PAQR4 has a tumorigenic effect in human breast cancers in association with reduced CDK4 degradation

PAQR4 has a tumorigenic effect in human breast cancers in association with reduced CDK4 degradation Abstract Progestin and adipoQ receptor 4 (PAQR4) is a member of the PAQR family, and the members within this family are involved in the regulation of a number of biological processes including metabolism and cancer development. The potential function of PAQR4 in human cancers is unknown. Analysis of ONCOMINE database reveals that PAQR4 is highly expressed in human breast cancers. We confirmed this finding by analyzing 82 human breast cancers samples. PAQR4 mRNA level was significantly upregulated in human breast cancer samples compared with their corresponding para-cancerous histological normal tissues (P < 0.0001). The mRNA level of PAQR4 was negatively correlated with disease-free survival (P < 0.0001) and overall survival of the patients (P = 0.001). Knockdown of PAQR4 in human breast cancer cells SUM159 and MCF7 suppressed cell proliferation. In contrast, overexpression of PAQR4 in SUM159 cells enhanced cell proliferation and colony formation. In a tumor xenograft model, overexpression of PAQR4 promoted tumor growth of SUM159 cells in vivo, while PAQR4 knockdown suppressed the tumor growth. PAQR4 was able to negatively regulate cyclin-dependent kinases 4 (CDK4) protein level in the breast cancer cells. Knockdown of PAQR4 accelerated degradation of CDK4 together with upregulation of CDK4 polyubiquitination. On the other hand, overexpression of PAQR4 slowed down CDK4 protein degradation and reduced CDK4 polyubiquitination. Collectively, these data at the cellular, animal and human levels indicate that PAQR4 has a tumorigenic effect on human breast cancers, and such effect is associated with a modulatory activity of PAQR4 on protein degradation of CDK4. Introduction Breast cancer is one of the most common cancers worldwide. According to Cancer Facts & Statistics 2015 reports, an estimated 231 840 new cases of invasive breast cancer are expected to be diagnosed among women in the USA during 2015. Excluding cancers of the skin, breast cancer is the most frequently diagnosed cancer in women. In addition, Breast cancer ranks second as a cause of cancer death in women (after lung cancer), an estimated 40 730 breast cancer deaths (40 290 women, 440 men) are expected in 2015 (1). Breast cancer is commonly treated with various combinations of radiation therapy, chemotherapy, hormone therapy (e.g. selective estrogen receptor modifiers, aromatase inhibitors, ovarian ablation) and/or targeted therapy (2–%6). Cyclin-dependent kinases 4 and 6 (CDK4 and CDK6), which are activated by D-type cyclins, are essential proteins important for cell proliferation as G1-specific serine/threonine kinases and have been targeted for cancer therapy (7–%9). The expression of cyclin D1 and activation of CDK4 and CDK6 can drive proliferation of breast cancer cells (10,11).Therapeutic agents that specifically inhibit CDK4 and CDK6 have been proven to be effective for breast cancers (12,13). Progestin and adipoQ receptor 4 (PAQR4) belongs to the PAQR family, which consists of 11 members (PAQR1 to PAQR11) in the human genome (14). All the members within this family are predicted to contain seven transmembrane domains with a topology distinct from the traditional GPCR (G-protein coupled receptor) proteins (14). The physiological roles and molecular functions of the PAQR family have been explored. In particular, the function of PAQR3, a close homologue of PAQR4, has been extensively investigated. As a new member of tumor suppressors, PAQR3 was downregulated in different types of human cancers including colon cancer, gastric cancer, bladder cancer, liver cancer, osteosarcoma, breast cancer and laryngeal squamous cell carcinoma (15–20). PAQR3 is found to negatively regulate Raf-1, a key kinase in the Ras-Raf-Mek-ERK signaling pathway (21). PAQR3 negatively modulates angiogenesis of endothelial cells (22). It also functionally interacts with p53 to modulate cancer formation and epithelial-mesenchymal transition (23). PAQR3 also suppresses AKT activation (24,25). However, the function of PAQR4 has been elusive. In this study, we explored the potential functions of PAQR4 in breast cancers. Material and methods cell culture Human breast cancer cell lines SUM159 and MCF7 were cultured in Dulbecco’s modified Eagle’s medium containing 10% FBS (Invitrogen, Grand Island, NY) and were incubated in a cell incubator at 37°C under 5% CO2. All the cells were purchased from the Cell Bank of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences in early 2016. The cells were authenticated by DNA fingerprinting in the Cell Bank at a regular basis. Lentivirus packaging and infection For lentiviral packaging, HEK293T cells (7 × 106) were seeded in 15 cm cell culture dish, incubated for 24 h and then transfected with 46 μg of lentivirus plasmids. The virus-containing medium was collected and filtered through 0.45 μm filter (Millipore, MA). Afterwards, the filtered supernatants were centrifuged at 20 000 rpm, 4°C for 2 h. The precipitate was suspended in 100 μl Dulbecco’s modified Eagle’s medium and subpackaged in 1.5 ml tubes. For lentivirus infections, the cells (SUM159/MCF7) were cultured in six-well plates for infection by moderate virus-contained Dulbecco’s modified Eagle’s medium with polybrene (Sigma–Aldrich, St. Louis, MO, 4 μg/ml), incubated at 37°C for 8 h and replaced by Dulbecco’s modified Eagle’s medium with 10% FBS. After incubation for 48 h, the infected cell lines were valuated the infection efficiency of lentiviral by real-time PCR. RNA isolation and quantitative RT-PCR The cells were lysed in Trizol reagent (Invitrogen, Carlsbad, CA). Total RNA was purified and reverse transcribed to cDNA according to the manufacturer’s instructions. The quantification of target gene transcripts was detected by ABI Prism 7900 sequence detection system (Applied Biosystems, CA). The primers were as follows: Human PAQR4: 5′-TACCTGCACAACGAACTGGG-3′and 5′-AAGAGGTGATAGAGCACGGAG-3′; Human CDK4: 5′-CTGGTGTTTGAGCATGTAGACC-3′and 5′-GATCCTTGATCGTTT CGGCTG-3′; Huma β-actin: 5′-CTGGAACGGTGAAGGTGACA-3′ and 5′-AAGGGACTTCCTGTAACAATGCA-3′. Cell proliferation assays MTT assay was performed according to the method described previously (26). SUM159 and MCF7 cells were seeded at a density of 4 × 103 cells/well into a 96-well culture plate and grown in 5% CO2 at 37°C for 24 h, 48 h and 72 h. Cell viability was measured by MTT (Sigma–Aldrich, MO) assay as follows: 20 µl of 5 mg/ml MTT was added to each well and incubated with cells at 37°C for 4 h. The MTT solution was then discarded, and 200 µl dimethyl sulfoxide was added to dissolve the formazan sediment. Finally, the optical density was detected using a microplate reader (Molecular Devices, CA) at an absorption wavelength of 490 nm. For colony formation assay, the cells were inoculated into six-well culture plate with 400 cells per well and cultured for 9–13 days. Then, the cells were stained with Crystal Violet for counting. Antibodies and immunoblotting The antibodies purchased were as follows: anti-HA antibody (Santa Cruz Biotechnology, Dallas, TX); anti-Flag and anti-α-tubulin antibody (Sigma–Aldrich); anti-CDK4 antibody (UpState Biotechnology, New York, NY). The protocol for immunoblotting has been described previously (21). Protein degradation analysis For cycloheximide (CHX, Sigma–Aldrich) assay, stable cells were cultured overnight, then treated with 100 μg/ml CHX and harvested at various time points. The cell lysates were subjected to immunoblotting. And the results were quantified by Quantityone software. For ubiquitination assay, stable cells were transfected with vector expressing HA-tagged ubiquitin and other plasmids as described in the figure legend and treated with or without 10 μM MG132 (BD Biosciences, San Jose, CA) for 6 h before the cells were