PHLDA2 regulates EMT and autophagy in colorectal cancer via the PI3K/AKT signaling pathway

In vitro data

Levels of PHLDA2 in CRC tissue, HCT116 cells, and SW480 cells

Protein and mRNA levels in CRC and adjacent tissue were assessed by IHC (CRC tissue, n=99; adjacent tissue, n=27) and RT-qPCR (n=29). Levels of PHLDA2 in CRC tissue were higher than in adjacent normal tissue at both the protein level (χ²=18.90, P < 0.001, Figure 1A–1C) and mRNA level (P < 0.001, Figure 1D). Using WB and PCR, we found that protein and mRNA levels of PHLDA2 were higher in HCT116 and SW480 cells than in the six other CRC cell lines (Figure 1E–1G); therefore, these cell lines were used for subsequent experiments in our study.

Figure 1. PHLDA2 expression in CRC tissue, adjacent normal tissue, and cell lines. (A–C) Immuno-histochemical staining and analysis of PHLDA2 protein in CRC tissue and adjacent normal tissue (magnification, ×100 and ×400). (D) RT-qPCR was used to detect mRNA expression levels of PHLDA2 in 29 CRC tissues and paired normal tissues. (E–G) RT-qPCR and western blot analyses were used to detect mRNA and protein expression of PHLDA2 in six CRC cell lines. Data are shown as mean ± SD; *P < 0.05, **P < 0.01, and ***P < 0.001.

PHLDA2 levels correlate with clinicopathological features

In order to measure the clinical significance of PHLDA2, we investigated the relationships among PHLDA2 expression and clinicopathological characteristics of CRC patients. As shown in Table 1, PHLDA2 expression correlated with lymphatic metastasis (P = 0.025) and TNM stage (P = 0.009). No difference was found for age, gender, or distant metastasis. These results suggest that PHLDA2 may promote CRC progression.

PHLDA2 knockdown inhibits proliferation of CRC cells

Since we selected HCT116 and SW480 for in vitro studies, we generated stably-transfected cells with low PHLDA2 expression. The highest knockout efficiency was exhibited by pL-sh-1 (Figure 2A, 2B). Lentivirus vector (sh-PHLDA2) strongly inhibited PHLDA2 protein levels in HCT116 (P < 0.001, Figure 2C) and SW480 (P < 0.01, Figure 2D) cells. To investigate the effect of PHLDA2 in CRC cells, we evaluated cell proliferation. The CCK8 assay demonstrated that low-expression of PHLDA2 inhibited HCT116 (P < 0.01, Figure 2E) and SW480 (P < 0.01, Figure 2F) cell growth. Colony formation assays revealed low-expression of that PHLDA2 suppresses the proliferation of HCT116 (P < 0.001, Figure 2G) and SW480 (P < 0.01, Figure 2H) cells. These results demonstrate that low-expression of PHLDA2 inhibits the proliferation of CRC cells.

Figure 2. Inhibition of PHLDA2 inhibits CRC cell proliferation. (A, B) RT-qPCR was used to assess the knockout efficiency of three pLVX-sh-PHLDA2 knockdown fragments in HCT116 and SW480 cells. (C, D) Western blot was used to assess the knockout efficiency of the sh-PHLDA2 lentivirus vector in HCT116 and SW480 cells. (E–H) Cell Counting Kit-8 (CCK8) and colony formation assays were used to assess cellular proliferation. Data are shown as mean ± SD; *P < 0.05, **P < 0.01, and ***P < 0.001.

PHLDA2 knockdown in CRC cells inhibits migration and invasion by downregulation of EMT

To assess the effect of PHLDA2 on migration and invasion of CRC cells, we performed Transwell and Matrigel assays. Invasion and migration by HCT116 (P < 0.01, Figure 3A) and SW480 (P < 0.01, Figure 3B) cells were reduced by sh-PHLDA2. Sh-PHLDA2 also reduced the levels of EMT-related proteins including; N-cadherin, Vimentin, β-catenin, and MMP2. In contrast, sh-PHLDA2 increased E-cadherin levels in CRC cells (Figure 3C–3E). Therefore, a reduction in PHLDA2 levels inhibits invasion and migration by CRC cells through effects on EMT.

Figure 3. Low PHLDA2 expression inhibits CRC cellular migration and invasion by down regulation of EMT. (A, B) Invasion and migration of HCT116 and SW480 cells were measured by flow cytometry. (C) Western blots showing the levels of EMT-associated proteins; Vimentin, N-cadherin, E-cadherin, β-catenin, and MMP2. (D, E) Protein expression and sub-localization of E-cadherin as visualized by immunofluorescence. Data are shown as mean ± SD; *P < 0.05, ** P< 0.01, and ***P < 0.001.

