suppressing β-catenin signaling (11). PLD hydrolyzes phosphatidylcholine to generate bioactive lipid phosphatidic acid (PA) and is upregulated in various human cancers, including colorectal cancer (12). Although PLD has been implicated in many survival and proliferation signals (13), the molecular mechanisms linking RB1/E2F1 and the PLD1 pathway in colorectal cancer have not been addressed. As the RB1/E2F1, PI3K/Akt, and Wnt/β-catenin pathways intersect in colorectal cancer, we investigated whether PLD1 acts as a modulator of functional interaction among the pathways in intestinal tumorigenesis.

Apc^min/+Pld1^−/− and Apc^min/+Pld1^Tg mice were generated as described previously (11). Apc^Min/+Pld1^−/−Pld1^Tg mice were generated by interbreeding mice carrying Pld1^Tg with Apc^Min/+Pld1^−/− mice. An azoxymethane (AOM)/dextran sodium sulfate (DSS)–induced mice were treated with 10 mg/kg PLD1 inhibitor (VU0155069, Cayman Chemical) as described previously (11). Animal studies were approved by the Institutional Animal Care Committee of Pusan National University (Busan, Republic of Korea).

Fifty-five human colon tumor and adjacent normal tissues for q-PCR analysis were obtained from Pusan National University Hospital (Busan, Republic of Korea), a member of the National Biobank of Korea, which is supported by the Ministry of Health and Welfare. All samples derived from the National Biobank of Korea were obtained with informed consent under Institutional Review Board–approved protocols. For immunohistochemical analysis, tissue microarray (TMA) slides were purchased from SuperBioChips (CDA2, CDA3, and CD4) and US Biomax (CC05-01-002). Each array includes more than 178 cases of normal, reactive, premalignant, and malignant tissues of the colon (various grades and stages). Overall survival was calculated from the date of treatment start to the date of death. Survival curves were constructed using the Kaplan–Meier methodology. Log-rank tests were used to assess differences in tumor characteristics.

Anti-NOXA (Imgenex), anti-RB1, anti-p-Akt, anti-p27KIP1, and anti-Bim antibodies (Santa Cruz Biotechnology) were used as the primary antibodies. The anti-PLD1 antibody was generated as described previously (14).

HCT116 p53^+/+ and p53^−/− isogenic cell lines were kindly gifted by Dr. Bert Vogelstein (Johns Hopkins University, Baltimore. MD). MEFs from E2F1^+/+ and E2F1^−/− mice were kindly provided by Dr. Han Woong Lee (Yonsei University, Seoul, South Korea). The human colorectal cancer cells: SW480 (ATCC CCL-228, passage number 04 - 18) and DLD1 (ATCC CCL-221, passage number 03 - 17) were obtained from the ATCC. Cell lines purchased from ATCC were analyzed by STR profiling. Tumorspheres were grown in B27 (Invitrogen) supplemented with 20 μg/mL bFGF and 20 μg/mL EGF (Sigma), and penicillin/streptomycin on ultra-low attachment culture dishes (Corning), as a sphere culture condition.

Apoptotic cell death was measured by APC-conjugated Anti-Annexin V Apoptosis Detection Kit I (BD Biosciences). The terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling (TUNEL) assay was performed using In Situ Cell Death Detection Kit, POD (Roche), according to the manufacturer's protocol.

Cell lysates were analyzed by immunoprecipitation and/or immunoblotting as described previously (11). Enhanced chemiluminescence was used for detection of the signal. The following antibodies were used: anti-α-tubulin, anti-c-Myc, anti-RB1, anti-phospho-RB1, anti-Bim, anti-p27KIP1, anti-p73, anti-HA, anti-mouse-IgG, and anti-rabbit-IgG (Santa Cruz Biotechnology); anti-NOXA (Thermo Fisher Scientific); TopBP1 (Abnova); anti-E2F1, anti-FLAG, and anti-phospho-serine (Sigma); anti-active caspase-3, anti-phospho-Akt, and anti-Akt1 (Cell Signaling Technology).

The phycoerythrin (PE)-conjugated anti-CD133 (Miltenyi Biotec) or allophycocyanin-conjugated anti-CD44 (BD Biosciences) antibodies were used for FACS analysis. To obtain CD133⁺CD44⁺ or CD133^–CD44^– cells, xenografted DLD1 tumor cells stained with PE-conjugated anti-CD133 and allophycocyanin-conjugated anti-CD44 antibody (both 1:40) were subjected to cell sorting performed by the FACS.