harvested. The lysates were immunoprecipated and then immunoblotted with the antibodies as indicated. PAQR4 gene deletion by CRISPR/Cas9 technology The sgRNA sequence to delete PAQR4 gene was designed according to the website http://crispr.mit.edu. The sequences of sgRNA were as follows: sgRNA-1, 5′-AGTTCGTGCTGACCGGGTACCGG-3′; sgRNA-2, 5′-TCAATAAGTTCGTGCTGACCGGG-3′ with both of them targeting on exon 1 of the human PAQR4 gene. The sgRNA oligo duplex was treated with T4 polynucleotide kinase (Thermo Scientific, MA) and the annealed oligo was inserted into lentiCRISPRv2 (from Addgene) and then used to transform Stbl3 bacteria (Transgene, Strasbourg, France). The plasmids containing the correct insert were confirmed by DNA sequencing. Lentivirus packaging and infection has already been described in the previous section. Following viral infection for at least 3 days, the genomic DNA of the cells was isolated and used in PCR to amplify the targeted region using a high fidelity enzyme KOD polymerase (TOYOBO,Osaka, Japan), followed by DNA sequencing to confirm disruption of PAQR4 gene. Nude mice xenograft model All animals were maintained and used in accordance with the guidelines of the Institutional Animal Care and Use Committee of the Institute for Nutritional Sciences. All of the experimental procedures were carried out in accordance with the Chinese Academy of Sciences ethics commission with an approval number 2010-AN-8. Mice were maintained on a 12 h light/dark cycle at 25°C. The cells in the logarithmic phase of growth were trypsinized, centrifuged and rinsed with PBS three times. The nude mice (4 weeks old, female) was injected subcutaneously with a clonal population of SUM159 cell (2.6 × 106 cells) or MCF7 cell (5 × 106 cells) in the lower right leg. Xenograft tumor sizes were analyzed by measuring two perpendicular diameters with digital calibers every other day and calculated according to the formula: 0.5 × length × width2. Patients and samples This study was approved by the Ethics Committee of the Zhejiang Province Cancer Hospital, Hangzhou, Zhejiang, China. All patients provided their full consent to participate in the study. The patients enrolled in this study underwent curative surgery without prior treatments. Tissue specimens were examined separately by two pathologists under double-blinded conditions without prior knowledge of the clinical status of the specimens. The patients’ medical records were reviewed to obtain data including age at diagnosis, sex, tumor location, tumor size (diameter), lymph node metastasis, histology, tumor invasion and TNM stage according to the guidelines of American Joint Committee. Detailed clinical histopathological factors were presented in Table 1. For the measurement of prognosis, we analyzed the clinical data concerning disease-free survival (DFS) and overall survival (OS), defined as the time from surgery to first recurrence or death, respectively. All recruited patients had been followed up periodically until the due date. Clinical follow-up results revealed that the mean follow-up duration was 69 months and the range was 3–109 months. Table 1. Correlation between relative PAQR4 expression level and clinicopathologic parameters of the patients Variables  N  PAQR4 mRNA level  χ2 (P-value)  Low/normal, n (%)  High, n (%)  Age (years)   <50  50  13 (26.0)  37 (74.0)  0.661   ≥50  32  11 (34.4)  21 (65.6)  (0.416)  Tumor location   Left  38  11 (28.9)  27 (71.1)  0.004   Right  44  13 (29.5)  31 (70.5)  (0.953)  Tumor size   ≤2 cm  39  13 (33.3)  26 (66.7)  0.594   >2 cm  43  11 (25.6)  32 (74.4)  (0.441)  Histology   Moderately/well  34  20 (58.8)  14 (41.2)  24.508   Poorly  48  4 (8.3)  44 (91.7)  (7.400E-07)  T stage   I/II  67  20 (29.9)  47 (70.1)  0.060   III/IV  15  4 (26.7)  11 (73.3)  (0.806)  Lymph node status   Negative  38  18 (47.4)  20 (52.6)  11.207   Positive  44  6 (13.6)  38 (86.4)  (0.001)  Distant metastasis   Negative  82  24 (29.3)  58 (70.7)  82.000   Positive  0  N/A  N/A  (N/A)  TNM stage   I/II  55  19 (34.5)  36 (65.5)  2.247   III/IV  27  5 (18.5)  22 (81.5)  (0.134)  ER status   Negative  39  9 (23.1)  30 (76.9)  1.377   Positive  43  15 (34.9)  28 (65.1)  (0.241)  Her-2 status   Negative  25  15 (60.0)  10 (40.0)  16.407   Positive  57  9 (15.8)  48 (84.2)  (5.108E-05)  p53 status   Negative  25  10 (40.0)  15 (60.0)  2.001   Positive  57  14 (24.6)  43 (75.4)  (0.157)  PR status   Negative  43  11 (25.6)  32 (74.4)  0.594   Positive  39  13 (31.7)  28 (68.3)  (0.441)  Variables  N  PAQR4 mRNA level  χ2 (P-value)  Low/normal, n (%)  High, n (%)  Age (years)   <50  50  13 (26.0)  37 (74.0)  0.661   ≥50  32  11 (34.4)  21 (65.6)  (0.416)  Tumor location   Left  38  11 (28.9)  27 (71.1)  0.004   Right  44  13 (29.5)  31 (70.5)  (0.953)  Tumor size   ≤2 cm  39  13 (33.3)  26 (66.7)  0.594   >2 cm  43  11 (25.6)  32 (74.4)  (0.441)  Histology   Moderately/well  34  20 (58.8)  14 (41.2)  24.508   Poorly  48  4 (8.3)  44 (91.7)  (7.400E-07)  T stage   I/II  67  20 (29.9)  47 (70.1)  0.060   III/IV  15  4 (26.7)  11 (73.3)  (0.806)  Lymph node status   Negative  38  18 (47.4)  20 (52.6)  11.207   Positive  44  6 (13.6)  38 (86.4)  (0.001)  Distant metastasis   Negative  82  24 (29.3)  58 (70.7)  82.000   Positive  0  N/A  N/A  (N/A)  TNM stage   I/II  55  19 (34.5)  36 (65.5)  2.247   III/IV  27  5 (18.5)  22 (81.5)  (0.134)  ER status   Negative  39  9 (23.1)  30 (76.9)  1.377   Positive  43  15 (34.9)  28 (65.1)  (0.241)  Her-2 status   Negative  25  15 (60.0)  10 (40.0)  16.407   Positive  57  9 (15.8)  48 (84.2)  (5.108E-05)  p53 status   Negative  25  10 (40.0)  15 (60.0)  2.001   Positive  57  14 (24.6)  43 (75.4)  (0.157)  PR status   Negative  43  11 (25.6)  32 (74.4)  0.594   Positive  39  13 (31.7)  28 (68.3)  (0.441)  Chi-square test was used for statistical analyses. P < 0.05 is considered statistically significant. ER, estrogen receptor; Her-2, Human epidermal growth factor receptor 2; PAQR4, Progestin and adipoQ receptor; PR, progesterone receptor; TNM, tumor, lymph node, metastasis. View Large Statistical analysis Statistical significance was assessed using Student’s t-test. All results were expressed as the mean ± standard deviation (SD). Values of P < 0.05 were considered statistically significant. Results PAQR4 is significantly upregulated in primary breast cancer tissues and correlated with the prognosis of the patients To investigate the potential role of PAQR4 in human cancers, we first analyzed ONCOMINE database and found that PAQR4 is mainly upregulated in breast cancers (Figure 1A). PAQR4 expression is upregulated (tumor versus normal) in 16 out of 43 breast cancer dataset using the threshold of >2-fold change and P-value < 0.0001 (Figure 1A). We next characterized the expression status of PAQR4 transcript in 82 patients with primary breast cancer. The mRNA level of PAQR4 was determined in both the primary breast cancer samples together with their corresponding para-cancerous histological normal tissue (PCHNT). Intriguingly, we found that PAQR4 mRNA level was significantly elevated in most of the cancer samples compared with PCHNT. Using the standard of over 2.5-fold changes, PAQR4 mRNA was significantly higher in 58 tumors (70.7%, P < 0.0001) than the paired PCHNT samples (Figure 1B and Table 1). The mRNA level of PAQR4 was also associated with a few clinical characteristics of the patients such as histology (P < 0.0001), lymph node metastasis (P = 0.001) (Table 1). In addition, the PAQR4 mRNA level was closely correlated with the status of HER2 expression (γ = 0.447, P < 0.0001) (Table 1). Figure 1. View largeDownload slide Expression of PAQR4 is upregulated in human breast cancer samples and correlated with patient survival (A) Analysis of PAQR4 expression profile in ONCOMINE database. Summary of PAQR4 mRNA expression in various human cancers compared with normal tissues using the threshold of >2-fold change and P < 0.0001. Red: upregulated in cancer; blue: downregulated in cancer. (B) The relative ratio of PAQR4 mRNA in 82 human breast cancer samples versus PCHNT samples. The tumor/PCHNT ratio < 2.5 is considered as low/normal expression. (C, D) Correlation of PAQR4 expression level with survival of breast cancer patients. Kaplan–Meier curves of DFS, shown in C, and OS shown in D in post surgery patients with breast cancers according to the expression level of PAQR4. Figure 1. View largeDownload slide Expression of PAQR4 is upregulated in human breast cancer samples and correlated with patient survival (A) Analysis of PAQR4 expression profile in ONCOMINE database. Summary of PAQR4 mRNA expression in various human cancers compared with normal tissues using the threshold of >2-fold change and P < 0.0001. Red: upregulated in cancer; blue: downregulated in cancer. (B) The relative ratio of PAQR4 mRNA in 82 human breast cancer samples versus PCHNT samples. The tumor/PCHNT ratio < 2.5 is considered as low/normal expression. (C, D) Correlation of PAQR4 expression level with survival of breast cancer patients. Kaplan–Meier curves of DFS, shown in C, and OS shown in D in post surgery patients with breast cancers according to the expression level of PAQR4. We next investigated the association of PAQR4 expression level with the survival of the patients. Both DFS and OS were determined. DFS was defined as the time from surgery to first recurrence, and OS was the time from surgery to death. We found that the average duration of DFS in patients with PAQR4 upregulation in the tumors was significantly shorter than those with normal/low expression of PAQR4 in the tumors (Figure 1C). The average DFS in patients with high expression of PAQR4 was 60.9 months versus 106.0 months in patients with normal/low expression of PAQR4 (P < 0.0001). In addition, the average OS was also different between the patients with high expression of PAQR4 and those with normal/low expression (P = 0.001). Collectively, these data indicate that the expression level of PAQR4 is significantly upregulated in human breast cancers, and such upregulation is associated with poor prognosis of the patients. Knockdown of PAQR4 suppresses proliferation of breast cancer cells We next investigated whether PAQR4 has a direct effect on the growth of human breast cancer cells including MCF7 and SUM159 cell lines. We established stable cell clones with endogenous PAQR4 being silenced by a PAQR4-specific shRNA. The efficiency of PAQR4 knockdown was confirmed by quantitative RT-PCR (Figure 2A and B). We next investigated the cell proliferation rate of these cells by MTT assay. As shown in Figure 2C and D, the cell growth rate was significantly reduced by PAQR4 knockdown in comparison with the control cells. Colony formation was also used to investigate the potential of tumorigenesis of the breast cancer cells. We found that PAQR4 knockdown profoundly reduced colony formation in MCF7 and SUM159 cells (Figure 2E and F). Collectively, these data indicate that silencing of PAQR4 suppresses growth of breast cancer cells. Figure 2. View largeDownload slide PAQR4 affects cell proliferation and colony formation of breast cancer cells (A, B) The mRNA level of PAQR4 in MCF7 and SUM159 cells expressing control shRNA (mock) or PAQR4-specific shRNA (shPAQR4) were detected by quantitative RT-PCR. (C, D) Effect of PAQR4 knockdown on cell proliferation. The breast cancer cells as in A and B were used to determine the cell proliferation rate by MTT assay at the indicated time point. (E, F) Effect of PAQR4 knockdown on colony formation. SUM159 or MCF7 cells as in A and B were seeded into six-well plate with 400 cells per well and cultured for 11 and 13 days, respectively and then used in crystal violet staining and colony counting. (G) The expression level of PAQR4 in control vector (mock) or PAQR4-overexpressing (PAQR4-OV) SUM159 breast cancer cells as detected by quantitative RT-PCR. (H) Effect of PAQR4 overexpression on the cell proliferation rate as determined by MTT assay with the cells as in G. (I) Effect of PAQR4 overexpression on colony formation. The cells as in G were seeded into six-well plate with 400 cells per well and cultured for 9 days, followed by crystal violet staining and colony counting. All data are shown as mean ± SD, **P < 0.01 and ***P < 0.001. Figure 2. View largeDownload slide PAQR4 affects cell proliferation and colony formation of breast cancer cells (A, B) The mRNA level of PAQR4 in MCF7 and SUM159 cells expressing control shRNA (mock) or PAQR4-specific shRNA (shPAQR4) were detected by quantitative RT-PCR. (C, D) Effect of PAQR4 knockdown on cell proliferation. The breast cancer cells as in A and B were used to determine the cell proliferation rate by MTT assay at the indicated time point. (E, F) Effect of PAQR4 knockdown on colony formation. SUM159 or MCF7 cells as in A and B were seeded into six-well plate with 400 cells per well and cultured for 11 and 13 days, respectively and then used in crystal violet staining and colony counting. (G) The expression level of PAQR4 in control vector (mock) or PAQR4-overexpressing (PAQR4-OV) SUM159 breast cancer cells as detected by quantitative RT-PCR. (H) Effect of PAQR4 overexpression on the cell proliferation rate as determined by MTT assay with the cells as in G. (I) Effect of PAQR4 overexpression on colony formation. The cells as in G were seeded into six-well plate with 400 cells per well and cultured for 9 days, followed by crystal violet staining and colony counting. All data are shown as mean ± SD, **P < 0.01 and ***P < 0.001. Overexpression of PAQR4 increases cell proliferation in breast cancer cells We further analyzed the function of PAQR4 on cell proliferation by establishing a SUM159 cell line with overexpression of PAQR4 using a lentivirus-based method. The expression of PAQR4 was confirmed by quantitative RT-PCR (Figure 2G). Overexpression of PAQR4 could promote cell proliferation rate of SUM159 cells by MTT assay (Figure 2H). The capacity of colony formation was also enhanced by PAQR3 overexpression (Figure 2I). These data, therefore, further confirmed the positive effect of PAQR4 on the proliferation of human breast cancer cells. PAQR4 promotes the growth of the breast cancer cells in vivo In order to further elucidate the tumorigenic activity of PAQR4 in breast cancers, we next investigated the effects of PAQR4 on tumor growth using a xenograft model. SUM159 cells with stable expression of PAQR4 were implanted into the nude mice. The mice were sacrificed in 25 days when the tumor formation became obvious. The growth of the breast cancer cells in the mice as measured by tumor volume and tumor weight (Figure 3A to C) was significantly increased by PAQR4 overexpression. Figure 3. View largeDownload slide PAQR4 affects the growth of breast cancer cell xenografts in nude mice (A–C) The volume of the tumors (A) from nude mice inoculated with SUM159 cells expressing control vector (mock) or PAQR4 (PAQR4-OV). Representative images of the tumors isolated from the mice (B) and the weight of the tumors (C). (D–F) The volume of the tumors (D) from nude mice inoculated with MCF7 cells expressing control vector (mock) or shPAQR4. Representative images of the tumors isolated from the mice (E) and the weight of the tumors from the two groups of mice (F). All the data are shown as mean ± SD, *P < 0.05, **P < 0.01 and ***P < 0.001. Figure 3. View largeDownload slide PAQR4 affects the growth of breast cancer cell xenografts in nude mice (A–C) The volume of the tumors (A) from nude mice inoculated with SUM159 cells expressing control vector (mock) or PAQR4 (PAQR4-OV). Representative images of the tumors isolated from the mice (B) and the weight of the tumors (C). (D–F) The volume of the tumors (D) from nude mice inoculated with MCF7 cells expressing control vector (mock) or shPAQR4. Representative images of the tumors isolated from the mice (E) and the weight of the tumors from the two groups of mice (F). All the data are shown as mean ± SD, *P < 0.05, **P < 0.01 and ***P < 0.001. We also investigated the effect of PAQR4 knockdown on tumor growth in vivo. MCF7 cells with knockdown of PAQR4 were inoculated into the nude mice. In agreement with the overexpression data, the tumor volume and tumor volume were all significantly reduced by PAQR4 knockdown (Figure 3D to F). These observations, therefore, clearly indicated that PAQR4 has a strong tumorigenic effect to in vivo. PAQR4 reduces ubiquitination and degradation of CDK4 in breast cancer cells Our preliminary studies revealed that PAQR4 reduces ubiquitination and degradation of CDK4, a critical molecule in controlling G1-to-S transition (Y. Chen, unpublished). We therefore explored the effect of PAQR4 on CDK4 in breast cancer cells. As shown in Figure 4A, overexpression of PAQR4 could elevate the protein level of CDK4. In contrast, knockdown of PAQR4 reduced the protein level of CDK4. Such changes of CDK4 protein level was not caused by changes of CDK4 mRNA level (Figure 4B). We next performed CHX experiments to determine the half-life of CDK4. Silencing of PAQR4 accelerated the degradation rate of CDK4 in the breast cancer cells (Figure 4C and D). In contrast, overexpression of PAQR4 delayed the half-life of CDK4 protein in SUM159 cells (Figure 4E). Next, we investigated the ubiquitination level of CDK4 in the presence and absence of MG132 (a proteasome inhibitor) in breast cancer cells. Knockdown of PAQR4 enhanced ubiquitination of CDK4 in the presence of MG132 in both MCF7 and SUM159 cells (Figure 4F and G). However, overexpression of PAQR4 could reduce ubiquitination of CDK4 in SUM159 cells (Figure 4H). These data, therefore, indicate that PAQR4 is able to modulate ubiquitination and degradation of CDK4 protein in breast cancer cells. Figure 4. View largeDownload slide PAQR4 reduces degradation and polyubiquitination of CDK4 protein (A) PAQR4 regulates the protein level of CDK4. SUM159 and MCF7 cells were infected with lentivirus that had overexpression of PAQR4 or knockdown of PAQR4 by a PAQR4-specific shRNA, respectively. The cells were plated in 12-well plate, cultured for 24 h and then harvested for immunoblotting (IB) with the antibodies as indicated. (B) Effect of PAQR4 on CDK4 expression. The mRNA levels of PAQR4 and CDK4 were measured by quantitative RT-PCR in SUM159 or MCF7 cells with overexpression or knockdown of PAQR4. (C–E) Effect of PAQR4 on the half-life of CDK4. The cells with knockdown or overexpression of PAQR4 were plated in 12-well plate, cultured for 24 h and then treated with 100 μg/ml CHX for various times and then harvested for immunoblotting with the antibodies as indicated. The immunoblot results (repeated for at least three times) were quantified by Quantity One software and were shown in the lower panels. (F–H) The cells were transfected with the plasmids as indicated and then treated with or without MG132 (10 μM) for 6 h before immunoprecipitation (IP) and immunoblotting (IB) using the antibodies as indicated. The data are shown as mean ± SD. **P < 0.01 and ***P < 0.001. Figure 4. View largeDownload slide PAQR4 reduces degradation and polyubiquitination of CDK4 protein (A) PAQR4 regulates the protein level of CDK4. SUM159 and MCF7 cells were infected with lentivirus that had overexpression of PAQR4 or knockdown of PAQR4 by a PAQR4-specific shRNA, respectively. The cells were plated in 12-well plate, cultured for 24 h and then harvested for immunoblotting (IB) with the antibodies as indicated. (B) Effect of PAQR4 on CDK4 expression. The mRNA levels of PAQR4 and CDK4 were measured by quantitative RT-PCR in SUM159 or MCF7 cells with overexpression or knockdown of PAQR4. (C–E) Effect of PAQR4 on the half-life of CDK4. The cells with knockdown or overexpression of PAQR4 were plated in 12-well plate, cultured for 24 h and then treated with 100 μg/ml CHX for various times and then harvested for immunoblotting with the antibodies as indicated. The immunoblot results (repeated for at least three times) were quantified by Quantity One software and were shown in the lower panels. (F–H) The cells were transfected with the plasmids as indicated and then treated with or without MG132 (10 μM) for 6 h before immunoprecipitation (IP) and immunoblotting (IB) using the antibodies as indicated. The data are shown as mean ± SD. **P < 0.01 and ***P < 0.001. CRISPR/Cas9-mediated deletion of PAQR4 retarded growth and reduced CDK4 protein level in MCF7 cells In order to further investigate the effect of PAQR4 on human breast cancer cells, we established MCF7 cells with deletion of endogenous PAQR4 by CRISPR/Cas9 technology. The efficiency of PAQR4 deletion was confirmed by sequencing of the targeted genomic region. We found that the sequences following the sgRNA guide sequence were disrupted, confirming successful deletion of PAQR4 gene in these cells (Supplementary Figures S1 and S2, available at Carcinogenesis Online). We investigated the cell proliferation rate of these cells by MTT and colony formation assays. As shown in Figure 5A and B, the cell growth rate was significantly reduced by PAQR4 deletion in comparison with the parental cells. Next, we explored the effect of PAQR4 deletion on CDK4 protein in MCF7 cells. As shown in Figure 5C, deletion of PAQR4 reduced the steady state protein level of CDK4. We then performed CHX experiments to determine the half-life of CDK4 protein. Deletion of PAQR4 accelerated the degradation rate of CDK4 in MCF7 cells (Figure 5D). Furthermore, deletion of PAQR4 in MCF7 cells could reduce the growth of tumor xenografts in nude mice (Supplementary Figure S3, available at Carcinogenesis Online). These data, therefore, confirmed that PAQR4 is able to positively regulate the growth of breast cancer cells both in vitro and in vivo. Figure 5. View largeDownload slide CRISPR/Cas9-mediated disruption of PAQR4 gene affects cell growth and CDK4 protein in MCF7 cells (A) Effect of PAQR4 deletion on cell proliferation. The cell proliferation rate of parental or PAQR4-deleted MCF7 cells was determined by MTT assay at the indicated time point. (B) Effect of PAQR4 deletion on colony formation. Parental or PAQR4-deleted MCF7 cells were seeded into six-well plate with 300 cells per well and cultured for 11 days, followed by crystal violet staining and colony counting. (C) PAQR4 regulates the protein level of CDK4. Parental or PAQR4-deleted MCF7 cells were harvested for immunoblotting with the antibodies as indicated. (D) Effect of PAQR4 on the half-life of CDK4 protein. Parental or PAQR4-deleted MCF7 cells were plated in 12-well plate and treated with 100 μg/ml CHX for various times and then harvested for immunoblotting with the antibodies as indicated. The immunoblot results (repeated for three times) were quantified by Quantity One software, and the quantitation is shown in the lower panel. All data are shown as mean ± SD, **P < 0.01 and ***P < 0.001. Figure 5. View largeDownload slide CRISPR/Cas9-mediated disruption of PAQR4 gene affects cell growth and CDK4 protein in MCF7 cells (A) Effect of PAQR4 deletion on cell proliferation. The cell proliferation rate of parental or PAQR4-deleted MCF7 cells was determined by MTT assay at the indicated time point. (B) Effect of PAQR4 deletion on colony formation. Parental or PAQR4-deleted MCF7 cells were seeded into six-well plate with 300 cells per well and cultured for 11 days, followed by crystal violet staining and colony counting. (C) PAQR4 regulates the protein level of CDK4. Parental or PAQR4-deleted MCF7 cells were harvested for immunoblotting with the antibodies as indicated. (D) Effect of PAQR4 on the half-life of CDK4 protein. Parental or PAQR4-deleted MCF7 cells were plated in 12-well plate and treated with 100 μg/ml CHX for various times and then harvested for immunoblotting with the antibodies as indicated. The immunoblot results (repeated for three times) were quantified by Quantity One software, and