PHLDA2 knockdown induces cell cycle arrest and increases apoptosis of CRC cells

Analysis of cell cycle distribution and apoptosis of HCT116 and SW480 cells showed that knockdown of PHLDA2 inhibition stalled cells in G1/S phase (P < 0.001, Figure 4A) and increased apoptosis (Figure 4B). Western blot demonstrated an increase in the expression of apoptosis-related proteins, including Cleaved caspase-3, Cleaved PARP, and Bax. Furthermore, sh-PHLDA2 transfection decreased Bcl-2 levels in CRC cells (Figure 4C). These results suggest that inhibition of PHLDA2 induces cell cycle arrest and apoptosis of HCT116 and SW480 cells.

Figure 4. Inhibition of PHLDA2 induces cell cycle arrest in G1/S phase and increases apoptosis in CRC cells. (A, B) Apoptosis and cell cycle phase of transfected cells as measured by flow cytometry. (C) Western blots showing the levels of apoptosis-associated proteins (Cleaved caspase-3, Cleaved PARP, Bax, and Bcl-2) were assessed. Data are shown as mean ± SD; *P < 0.05, ** P< 0.01, and ***P < 0.001.

Low expression of PHLDA2 enhances autophagic flux in CRC cells

We found that PHLDA2 expression correlates negatively with apoptosis of CRC cells. Autophagy is a hallmark of cellular apoptosis; therefore, we assessed the effect of PHLDA2 on CRC cellular apoptosis. We used Transmission electron microscopy (TEM) to examine CRC cells and found stably-transfected with sh-PHLDA2 increased numbers of autophagosomes and autolysosomes in HCT116 (Figure 5A) and SW480 (Figure 5B) cells. Western blot analyses demonstrated an accumulation of ATG7, Beclin1, and lipid-bound LC3-II in cells with low PHLDA2 levels (Figure 5C, 5D), indicating an increase in the synthesis of autophagosomes. We also measured the levels of P62, a substrate of autophagy. As expected, inhibition of PHLDA2 decreased the expression of P62 protein (Figure 5C, 5D). Taken together, these results suggest that low PHLDA2 levels induce autophagy in CRC cells.

Figure 5. Low PHLDA2 expression enhances autophagic flux in CRC cells. (A, B) We used TEM (×20,000) to image HCT116 and SW480 cells and quantified the number of autophagic vacuoles (autophagosomes and autolysosomes) in subsets of 10 randomly-selected cells of each type. Red arrows, autophagic vacuoles; *P<0.05, ***P<0.001. (C, D) Expression of autophagy-associated proteins ATG7, Beclin1, LC3, and P62. Data are shown as mean ± SD; *P < 0.05, **P < 0.01, and ***P < 0.001.

PHLDA2-mediated autophagy contributes to apoptosis of CRC cells induced by low PHLDA2 expression

Inhibition of PHLDA2 increased autophagy (Figure 5A, 5B) and induced apoptosis (Figure 4B, 4C) of CRC cells. To determine whether PHLDA2-induced inhibition of apoptosis was related to autophagy, we used flow cytometry to assess cellular apoptosis with or without PHLDA2 inhibition, and with or without autophagy suppression by chloroquine (CQ). Results showed that inhibition of PHLDA2 increased apoptosis of HCT116 and SW480 cells. No obvious cytotoxicity was found in control cells treated with CQ alone (Figure 6A, 6B). CQ blocked autophagic flux (Figure 6C–6E) and partially rescued apoptosis in cells with low PHLDA2 levels (Figure 6A, 6B). These data demonstrate that PHLDA2-induced apoptosis in CRC cell lines is associated with an increased autophagic flux.

Figure 6. PHLDA2-mediated autophagy contributes to apoptosis induced by PHLDA2 inhibition in of CRC cells. (A, B) We used flow cytometry to detect cellular apoptosis with or without PHLDA2 inhibition, as well as with or without autophagy suppression by chloroquine (CQ). (C–E) Protein levels of LC3 and p62 were assessed by western blot with or without PHLDA2 inhibition, as well as with or without autophagy suppression by CQ. Data are shown as mean ± SD; ns: no significant difference; *P < 0.05, **P < 0.01, and ***P < 0.001.