To determine the number of sphere-forming units, in vitro limiting dilution assays were performed as described previously (11, 15).

Microarray analysis was carried out by Macrogen Inc. Briefly, biotinylated complementary RNAs were amplified and purified using an Illumina RNA Amplification Kit (Ambion) according to the manufacturer's instructions. Labeled cRNA samples were hybridized to each HumanHT-12 expression v.4 bead array for 16 to 18 hours at 58°C, according to the manufacturer's instructions (Illumina). Detection of array signal was carried out using FluoroLink streptavidin-Cy3 (GE Healthcare Bio-Sciences). Arrays were scanned with an Illumina BeadArray Reader confocal scanner, and the scanned images were analyzed using an Illumina BeadStudio v3.1.3 software (Gene Expression Module v3.3.8). All data normalization and selection of fold-changed probes were performed using GeneSpringGX 7.3 (Agilent Technologies). We performed data transformation (set measurements <0.01–0.01) and per chip (normalize to 75th percentile) normalization. Probes that were changed not less than 1.25-fold and 2-fold of ratio in shPLD1/shCTRL and shPLD1-shE2F1/shPLD1 groups were selected and considered as differentially expressed probes.

The mRNA and miRNA microarray data reported in this article are GSE55724 and GSE55771, respectively.

The primer sets are listed in Supplementary Tables S1–S3.

Data were analyzed using the paired t test or ANOVA test, and correlation coefficients were calculated using Spearman r. Colorectal cancer patient survival probability, defined as the time from colon resection to death or the date of last follow-up, was analyzed using Kaplan–Meier method, and differences were evaluated using log-rank test. Immunohistochemical staining results were analyzed using χ² test. Statistical analysis was performed using Origin 8.0 and GraphPad Prism 5.0.

For detailed information, please refer to the Supplementary Materials and Methods.

Recently, we reported that intestinal epithelial cell (IEC)-specific PLD1 overexpression in Apc^Min/+ mice accelerates intestinal tumorigenesis with increased proliferation and β-catenin level, compared with Apc^Min/+ mice, whereas loss of PLD1 in Apc^Min/+ mice suppresses the intestinal tumorigenesis based on tumor number, tumor size, and survival probability (11). Evasion of apoptosis is a hallmark of cancer and contributes both to the development of the tumor and the refractory nature of tumors to treatment (16). Although PLD1 is known to protect apoptosis in vitro (17, 18), the role of PLD1 in the regulation of apoptosis in vivo mice models has not yet been described. Thus, we examined whether PLD1 regulates apoptotic function in vivo tumor model using Apc^Min/+ mice. The proportions of TUNEL-positive apoptotic cells in intestinal tumors of 14-, 16-, and 20-week-old Apc^Min/+Pld1^−/− mice were remarkably higher than in Apc^Min/+ control mice (Fig. 1A). IEC-specific PLD1 overexpression in Apc^Min/+Pld1^−/− mice (Apc^Min/+Pld1^−/−Pld1^Tg) significantly reduced the proportions of the apoptotic cells induced in Apc^Min/+Pld1^−/− mice (Fig. 1A). In accordance with the result, the mortality of Apc^Min/+Pld1^−/−Pld1^Tg mice was significantly increased relative to Apc^Min/+Pld1^−/− mice, whereas Apc^Min/+Pld1^−/− mice exhibit an increased survival rate compared with Apc^Min/+ littermate controls (Fig. 1B). Serum deprivation under tumor microenvironment simulates the harsh growth environments encountered by neoplastic cells prior to vascularization and restoration of nutrient supply within the tumor mass. PLD1 depletion increased apoptosis of the HCT116 cells in the presence of 0%, 0.5%, or 1% of serum but not 3% or 10% of serum (Fig. 1C). Ectopic expression of PLD1 in PLD1-depleted cells protected the apoptosis induced by serum deprivation (Fig. 1C). These results are comparable with those of Apc^Min/+mice. Moreover, PLD1 inhibition also showed that the results are comparable with those of PLD1 depletion (Supplementary Fig. S1A). The serum deprivation significantly increased the formation of PA, a product of PLD activity, which was suppressed by PLD1 inhibitor (VU0155069; Supplementary Fig. S1B), suggesting that serum deprivation–generated PA may prevent cancer cells from apoptosis. Taken together, these results indicate that PLD1 plays a crucial role in the modulation of apoptosis in vivo and in vitro under tumor microenvironment.