the quantitation is shown in the lower panel. All data are shown as mean ± SD, **P < 0.01 and ***P < 0.001. Discussion Our studies have provided compelling evidence that PAQR4 has a tumorigenic function in human breast cancer cells via studies in vitro, in vivo and in human samples. At the cellular level, PAQR4 promotes the cell proliferation and tumorigenicity of human breast cancer cells. At the animal level, PAQR4 strongly enhances the growth of breast cancers in nude mice. Mechanistically, PAQR4 regulates the steady state level of CDK4, a critical protein that controls G1-to-S transition of the cell cycle. PAQR4 reduces ubiquitination and degradation rate of CDK4 in breast cancer cells and such regulation likely underlies its tumor-promoting activity in these cells. At the clinical level, PAQR4 expression level was robustly upregulated in human breast cancer samples as compared with the adjacent normal tissues. Furthermore, the expression status of PAQR4 was associated with the survival of the breast cancer patients. Due to potential significance of PAQR4 in regulating the tumorigenicity of breast cancers, it will be important to further explore the functions of PAQR4 in the context of breast cancer development. Firstly, it will be important to further elucidate the molecular mechanisms associated with the tumor-promoting activity of PAQR4 in breast cancer. Next, it will be important to investigate the functional interaction between PAQR4 and Her2. Her2 has stood out as an important target for the therapy of breast cancers in recent years. We found that the expression level of PAQR4 is closely correlated with HER2 expression in human breast cancer samples. High expression of PAQR4 is closely associated with positive HER2 expression (Table 1, P < 0.0001). It is currently unknown how PAQR4 expression is associated with HER2. In addition, the studies with the patient samples revealed that high expression of PAQR4 is associated with increased metastasis of breast cancer into the lymph nodes (Table 1, P = 0.001). It will be necessary to determine whether PAQR4 has a regulatory role on the migration and metastasis of breast cancer cells in the future. Finally, it will be of paramount significance to explore whether perturbation of PAQR4 function can be used as a new strategy to treat breast cancers. Discovery of chemicals or other means to suppress the tumorigenic activity of PAQR4 will have a beneficial effect on inhibiting tumor cell proliferation. Future studies at these directions will undoubtedly bring new hopes to breast cancer patients. Supplementary material Supplementary data are available at Carcinogenesis online. Funding This work was supported by research grants from National Natural Science Foundation of China (31630036 and 81390350 to Y.C.), Ministry of Science and Technology of China National Key R&D Program of China (2016YFA0500103 to Y.C.), Chinese Academy of Sciences (XDA12010102 and QYZDJ-SSW-SMC008 and ZDRW-ZS-2016–8 to Y.C.). Conflict of Interest Statement: The authors declare no conflicts of interest. Abbreviations CDK4 cyclin-dependent kinases 4 CHX cycloheximide DFS disease-free survival OS overall survival PAQR4 progestin and adipoQ receptor 4 PCHNT para-cancerous histological normal tissue SD standard deviation Acknowledgments We are grateful to Renxu Chang for providing SUM159 cells. We thank Dongxian Guan for advice with the mouse model. References 1. Siegel, R.L.et al.   ( 2015) Cancer statistics, 2015. CA. Cancer J. Clin ., 65, 5– 29. Google Scholar CrossRef Search ADS PubMed  2. Early Breast Cancer Trialists’ Collaborative Group. ( 2005) Effects of chemotherapy and hormonal therapy for early breast cancer on recurrence and 15-year survival: an overview of the randomised trials. Lancet , 365, 1687– 1717. CrossRef Search ADS PubMed  3. Baselga, J.et al.   ( 2012) Everolimus in postmenopausal hormone-receptor-positive advanced breast cancer. N. Engl. J. Med ., 366, 520– 529. Google Scholar CrossRef Search ADS PubMed  4. 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PAQR4 has a tumorigenic effect in human breast cancers in association with reduced CDK4 degradation

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Abstract

Abstract Progestin and adipoQ receptor 4 (PAQR4) is a member of the PAQR family, and the members within this family are involved in the regulation of a number of biological processes including metabolism and cancer development. The potential function of PAQR4 in human cancers is unknown. Analysis of ONCOMINE database reveals that PAQR4 is highly expressed in human breast cancers. We confirmed this finding by analyzing 82 human breast cancers samples. PAQR4 mRNA level was significantly upregulated in human breast cancer samples compared with their corresponding para-cancerous histological normal tissues (P < 0.0001). The mRNA level of PAQR4 was negatively correlated with disease-free survival (P < 0.0001) and overall survival of the patients (P = 0.001). Knockdown of PAQR4 in human breast cancer cells SUM159 and MCF7 suppressed cell proliferation. In contrast, overexpression of PAQR4 in SUM159 cells enhanced cell proliferation and colony formation. In a tumor xenograft model, overexpression of PAQR4 promoted tumor growth of SUM159 cells in vivo, while PAQR4 knockdown suppressed the tumor growth. PAQR4 was able to negatively regulate cyclin-dependent kinases 4 (CDK4) protein level in the breast cancer cells. Knockdown of PAQR4 accelerated degradation of CDK4 together with upregulation of CDK4 polyubiquitination. On the other hand, overexpression of PAQR4 slowed down CDK4 protein degradation and reduced CDK4 polyubiquitination. Collectively, these data at the cellular, animal and human levels indicate that PAQR4 has a tumorigenic effect on human breast cancers, and such effect is associated with a modulatory activity of PAQR4 on protein degradation of CDK4. Introduction Breast cancer is one of the most common cancers worldwide. According to Cancer Facts & Statistics 2015 reports, an estimated 231 840 new cases of invasive breast cancer are expected to be diagnosed among women in the USA during 2015. Excluding cancers of the skin, breast cancer is the most frequently diagnosed cancer in women. In addition, Breast cancer ranks second as a cause of cancer death in women (after lung cancer), an estimated 40 730 breast cancer deaths (40 290 women, 440 men) are expected in 2015 (1). Breast cancer is commonly treated with various combinations of radiation therapy, chemotherapy, hormone therapy (e.g. selective estrogen receptor modifiers, aromatase inhibitors, ovarian ablation) and/or targeted therapy (2–%6). Cyclin-dependent kinases 4 and 6 (CDK4 and CDK6), which are activated by D-type cyclins, are essential proteins important for cell proliferation as G1-specific serine/threonine kinases and have been targeted for cancer therapy (7–%9). The expression of cyclin D1 and activation of CDK4 and CDK6 can drive proliferation of breast cancer cells (10,11).Therapeutic agents that specifically inhibit CDK4 and CDK6 have been proven to be effective for breast cancers (12,13). Progestin and adipoQ receptor 4 (PAQR4) belongs to the PAQR family, which consists of 11 members (PAQR1 to PAQR11) in the human genome (14). All the members within this family are predicted to contain seven transmembrane domains with a topology distinct from the traditional GPCR (G-protein coupled receptor) proteins (14). The physiological roles and molecular functions of the PAQR family have been explored. In particular, the function of PAQR3, a close homologue of PAQR4, has been extensively investigated. As a new member of tumor suppressors, PAQR3 was downregulated in different types of human cancers including colon cancer, gastric cancer, bladder cancer, liver cancer, osteosarcoma, breast cancer and laryngeal squamous cell carcinoma (15–20). PAQR3 is found to negatively regulate Raf-1, a key kinase in the Ras-Raf-Mek-ERK signaling