The effect of PHLDA2 on autophagy and EMT is partially dependent upon the PI3K/AKT signaling pathway

A previous study reported that PHLDA2 inhibited the phosphorylation of AKT [6]. In view of the contributions of the PI3K/AKT pathway to autophagy and EMT, we measured PI3K, AKT and p-AKT^S473 levels in HCT116 and SW480 cells stably-transfected with sh-PHLDA2 and sh-NC. Our results showed that PHLDA2 inhibition decreased the levels of PI3K and p-AKT^S473, but had no effect on AKT levels (Figure 7A, 7B), suggesting that the PI3K/AKT pathway may be involved in PHLDA2-induced autophagy and EMT. To further investigate the underlying molecular basis for PHLDA2-mediated autophagy and EMT, we measured the levels of mTOR, p-mTOR, and GSK3β. We found that low levels of PHLDA2 promoted higher levels of GSK3β and lower levels of p-mTOR in HCT116 and SW480 cells, but did not affect mTOR levels (Figure 7A, 7B). These results indicate that PHLDA2-induced autophagy and EMT partially depend on the PI3K/AKT/mTOR and PI3K/AKT/GSK3β pathways, respectively.

Figure 7. The effects of PHLDA2 on autophagy and EMT are partially dependent upon the PI3K/AKT signaling pathway in CRC cells. (A, B) Levels of PI3K, AKT, p-AKT^S473, mTOR, p-mTOR, and GSK3β proteins were assessed in stably-transfected HCT116 and SW480 cells. Data are shown as mean ± SD; ns: no statistically significant difference; *P < 0.05, **P < 0.01.

In vivo data

PHLDA2 modulates tumorigenesis

In the above in vitro experiments, we demonstrated that PHLDA2 promoted CRC cell growth, inhibited apoptosis, promoted EMT, and inhibited autophagy partly through the PI3K/AKT signaling pathway. We next tested the role of PHLDA2 in vivo. Stably-transfected HCT116 and SW480 cells were injected subcutaneously into the left flank of nude mice. We found that inhibition of PHLDA2 reduced the proliferation of HCT116 and SW480 cells in this mouse tumor model (Figure 8A–8D) and inhibited the expression of KI67 protein (Figure 8E, 8F). These results are consistent with the finding that inhibition of PHLDA2 inhibits cell proliferation in vitro (Figure 2E–2H), suggesting that PHLDA2 promotes CRC progression in vivo and vitro.

Figure 8. PHLDA2 modulates tumorigenesis autophagy and apoptosis in vivo. (A–D) The volume and growth curve of nude mouse tumors. (E, F) Levels of KI67 proteins in tumors from nude mice. *P < 0.05, ** P< 0.01, and ***P<0.001.

Patient specimens

In this study, 29 fresh CRC samples and paired adjacent benign tissue samples were randomly collected from the First Affiliated Hospital of Chongqing Medical University (Chongqing, China from June to December, 2017). RNA was extracted and detected by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Ninety-nine paraffin sections of CRC and 27 normal tissues were obtained from the Department of Pathology of the First Affiliated Hospital of Chongqing Medical University (June, 2012 to September, 2013) and were used to measure protein levels. Informed consent was signed by all patients, and the study was supported by the Ethics Committee of the First Affiliated Hospital of Chongqing Medical University.

Cell culture and transfection

Human CRC cell lines (Caco2, HT29, SW480, LOVO, HCT116, and SW620) were purchased from the American Type Culture Collection (Manassas, VA, USA). All cells were cultured in RPMI-1640 (Gibco, CA) supplemented with 10% fetal bovine serum (FBS) (PAN, Germany) with standard conditions at 37 °C, 5% CO2, and a humidified atmosphere. To inhibit autophagy, cells were treated with 10 nm chloroquine (CQ, Sigma-Aldrich, USA) for 12 h. Short hairpin RNA (shRNA) in the pLVX-shRNA1 plasmid vector targeting PHLDA2; (pLVX-sh-PHLDA2; sh1, 5‘-GCAAGTACGTGTACTTCACCA-3’; sh2, 5‘-GACCACAAGGAG ATCGACTTC-3’; sh3, 5‘-GCGCTCATCGATTTCCAGAAC-3’) and its negative control pLVX-shRNA1 (sh-NC) were purchased from GeneCopeia, Inc. (Rockville, MD, USA). The HCT116 cells were transfected using Lipofectamine 2000 (Invitrogen Preservation, Carlsbad, CA, USA) according to the manufacturer's protocol. The plasmid with the highest knockdown efficiency (pL-sh-1) and the negative control plasmid (pL-sh-NC) were selected and packaged into lentivirus (sh-PHLDA2 and sh-NC) according to the manufacturer's protocol, and then transfected into HCT116 and SW480 cells. Knockdown efficiency was assessed by western blot (WB).