The antiapoptotic survival signals generated by PLD are known to be mediated, at least in part, by suppression of the p53 response pathway (19, 20). Thus, we examined the role of p53 in PLD1-mediated apoptotic regulation using HCT116 p53-positive and -negative isogenic cell lines. Contrary to our expectation, depletion and inhibition of PLD1 induced apoptosis independent of p53 (Fig. 2A). Recently, we performed an mRNA microarray in DLD1 and HCT116 cells under serum-deprived conditions (11). The genes commonly up- or downregulated in PLD1 inhibitor–treated or PLD1-depleted cells were visualized by scatter diagrams (Fig. 2B). PLD1 inactivation increased expression of the positive regulators of apoptosis (Bim, p73, NOXA, p27KIP1, DR5, Cyp26b1, p14ARF, and Nr4A3) and endoplasmic reticulum stress (CHOP, GCLC, Mif1, and Sos2), but decreased the expression of RB1 and cell cycle progression–related genes (Fig. 2B; Supplementary Fig. S2). PLD1 inhibition suppressed the protein level of endogenous RB1 in HCT116 cells, whereas PLD1 inhibition–induced apoptosis under serum starvation was prevented by ectopic expression of RB1 (Fig. 2C). Moreover, depletion of RB1 induced apoptosis in serum-deprived cells, whereas depletion of both RB1 and PLD1 did not further promote apoptosis compared with that of PLD1 depletion, suggesting that RB1 repression may be involved in the downstream regulation of apoptosis by PLD1 depletion (Fig. 2D). Apparently, contradicting its role as a classical tumor suppressor protein, colorectal cancer cells retain the expression of RB1; they express high levels of RB1 compared with adjacent normal tissue, and loss or mutations of the RB1 gene are rare (7, 21). Ablation of PLD1 in Apc^Min/+mice showed decreased level of RB1 in the intestinal tumors, whereas IEC-specific PLD1 overexpression recovered the levels of RB1 decreased in the tumor tissues from Apc^Min/+Pld1^−/− mice (Fig. 2E). In addition, PLD1 inhibition suppressed the expression of RB1 in the tumor tissues from Apc^Min/+mice (Supplementary Fig. S3). Moreover, ablation of PLD1 in an AOM/DSS–induced mouse colon cancer model decreased the level of RB1 relative to that of control mice (Supplementary Fig. S4). Collectively, these data suggest that PLD1 inactivation–mediated apoptosis is intimately associated with downregulation of RB1.

We further examined how PLD1 regulates the level of RB1. Depletion and inhibition of PLD1 decreased the mRNA level of RB1, which was not affected by Akt1 and E2F1 depletion (Supplementary Fig. S5A). RB1 is epigenetically silenced by hypermethylation of the CpG island within its promoter region (22). Histone methyltransferases, which bind to methylated CpG dinucleotides, not only recruit histone deacetylase, but also facilitate the methylation of H3 lysine 9, thereby reinforcing an inactive chromatin state (23). PLD1 inactivation did not affect DNA methylation (Supplementary Fig. S5B) or the binding of histone methyltransferase (H3K9me2) and acetyltransferase (H3K9ac) to the RB1 promoter (Supplementary Fig. S5C). Moreover, targeting PLD1 did not affect the promoter activity of RB1 (Supplementary Fig. S5D). miRNAs silence gene expression by binding to the 3′ untranslated regions (UTR) of target miRNAs, inhibiting their translation or marking them for degradation (24). Targeting of PLD1 abolished the luciferase activity of RB1 3′ UTR reporter (Fig. 3A), suggesting the involvement of miRNA in the regulation of RB1 expression by PLD1. miRNA microarray analysis showed that a total of 61 miRNAs displayed at least a 2-fold change in PLD1 inhibitor-treated DLD1 cells (GSE55771). On the basis of the bioinformatic approach, we found that miR-192 and -4465 upregulated by PLD1 inhibition have a putative target site in the 3′ UTR of RB1 that is conserved in various species (Fig. 3B). PLD1 inactivation significantly increased the expression of these miRNAs (Fig. 3C). Deletion of the putative binding sites of the miRNA recovered the decrease in luciferase activity of RB1 3′ UTR that occurred in response to PLD1 inactivation (Fig. 3D). Their precursor (pre)-miR inhibited RB1 expression (Supplementary Fig. S6), whereas anti-miR recovered the luciferase activity of RB1 3′ UTR decreased by the targeting of PLD1 (Fig. 3E), suggesting that PLD1 inhibition downregulates RB1 expression at the posttranscriptional level via miR-192 and -4465. Moreover, the expression of RB1 showed a statistically inverse correlation with the expression of these miRNAs in colorectal cancer tissues (Fig. 3F), suggesting in vivo relevance. These data suggest that targeting PLD1 downregulates RB1 via miR-192 and -4465.