pathway (21). PAQR3 negatively modulates angiogenesis of endothelial cells (22). It also functionally interacts with p53 to modulate cancer formation and epithelial-mesenchymal transition (23). PAQR3 also suppresses AKT activation (24,25). However, the function of PAQR4 has been elusive. In this study, we explored the potential functions of PAQR4 in breast cancers. Material and methods cell culture Human breast cancer cell lines SUM159 and MCF7 were cultured in Dulbecco’s modified Eagle’s medium containing 10% FBS (Invitrogen, Grand Island, NY) and were incubated in a cell incubator at 37°C under 5% CO2. All the cells were purchased from the Cell Bank of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences in early 2016. The cells were authenticated by DNA fingerprinting in the Cell Bank at a regular basis. Lentivirus packaging and infection For lentiviral packaging, HEK293T cells (7 × 106) were seeded in 15 cm cell culture dish, incubated for 24 h and then transfected with 46 μg of lentivirus plasmids. The virus-containing medium was collected and filtered through 0.45 μm filter (Millipore, MA). Afterwards, the filtered supernatants were centrifuged at 20 000 rpm, 4°C for 2 h. The precipitate was suspended in 100 μl Dulbecco’s modified Eagle’s medium and subpackaged in 1.5 ml tubes. For lentivirus infections, the cells (SUM159/MCF7) were cultured in six-well plates for infection by moderate virus-contained Dulbecco’s modified Eagle’s medium with polybrene (Sigma–Aldrich, St. Louis, MO, 4 μg/ml), incubated at 37°C for 8 h and replaced by Dulbecco’s modified Eagle’s medium with 10% FBS. After incubation for 48 h, the infected cell lines were valuated the infection efficiency of lentiviral by real-time PCR. RNA isolation and quantitative RT-PCR The cells were lysed in Trizol reagent (Invitrogen, Carlsbad, CA). Total RNA was purified and reverse transcribed to cDNA according to the manufacturer’s instructions. The quantification of target gene transcripts was detected by ABI Prism 7900 sequence detection system (Applied Biosystems, CA). The primers were as follows: Human PAQR4: 5′-TACCTGCACAACGAACTGGG-3′and 5′-AAGAGGTGATAGAGCACGGAG-3′; Human CDK4: 5′-CTGGTGTTTGAGCATGTAGACC-3′and 5′-GATCCTTGATCGTTT CGGCTG-3′; Huma β-actin: 5′-CTGGAACGGTGAAGGTGACA-3′ and 5′-AAGGGACTTCCTGTAACAATGCA-3′. Cell proliferation assays MTT assay was performed according to the method described previously (26). SUM159 and MCF7 cells were seeded at a density of 4 × 103 cells/well into a 96-well culture plate and grown in 5% CO2 at 37°C for 24 h, 48 h and 72 h. Cell viability was measured by MTT (Sigma–Aldrich, MO) assay as follows: 20 µl of 5 mg/ml MTT was added to each well and incubated with cells at 37°C for 4 h. The MTT solution was then discarded, and 200 µl dimethyl sulfoxide was added to dissolve the formazan sediment. Finally, the optical density was detected using a microplate reader (Molecular Devices, CA) at an absorption wavelength of 490 nm. For colony formation assay, the cells were inoculated into six-well culture plate with 400 cells per well and cultured for 9–13 days. Then, the cells were stained with Crystal Violet for counting. Antibodies and immunoblotting The antibodies purchased were as follows: anti-HA antibody (Santa Cruz Biotechnology, Dallas, TX); anti-Flag and anti-α-tubulin antibody (Sigma–Aldrich); anti-CDK4 antibody (UpState Biotechnology, New York, NY). The protocol for immunoblotting has been described previously (21). Protein degradation analysis For cycloheximide (CHX, Sigma–Aldrich) assay, stable cells were cultured overnight, then treated with 100 μg/ml CHX and harvested at various time points. The cell lysates were subjected to immunoblotting. And the results were quantified by Quantityone software. For ubiquitination assay, stable cells were transfected with vector expressing HA-tagged ubiquitin and other plasmids as described in the figure legend and treated with or without 10 μM MG132 (BD Biosciences, San Jose, CA) for 6 h before the cells were harvested. The lysates were immunoprecipated and then immunoblotted with the antibodies as indicated. PAQR4 gene deletion by CRISPR/Cas9 technology The sgRNA sequence to delete PAQR4 gene was designed according to the website http://crispr.mit.edu. The sequences of sgRNA were as follows: sgRNA-1, 5′-AGTTCGTGCTGACCGGGTACCGG-3′; sgRNA-2, 5′-TCAATAAGTTCGTGCTGACCGGG-3′ with both of them targeting on exon 1 of the human PAQR4 gene. The sgRNA oligo duplex was treated with T4 polynucleotide kinase (Thermo Scientific, MA) and the annealed oligo was inserted into lentiCRISPRv2 (from Addgene) and then used to transform Stbl3 bacteria (Transgene, Strasbourg, France). The plasmids containing the correct insert were confirmed by DNA sequencing. Lentivirus packaging and infection has already been described in the previous section. Following viral infection for at least 3 days, the genomic DNA of the cells was isolated and used in PCR to amplify the targeted region using a high fidelity enzyme KOD polymerase (TOYOBO,Osaka, Japan), followed by DNA sequencing to confirm disruption of PAQR4 gene. Nude mice xenograft model All animals were maintained and used in accordance with the guidelines of the Institutional Animal Care and Use Committee of the Institute for Nutritional Sciences. All of the experimental procedures were carried out in accordance with the Chinese Academy of Sciences ethics commission with an approval number 2010-AN-8. Mice were maintained on a 12 h light/dark cycle at 25°C. The cells in the logarithmic phase of growth were trypsinized, centrifuged and rinsed with PBS three times. The nude mice (4 weeks old, female) was injected subcutaneously with a clonal population of SUM159 cell (2.6 × 106 cells) or MCF7 cell (5 × 106 cells) in the lower right leg. Xenograft tumor sizes were analyzed by measuring two perpendicular diameters with digital calibers every other day and calculated according to the formula: 0.5 × length × width2. Patients and samples This study was approved by the Ethics Committee of the Zhejiang Province Cancer Hospital, Hangzhou, Zhejiang, China. All patients provided their full consent to participate in the study. The patients enrolled in this study underwent curative surgery without prior treatments. Tissue specimens were examined separately by two pathologists under double-blinded conditions without prior knowledge of the clinical status of the specimens. The patients’ medical records were reviewed to obtain data including age at diagnosis, sex, tumor location, tumor size (diameter), lymph node metastasis, histology, tumor invasion and TNM stage according to the guidelines of American Joint Committee. Detailed clinical histopathological factors were presented in Table 1. For the measurement of prognosis, we analyzed the clinical data concerning disease-free survival (DFS) and overall survival (OS), defined as the time from surgery to first recurrence or death, respectively. All recruited patients had been followed up periodically until the due date. Clinical follow-up results revealed that the mean follow-up duration was 69 months and the range was 3–109 months. Table 1. Correlation between relative PAQR4 expression level and clinicopathologic parameters of the patients Variables  N  PAQR4 mRNA level  χ2 (P-value)  Low/normal, n (%)  High, n (%)  Age (years)   <50  50  13 (26.0)  37 (74.0)  0.661   ≥50  32  11 (34.4)  21 (65.6)  (0.416)  Tumor location   Left  38  11 (28.9)  27 (71.1)  0.004   Right  44  13 (29.5)  31 (70.5)  (0.953)  Tumor size   ≤2 cm  39  13 (33.3)  26 (66.7)  0.594   >2 cm  43  11 (25.6)  32 (74.4)  (0.441)  Histology   Moderately/well  34  20 (58.8)  14 (41.2)  24.508   Poorly  48  4 (8.3)  44 (91.7)  (7.400E-07)  T stage   I/II  67  20 (29.9)  47 (70.1)  0.060   III/IV  15  4 (26.7)  11 (73.3)  (0.806)  Lymph node status   Negative  38  18 (47.4)  20 (52.6)  11.207   Positive  44  6 (13.6)  38 (86.4)  (0.001)  Distant metastasis   Negative  82  