RNA extraction and reverse transcription-quantitative polymerase chain reaction (RT-qPCR)

RT-qPCR experiments were performed as previously described [38]. The primers used in this study were: PHLDA2, forward, 5'-GCGAGGGCGAGTTGGAGAA-3′, and reverse, 5'-CCACCTTGAGGATGGAGTGGAA-3′, GAPDH, forward, 5'-TGACTTCAACAGCGACA CCCA-3 ' and reverse, 5'-CACCCTGTTG CTGTAGCCAAA-3'. The qPCR assays were performed with TB Green™ Premix Ex Taq (Takara Biotechnology Co., Ltd.). The qPCR conditions were as follows: Pre-denaturation at 95 ^oC for 30 s, followed by 40 cycles at 95 ^oC for 5 s, with annealing and extension at 60 ^oC for 30 s. GAPDH was uses as the internal reference. The relative mRNA expression of target genes was measured by the 2-ΔΔCq method [39].

Immunohistochemistry (IHC) and immunofluorescence (IF)

IHC staining of CRC tissue samples and xenografted tumors was performed as described previously [40]. Sections were incubated with primary antibody PHLDA2 (Bs-6884R; 1:100), KI67 (ab15580; 1:200). The tissue sections were imaged with a Leica microscope imaging system (Leica Microsystems GmbH, Wetzlar, Germany). Results were analyzed as previously described [41].

For IF staining, cells were cultured on glass coverslips in 6-well plates, fixed with 4% paraformaldehyde, treated with 0.5% Triton X-100 (Beyotime, China), blocked with 1% goat serum albumin, and incubated with primary antibody E-cadherin (20874-1-AP, 1:50) overnight at 4 °C. After treatment with secondary antibody (SA00009-2 Cy3-conjugated goat anti-rabbit), cell nuclei were stained with DAPI (Sigma-Aldrich; Merck KGaA). Images were captured with a fluorescence microscope (× 400, Leica Microsystems GmbH).

Cell proliferation assay

Cell proliferation was measured with a Cell Counting Kit-8 (CCK8) [42]. Briefly, stably-transfected HCT116 and SW480 cells were inoculated into 96-well plates at a density of 2,000 cells per well and cultured at 37 °C in 5% CO₂. The CCK-8 reagent was added at 0, 24, 48, and 72 h. Absorbance at 450 nm was detected with a microplate reader (Bio-Rad Laboratories, Inc.).

The colony formation assay was performed as previously described [43]. Briefly, HCT116 and SW480 cells were inoculated into 6-well plates at a density of 500 per well and cultured for two weeks. The cells were fixed with 4% paraformaldehyde for 30 min, stained with 1% crystal purple for 20 min, and washed three times with PBS. The numbers of clones (> 50 cells/colony) were counted with an inverted phase contrast microscope (Leica, Wetzlar, Germany).

Cell migration and invasion assays

Cell migration and invasion assays were performed with Transwell chambers (Corning Inc., Corning, NY, USA). For the migration assay, the stably-transfected cell lines (HCT116 and SW480) were digested with trypsin and suspended (2.5 × 10⁵ cells/ml) in RPMI-1640 medium. Cell suspensions (200 μl) were added to the upper chamber, and 700 μl of RPMI-1640 containing 10% FBS added to the lower chamber. After 48 h, the migrated cells were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet for 20 min at room temperature. Subsequently, the cells from five random fields were counted with a microscope (× 200 magnification; Leica Microsystems GmbH, Wetzlar, Germany). For the invasion assay, Matrigel (BD Biosciences) was diluted with RPMI-1640 at a 1:8 ratio. A 100 μl aliquot of the mixture was added to the upper chambers and incubated at 37 ^oC for 5 h to solidify. A 100 μl RPMI-1640 aliquot containing 1 × 10⁵ cells was added to the upper chambers with the subsequent steps identical to the migration assay.

Cell cycle and apoptosis assays

For the cell cycle assay, cell culture supernatants were discarded, stably-transfected cells (HCT-116 and SW480) were digested with trypsin and washed with PBS. Then, the cells were fixed in 70% iced ethanol, prepared in advance for 24 h at 4 ^oC. After centrifugation, RNase A (10 mg/ml; Sigma-Aldrich; Merck KGaA) was added to the cells and incubated for 30 min at 37 ^oC in 5% CO₂. The cells were then stained with propidium iodide (PI; Beyotime Institute of Biotechnology, Shanghai, China). Cell-cycle was detected by flow cytometry (BD Biosciences, San Jose, CA, USA) and analyzed by FlowJo version 7.6 software (FlowJo LLC, Ashland, OR, USA).