The E2F1 transcription factor can promote proliferation or apoptosis when activated and is a key downstream target of the RB1. The interaction of RB1 with E2F1 is known to inhibit E2F1 transactivation (25). Interestingly, PLD1 inactivation–induced apoptosis was suppressed by the depletion or ablation of E2F1, and this repression was recovered by the ectopic expression of E2F1 (Fig. 4A and B). The PI3K/Akt signaling pathway regulates E2F1 through the E2F1-interacting protein, TopBP1 (topoisomerase II β binding protein), which inhibits E2F1-dependent apoptosis (26). Phosphorylation of TopBP1 by Akt is crucial to interaction of TopBP1 with E2F1 and its repression. PLD1 inactivation greatly decreased the phosphorylation of Akt/TopBP1, RB1 expression, and the TopBP1–E2F1 and RB1–E2F1 interaction (Fig. 4C). In addition, the expression of PLD1 or ca-Akt1 increased the phosphorylation of TopBP1 and the interaction of TopBP1 with E2F1 (Fig. 4D). Targeting of PLD1 in Apc^Min/+and AOM/DSS mouse models reduced the phosphorylation of Akt, RB1 expression, and the interaction of E2F1 with RB1 or TopBP1 (Fig. 4E). IEC-specific PLD1 overexpression recovered the levels of p-Akt decreased in the tumor tissues from Apc^Min/+Pld1^−/− mice (Fig. 4F). Moreover, IEC-specific PLD1 overexpression in Apc^Min/+ mice (Apc^Min/+Pld1^Tg) increased the levels of RB1 and p-Akt in the intestinal tumors, relative to Apc^Min/+ (Supplementary Fig. S7). In addition, PLD1 inhibition in the tumor tissues from Apc^Min/+or AOM/DSS mice decreased the level of p-Akt (Supplementary Fig. S8A and S8B). Taken together, these results suggest that both RB1–E2F1 and Akt–TopBP1–E2F1 signaling pathways are involved in PLD1-mediated apoptotic regulation.

Next, we examined potential downstream apoptotic effectors of the PLD1–E2F1 axis using microarray analysis in PLD1- and E2F1-depleted cells. Expression of some genes increased by PLD1 depletion was decreased by E2F1 depletion in two colorectal cancer cells (Fig. 5A). These genes are assumed to be targets of E2F1. A recent study identified a subset of E2F1 target genes required for E2F1-induced apoptosis that is specifically repressed by the PI3K/Akt signaling pathway (27). PLD1 inhibition significantly enhanced the expression of the proapoptotic E2F1 target genes (NOXA, CHOP, Nr4a3, p27KIP1, p73, and Bim) in serum-deprived HCT116 cells and that this enhancement was abolished by the depletion of E2F1 or expression of ca-Akt1 (Fig. 5B and C). Moreover, PLD1 inactivation in Apc^Min/+ mice enhanced the expression of the proapoptotic E2F1 target genes (Fig. 5D), suggesting in vivo relevance. Moreover, PLD1 inhibition enhanced the binding of E2F1 to promoters of its target genes, which was decreased by the expression of RB1 and ca-Akt1 (Fig. 5E). Furthermore, depletion of E2F1 target genes impaired PLD1 inhibition or E2F1-induced apoptosis under serum-deprived conditions (Fig. 5F; Supplementary Fig. S9). Taken together, these results suggest that PLD1 regulates the E2F1-dependent apoptotic program in vivo and in vitro.