24 (29.3)  58 (70.7)  82.000   Positive  0  N/A  N/A  (N/A)  TNM stage   I/II  55  19 (34.5)  36 (65.5)  2.247   III/IV  27  5 (18.5)  22 (81.5)  (0.134)  ER status   Negative  39  9 (23.1)  30 (76.9)  1.377   Positive  43  15 (34.9)  28 (65.1)  (0.241)  Her-2 status   Negative  25  15 (60.0)  10 (40.0)  16.407   Positive  57  9 (15.8)  48 (84.2)  (5.108E-05)  p53 status   Negative  25  10 (40.0)  15 (60.0)  2.001   Positive  57  14 (24.6)  43 (75.4)  (0.157)  PR status   Negative  43  11 (25.6)  32 (74.4)  0.594   Positive  39  13 (31.7)  28 (68.3)  (0.441)  Variables  N  PAQR4 mRNA level  χ2 (P-value)  Low/normal, n (%)  High, n (%)  Age (years)   <50  50  13 (26.0)  37 (74.0)  0.661   ≥50  32  11 (34.4)  21 (65.6)  (0.416)  Tumor location   Left  38  11 (28.9)  27 (71.1)  0.004   Right  44  13 (29.5)  31 (70.5)  (0.953)  Tumor size   ≤2 cm  39  13 (33.3)  26 (66.7)  0.594   >2 cm  43  11 (25.6)  32 (74.4)  (0.441)  Histology   Moderately/well  34  20 (58.8)  14 (41.2)  24.508   Poorly  48  4 (8.3)  44 (91.7)  (7.400E-07)  T stage   I/II  67  20 (29.9)  47 (70.1)  0.060   III/IV  15  4 (26.7)  11 (73.3)  (0.806)  Lymph node status   Negative  38  18 (47.4)  20 (52.6)  11.207   Positive  44  6 (13.6)  38 (86.4)  (0.001)  Distant metastasis   Negative  82  24 (29.3)  58 (70.7)  82.000   Positive  0  N/A  N/A  (N/A)  TNM stage   I/II  55  19 (34.5)  36 (65.5)  2.247   III/IV  27  5 (18.5)  22 (81.5)  (0.134)  ER status   Negative  39  9 (23.1)  30 (76.9)  1.377   Positive  43  15 (34.9)  28 (65.1)  (0.241)  Her-2 status   Negative  25  15 (60.0)  10 (40.0)  16.407   Positive  57  9 (15.8)  48 (84.2)  (5.108E-05)  p53 status   Negative  25  10 (40.0)  15 (60.0)  2.001   Positive  57  14 (24.6)  43 (75.4)  (0.157)  PR status   Negative  43  11 (25.6)  32 (74.4)  0.594   Positive  39  13 (31.7)  28 (68.3)  (0.441)  Chi-square test was used for statistical analyses. P < 0.05 is considered statistically significant. ER, estrogen receptor; Her-2, Human epidermal growth factor receptor 2; PAQR4, Progestin and adipoQ receptor; PR, progesterone receptor; TNM, tumor, lymph node, metastasis. View Large Statistical analysis Statistical significance was assessed using Student’s t-test. All results were expressed as the mean ± standard deviation (SD). Values of P < 0.05 were considered statistically significant. Results PAQR4 is significantly upregulated in primary breast cancer tissues and correlated with the prognosis of the patients To investigate the potential role of PAQR4 in human cancers, we first analyzed ONCOMINE database and found that PAQR4 is mainly upregulated in breast cancers (Figure 1A). PAQR4 expression is upregulated (tumor versus normal) in 16 out of 43 breast cancer dataset using the threshold of >2-fold change and P-value < 0.0001 (Figure 1A). We next characterized the expression status of PAQR4 transcript in 82 patients with primary breast cancer. The mRNA level of PAQR4 was determined in both the primary breast cancer samples together with their corresponding para-cancerous histological normal tissue (PCHNT). Intriguingly, we found that PAQR4 mRNA level was significantly elevated in most of the cancer samples compared with PCHNT. Using the standard of over 2.5-fold changes, PAQR4 mRNA was significantly higher in 58 tumors (70.7%, P < 0.0001) than the paired PCHNT samples (Figure 1B and Table 1). The mRNA level of PAQR4 was also associated with a few clinical characteristics of the patients such as histology (P < 0.0001), lymph node metastasis (P = 0.001) (Table 1). In addition, the PAQR4 mRNA level was closely correlated with the status of HER2 expression (γ = 0.447, P < 0.0001) (Table 1). Figure 1. View largeDownload slide Expression of PAQR4 is upregulated in human breast cancer samples and correlated with patient survival (A) Analysis of PAQR4 expression profile in ONCOMINE database. Summary of PAQR4 mRNA expression in various human cancers compared with normal tissues using the threshold of >2-fold change and P < 0.0001. Red: upregulated in cancer; blue: downregulated in cancer. (B) The relative ratio of PAQR4 mRNA in 82 human breast cancer samples versus PCHNT samples. The tumor/PCHNT ratio < 2.5 is considered as low/normal expression. (C, D) Correlation of PAQR4 expression level with survival of breast cancer patients. Kaplan–Meier curves of DFS, shown in C, and OS shown in D in post surgery patients with breast cancers according to the expression level of PAQR4. Figure 1. View largeDownload slide Expression of PAQR4 is upregulated in human breast cancer samples and correlated with patient survival (A) Analysis of PAQR4 expression profile in ONCOMINE database. Summary of PAQR4 mRNA expression in various human cancers compared with normal tissues using the threshold of >2-fold change and P < 0.0001. Red: upregulated in cancer; blue: downregulated in cancer. (B) The relative ratio of PAQR4 mRNA in 82 human breast cancer samples versus PCHNT samples. The tumor/PCHNT ratio < 2.5 is considered as low/normal expression. (C, D) Correlation of PAQR4 expression level with survival of breast cancer patients. Kaplan–Meier curves of DFS, shown in C, and OS shown in D in post surgery patients with breast cancers according to the expression level of PAQR4. We next investigated the association of PAQR4 expression level with the survival of the patients. Both DFS and OS were determined. DFS was defined as the time from surgery to first recurrence, and OS was the time from surgery to death. We found that the average duration of DFS in patients with PAQR4 upregulation in the tumors was significantly shorter than those with normal/low expression of PAQR4 in the tumors (Figure 1C). The average DFS in patients with high expression of PAQR4 was 60.9 months versus 106.0 months in patients with normal/low expression of PAQR4 (P < 0.0001). In addition, the average OS was also different between the patients with high expression of PAQR4 and those with normal/low expression (P = 0.001). Collectively, these data indicate that the expression level of PAQR4 is significantly upregulated in human breast cancers, and such upregulation is associated with poor prognosis of the patients. Knockdown of PAQR4 suppresses proliferation of breast cancer cells We next investigated whether PAQR4 has a direct effect on the growth of human breast cancer cells including MCF7 and SUM159 cell lines. We established stable cell clones with endogenous PAQR4 being silenced by a PAQR4-specific shRNA. The efficiency of PAQR4 knockdown was confirmed by quantitative RT-PCR (Figure 2A and B). We next investigated the cell proliferation rate of these cells by MTT assay. As shown in Figure 2C and D, the cell growth rate was significantly reduced by PAQR4 knockdown in comparison with the control cells. Colony formation was also used to investigate the potential of tumorigenesis of the breast cancer cells. We found that PAQR4 knockdown profoundly reduced colony formation in MCF7 and SUM159 cells (Figure 2E and F). Collectively, these data indicate that silencing of PAQR4 suppresses growth of breast cancer cells. Figure 2. View largeDownload slide PAQR4 affects cell proliferation and colony formation of breast cancer cells (A, B) The mRNA level of PAQR4 in MCF7 and SUM159 cells expressing control shRNA (mock) or PAQR4-specific shRNA (shPAQR4) were detected by quantitative RT-PCR. (C, D) Effect of PAQR4 knockdown on cell proliferation. The breast cancer cells as in A and B were used to determine the cell proliferation rate by MTT assay at the indicated time point. (E, F) Effect of PAQR4 knockdown on colony formation. SUM159 or MCF7 cells as in A and B were seeded into six-well plate with 400 cells per well and cultured for 11 and 13 days, respectively and then used in crystal violet staining and colony counting. (G) The expression level of PAQR4 in control vector (mock) or PAQR4-overexpressing (PAQR4-OV) SUM159 breast cancer cells as detected by quantitative RT-PCR. (H) Effect of PAQR4 