For cellular apoptosis, cell culture supernatants were collected and stably transfected cells (HCT-116 and SW480) were washed, digested, centrifuged, and diluted in PBS. Cells were stained with PI (BD Pharmingen; BD Biosciences) and annexin V-fluorescein isothiocyanate for 15 min in the dark. Apoptosis was examined by flow cytometry (BD Biosciences, San Jose, CA, USA). The apoptotic rate was assessed with BD FACS software.

Transmission electron microscopy (TEM)

For TEM, cells were washed, digested, centrifuged, collected, and fixed with 2.5% glutaraldehyde. Stably-transfected cells (HCT-116 and SW480) were embedded in Epon 812 resin and dehydrated in various concentrations of acetone. The specimens were cut into 1 μm slices and stained with sodium acetate and lead citrate. The cells were observed with a transmission electron microscope (JEM-1400Plus; JEOL, Tokyo, Japan). The number of autophagic vacuoles, including autophagosomes and autolysosomes, in each cell was quantified in 10 randomly-selected cells from each group [44].

Western blot (WB) analysis

Proteins were extracted from stably-transfected HCT-116 and SW480 cells, as well as from nude-mice tumors, and were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The proteins were transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were blocked with 5% skim milk for 1-2 h and incubated at 4 °C overnight with primary antibodies, and then with goat anti-rabbit or anti-mouse antibodies (1:5000; Abbkine, Redlands, CA, USA) at 37 °C for 1 h. The membranes were examined with a Bio-Rad gel imaging system (Bio-Rad, Hercules, CA, USA) with a WB kit (Advansta, Menlo Park, USA). Results were analyzed with Image J software (version 6.0; National Institutes of Health, Bethesda, MD, USA), normalized to GAPDH. The primary antibodies used for WB analysis included: PHLDA2 (14661-1-AP, 1:800), GAPDH (60004-1-Ig, 1:5000), E-cadherin (20874-1-AP, 1:5000), N-cadherin (22018-1-AP, 1:3000), and Vimentin (10366-1-AP, 1:2000) from Proteintech Group Inc. (Chicago, IL, USA); PI3K (YM3800, 1:1000), MMP2 (YT2798, 1:1,000), GSK3β (YT2082, 1:1,000), and β-catenin (YM6676, 1:1,000) purchased from Immunoway Biotechnology Company (Plano, TX, USA); P62 (ab109012, 1:5000), Bax (ab32503, 1:5000), Bcl-2 (ab32124, 1:2000), ATG7 (ab52472, 1:5000), Beclin-1 (ab207612, 1:2000), p-AKT^Ser473 (ab81283, 1:3000), AKT (ab8805, 1:1000), p-mTOR (ab109268, 1:1000), Cleaved caspase-3 (ab2302, 1:500), LC3 (ab51520, 1:3000), and mTOR (ab2732, 1:2000) from Abcam, Inc. (Cambridge, UK); cleaved PARP (#5625, 1:1000) from Cell Signaling Technology Inc. (Danvers, MA, USA).

In vivo tumorigenesis

Four week-old male BALB/c nude mice were purchased from Huafukang Biotechnology (Beijing, China) and housed in the Animal Experimental Center of Chongqing Medical University (Chongqing, China). All animal experiments were performed with the approval of the Ethical Committee of Chongqing Medical University with the use of experiment animals in accordance with the Guide for Care and Use of Laboratory Animals (US National Institutes of Health). The mice were randomized into four groups with five mice per group (HCT116-sh-NC, HCT116-sh-PHLDA2, SW480-sh-NC and SW480-sh-PHLDA2). Stably-transfected HCT116 and SW480 cells were digested, centrifuged, washed twice with PBS, and diluted in a 100 μl suspension with PBS containing 1×10⁶ cells. The suspensions were injected subcutaneously into the left flank of the nude mice to induce formation of xenografted tumors. The nude mice were maintained with normal feed and water for 21-30 days and then tumors were resected for analysis. Tumor volume was measured every three days using a Vernier caliper. Tumor volume was calculated using the formula: V = (length × width²)/2. Finally, the mice were anesthetized, euthanized, and humanely disposed of.

Statistical analysis

Except where otherwise noted, experiments were repeated at least three times. Statistical analyses were performed using SPSS 22.0 software (Chicago, IL, USA). The data are presented as mean ± SD. Briefly, the Student’s t-test or analysis of variance (ANOVA) were used to analyze the data. The Chi-square test was used to analyze the relationships among PHLDA2 expression and clinical characteristics of CRC patients. P-values < 0.05 were considered statistically significant.