We further investigated whether miR-192 and -4465 affect the E2F1 target gene expression and apoptosis. The anti-miR-192/4465 suppressed PLD1 inactivation–induced proapoptotic E2F1 target gene expression and apoptosis (Fig. 6A and B). Both pre-miR-192/4465 and Akt1 depletion further increased apoptosis under serum-deprived condition, compared with that of either expression (Fig. 6B), suggesting that both RB1 and Akt pathways are required for the promotion of apoptosis. We recently have reported that PLD1 is highly upregulated in the population of colon cancer–initiating cells (CC-IC; CD133⁺CD44⁺) and involved in self-renewal capacity. Interestingly, overexpression of RB1, ca-Akt1, or anti-miR-192/4465 significantly recovered CD133⁺CD44⁺ population and sphere-forming capacity decreased by PLD1 depletion (Fig. 6C and D). Although treatment of 5-fluorouracil and oxaliplatin, clinically used for colorectal cancer therapy, has a marginal effect on apoptosis of colorectal cancer cells under sphere culture, treatment of the drugs in PLD1-depleted cells increased the chemosensitivity (Fig. 6E). However, expression of RB1, ca-Akt1, and miR-192/4465 significantly reduced PLD1 depletion–induced chemosensitivity. PLD1-mediated cancer-initiating capacity is associated with upregulation of β-catenin and its target genes (12). Moreover, the decrease in expression of β-catenin and its target genes, in response to PLD1 depletion, was recovered by the overexpression of RB1, ca-Akt1, and anti-miR-192/4465 (Fig. 6F; Supplementary Fig. S10). Furthermore, their overexpression recovered the binding of β-catenin to the promoter of its target genes, which was decreased by PLD1 depletion (Fig. 6G). Taken together, these results suggest that PLD1 regulates apoptosis and the self-renewal capacity of CC-ICs via the miR-192/4465–RB1 axis and Akt pathway.

We next attempted to demonstrate the physiologic relevance of the relationship of PLD1 level with the expression of E2F1 target genes, RB1, and miR-192/4465. The mRNA expression in 55 colorectal cancer tissues exhibited elevated expression of RB1, reduction of proapoptotic E2F1 target genes, miR-4465, and miR-192 (Fig. 7A), compared with that of normal tissues. Moreover, expression of PLD1 and the E2F1 target genes showed a strong negative correlation, whereas a positive correlation of PLD1 with RB1 was noted in colorectal cancer tissues as analyzed by qRT-PCR (Fig. 7B). miR-192, but not miR-4465, showed a significant negative correlation with PLD1 level in colorectal cancer tissues (Fig. 7B). Consistently, elevated expression of PLD1 in colorectal cancer was found to be positively correlated with levels of RB1 and p-Akt, but negatively correlated with the E2F1 targets as analyzed by IHC (Fig. 7C). The tumors were interpreted as low when immunostaining was weak, as in the corresponding normal colonic mucosa, or when immunopositive cells represented <10% of the cancer cells. Factors of multivariate variables, the basic features, and the protein group using Cox proportional hazards model, show that survival difference between PLD1/p27KIP1/Bim/NOXA expression groups are, in a statistical significance, associated with tumor stage (Supplementary Table S4), but not with gender, organ, and age. Fitted survival function for the protein groups, in which tumor stage was considered, shows that a low level of PLD1 with high expression of E2F1 targets (PLD1^L/p27KIP1^H PLD1^L/Bim^H, and PLD1^L/NOXA^H) was significantly correlated with higher overall survival of colorectal cancer patients, and vice versa (Fig. 7D; Supplementary Fig. S11). Taken together, these data support an intimate association of PLD1 expression with levels of the E2F1 target genes in the survival of colorectal cancer patients and suggest a potential value of PLD1 and E2F1 targets as prognostic biomarkers and therapeutic targets of colorectal cancer.

In the current study, we demonstrate that PLD1 has a previously unrecognized role in regulating E2F1-dependent apoptosis, which is entangled with the RB1 and Akt/TopBP1 pathways and emerges as a potential therapeutic target in colorectal cancer. The numerous signaling molecules affecting cancer cells operate as nodes and branches of elaborate integrated circuits that are reprogrammed derivatives of the circuits operating in normal cells (16). Our findings show that PLD1 orchestrates the cross-talk between RB1–E2F1 and Akt–TopBP1 signaling pathways and contributes to apoptotic regulation and self-renewal of colorectal cancer. Targeting these signaling networks is particularly attractive, as almost all human cancers exhibit alterations in these pathways; however, key modulators and the mechanism underlying the functional interaction among the signaling circuits in colorectal cancer remains elusive. Our findings that PLD1-mediated apoptotic regulation occurs through E2F1 and RB1, but not p53, suggest the possibility that PLD1 might be a new regulator of RB1–E2F1 signaling circuits in colorectal cancer cells. PLD1 inactivation increases the transactivation of E2F1 and its apoptotic program via the suppression of both RB1 and Akt–TopBP1 pathways. Genetic and pharmacologic targeting of PLD1 in Apc^Min/+ mice greatly increased the expression of proapoptotic E2F1 target genes, which ultimately is involved in the attenuation of intestinal tumorigenesis. Indeed, a variety of cancer-relevant molecules and signaling pathways intersect with the Wnt pathway and may modulate it. We recently demonstrated that the inactivation of PLD1 downregulates β-catenin expression via E2F1-induced miR-4496 upregulation (12). Ultimately, PLD1 regulates tumor-initiating capacity through the E2F1–miR-4496–β-catenin signaling axis (11). miR-4496 is a new target of E2F1 and a crucial node responsible for mediation of the cross-talk between E2F1 and Wnt/β-catenin signaling. PLD1 inactivation increases the expression of PI3K-repressed E2F1 target genes via both downregulation of RB1 and inhibition of the PI3K/Akt1–TopBP1 pathway, which is followed by activation of the apoptotic program and suppression of Wnt/β-catenin signaling. E2F1 can function as both a tumor suppressor and an oncogene under different conditions (28). Unlike other human cancers, in most colorectal cancers, although Wnt/β-catenin signaling is activated, E2F1 activity is kept at a low level because there are no mutations in RB1 (29). Moreover, instead of acting as an oncogene, E2F1 in human colorectal cancer may function as a tumor suppressor (30). It has long been known that RB1 in colon carcinoma is not mutated, but overexpressed, and even amplified (7, 21). Thus, in the context of colorectal cancer, RB1 is more likely to act as an oncoprotein than a tumor suppressor. In this regard, strategies focused on the control of E2F1 proteins hold particular promise, as activation of E2F activity is the ultimate consequence of deregulation of the RB1 pathway. E2F1 activity is negatively regulated by PLD1; accordingly, retaining RB1 and amplifying PLD1 may enable colorectal cancer cells to select for mechanisms that limit the activity of E2F1 and tip the balance toward β-catenin–driven proliferation conditions that suppress E2F1 and enhance the activity of β-catenin. Interestingly, PLD1 inactivation reduced the expression of RB1 via miR-192 and -4465; thus, downregulation of RB1 by targeting PLD1 may release active E2F1 and induce E2F1 transactivation. This is the first evidence that PLD1 regulates the expression of β-catenin and RB1 at the posttranscriptional level via the miRNA; accordingly, PLD1 inactivation regulates E2F1-mediated apoptosis and cancer-initiating capacity by modulating RB1 expression and Akt pathway, E2F1 transactivation, and β-catenin signaling. Interestingly, the survival probability between PLD1 and the E2F1 target protein–expressed patient groups was significantly associated with tumor stage, suggesting that the proteins might be potential prognostic biomarkers in colorectal cancer patients. Collectively, our findings demonstrate that PLD1 plays a crucial role in apoptosis and cancer-initiating capacity, via regulation of the cross-talk among RB1–E2F1, PI3K/Akt–TopBP1, and Wnt/β-catenin pathways. These results provide a missing link between the signaling networks mediating cancer and may fill the gap in current knowledge regarding how the complexity of these pathways is delicately regulated in cancer. Considering the highly complex interactions among cancer-relevant pathways, targeting these pathways by PLD1 inhibition might be an effective therapy for the treatment of colorectal cancer.

No potential conflicts of interest were disclosed.

Conception and design: D.W. Kang, D.S. Min

Development of methodology: D.W. Kang, D.S. Min

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): D.W. Kang, S.W. Lee, W.C. Hwang, Y.-A. Suh, K.-Y. Choi, D.S. Min

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): D.W. Kang, S.W. Lee, W.C. Hwang, B.H. Lee, Y.-S. Choi, D.S. Min

Writing, review, and/or revision of the manuscript: D.W. Kang, S.W. Lee, K.-Y. Choi, D.S. Min

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): D.W. Kang, S.W. Lee, Y.-A. Suh, D.S. Min

Study supervision: D.S. Min

This study was supported by a National Research Foundation of Korea(NRF) grant funded by the Korean government (NRF-2015R1A2A1A05001884), a Translational Research Center for Protein Function Control grant (2016R1A5A11004694), a National R&D Program for Cancer Control grant from the Ministry for Health, Welfare, and Family Affairs (Republic of Korea; 0920050), and Korean Health Technology R&D Project, Ministry of Health & Welfare, Republic of Korea (HI06C0868).

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.