overexpression on the cell proliferation rate as determined by MTT assay with the cells as in G. (I) Effect of PAQR4 overexpression on colony formation. The cells as in G were seeded into six-well plate with 400 cells per well and cultured for 9 days, followed by crystal violet staining and colony counting. All data are shown as mean ± SD, **P < 0.01 and ***P < 0.001. Figure 2. View largeDownload slide PAQR4 affects cell proliferation and colony formation of breast cancer cells (A, B) The mRNA level of PAQR4 in MCF7 and SUM159 cells expressing control shRNA (mock) or PAQR4-specific shRNA (shPAQR4) were detected by quantitative RT-PCR. (C, D) Effect of PAQR4 knockdown on cell proliferation. The breast cancer cells as in A and B were used to determine the cell proliferation rate by MTT assay at the indicated time point. (E, F) Effect of PAQR4 knockdown on colony formation. SUM159 or MCF7 cells as in A and B were seeded into six-well plate with 400 cells per well and cultured for 11 and 13 days, respectively and then used in crystal violet staining and colony counting. (G) The expression level of PAQR4 in control vector (mock) or PAQR4-overexpressing (PAQR4-OV) SUM159 breast cancer cells as detected by quantitative RT-PCR. (H) Effect of PAQR4 overexpression on the cell proliferation rate as determined by MTT assay with the cells as in G. (I) Effect of PAQR4 overexpression on colony formation. The cells as in G were seeded into six-well plate with 400 cells per well and cultured for 9 days, followed by crystal violet staining and colony counting. All data are shown as mean ± SD, **P < 0.01 and ***P < 0.001. Overexpression of PAQR4 increases cell proliferation in breast cancer cells We further analyzed the function of PAQR4 on cell proliferation by establishing a SUM159 cell line with overexpression of PAQR4 using a lentivirus-based method. The expression of PAQR4 was confirmed by quantitative RT-PCR (Figure 2G). Overexpression of PAQR4 could promote cell proliferation rate of SUM159 cells by MTT assay (Figure 2H). The capacity of colony formation was also enhanced by PAQR3 overexpression (Figure 2I). These data, therefore, further confirmed the positive effect of PAQR4 on the proliferation of human breast cancer cells. PAQR4 promotes the growth of the breast cancer cells in vivo In order to further elucidate the tumorigenic activity of PAQR4 in breast cancers, we next investigated the effects of PAQR4 on tumor growth using a xenograft model. SUM159 cells with stable expression of PAQR4 were implanted into the nude mice. The mice were sacrificed in 25 days when the tumor formation became obvious. The growth of the breast cancer cells in the mice as measured by tumor volume and tumor weight (Figure 3A to C) was significantly increased by PAQR4 overexpression. Figure 3. View largeDownload slide PAQR4 affects the growth of breast cancer cell xenografts in nude mice (A–C) The volume of the tumors (A) from nude mice inoculated with SUM159 cells expressing control vector (mock) or PAQR4 (PAQR4-OV). Representative images of the tumors isolated from the mice (B) and the weight of the tumors (C). (D–F) The volume of the tumors (D) from nude mice inoculated with MCF7 cells expressing control vector (mock) or shPAQR4. Representative images of the tumors isolated from the mice (E) and the weight of the tumors from the two groups of mice (F). All the data are shown as mean ± SD, *P < 0.05, **P < 0.01 and ***P < 0.001. Figure 3. View largeDownload slide PAQR4 affects the growth of breast cancer cell xenografts in nude mice (A–C) The volume of the tumors (A) from nude mice inoculated with SUM159 cells expressing control vector (mock) or PAQR4 (PAQR4-OV). Representative images of the tumors isolated from the mice (B) and the weight of the tumors (C). (D–F) The volume of the tumors (D) from nude mice inoculated with MCF7 cells expressing control vector (mock) or shPAQR4. Representative images of the tumors isolated from the mice (E) and the weight of the tumors from the two groups of mice (F). All the data are shown as mean ± SD, *P < 0.05, **P < 0.01 and ***P < 0.001. We also investigated the effect of PAQR4 knockdown on tumor growth in vivo. MCF7 cells with knockdown of PAQR4 were inoculated into the nude mice. In agreement with the overexpression data, the tumor volume and tumor volume were all significantly reduced by PAQR4 knockdown (Figure 3D to F). These observations, therefore, clearly indicated that PAQR4 has a strong tumorigenic effect to in vivo. PAQR4 reduces ubiquitination and degradation of CDK4 in breast cancer cells Our preliminary studies revealed that PAQR4 reduces ubiquitination and degradation of CDK4, a critical molecule in controlling G1-to-S transition (Y. Chen, unpublished). We therefore explored the effect of PAQR4 on CDK4 in breast cancer cells. As shown in Figure 4A, overexpression of PAQR4 could elevate the protein level of CDK4. In contrast, knockdown of PAQR4 reduced the protein level of CDK4. Such changes of CDK4 protein level was not caused by changes of CDK4 mRNA level (Figure 4B). We next performed CHX experiments to determine the half-life of CDK4. Silencing of PAQR4 accelerated the degradation rate of CDK4 in the breast cancer cells (Figure 4C and D). In contrast, overexpression of PAQR4 delayed the half-life of CDK4 protein in SUM159 cells (Figure 4E). Next, we investigated the ubiquitination level of CDK4 in the presence and absence of MG132 (a proteasome inhibitor) in breast cancer cells. Knockdown of PAQR4 enhanced ubiquitination of CDK4 in the presence of MG132 in both MCF7 and SUM159 cells (Figure 4F and G). However, overexpression of PAQR4 could reduce ubiquitination of CDK4 in SUM159 cells (Figure 4H). These data, therefore, indicate that PAQR4 is able to modulate ubiquitination and degradation of CDK4 protein in breast cancer cells. Figure 4. View largeDownload slide PAQR4 reduces degradation and polyubiquitination of CDK4 protein (A) PAQR4 regulates the protein level of CDK4. SUM159 and MCF7 cells were infected with lentivirus that had overexpression of PAQR4 or knockdown of PAQR4 by a PAQR4-specific shRNA, respectively. The cells were plated in 12-well plate, cultured for 24 h and then harvested for immunoblotting (IB) with the antibodies as indicated. (B) Effect of PAQR4 on CDK4 expression. The mRNA levels of PAQR4 and CDK4 were measured by quantitative RT-PCR in SUM159 or MCF7 cells with overexpression or knockdown of PAQR4. (C–E) Effect of PAQR4 on the half-life of CDK4. The cells with knockdown or overexpression of PAQR4 were plated in 12-well plate, cultured for 24 h and then treated with 100 μg/ml CHX for various times and then harvested for immunoblotting with the antibodies as indicated. The immunoblot results (repeated for at least three times) were quantified by Quantity One software and were shown in the lower panels. (F–H) The cells were transfected with the plasmids as indicated and then treated with or without MG132 (10 μM) for 6 h before immunoprecipitation (IP) and immunoblotting (IB) using the antibodies as indicated. The data are shown as mean ± SD. **P < 0.01 and ***P < 0.001. Figure 4. View largeDownload slide PAQR4 reduces degradation and polyubiquitination of CDK4 protein (A) PAQR4 regulates the protein level of CDK4. SUM159 and MCF7 cells were infected with lentivirus that had overexpression of PAQR4 or knockdown of PAQR4 by a PAQR4-specific shRNA, respectively. The cells were plated in 12-well plate, cultured for 24 h and then harvested for immunoblotting (IB) with the antibodies as indicated. (B) Effect of PAQR4 on CDK4 expression. The mRNA levels of PAQR4 and CDK4 were measured by quantitative RT-PCR in SUM159 or MCF7 cells with overexpression or knockdown of PAQR4. (C–E) Effect of PAQR4 on the half-life of CDK4. The cells with knockdown or overexpression of PAQR4 were plated in 12-well plate, cultured for 24 h and then treated with 100 μg/ml CHX for various times and then harvested for immunoblotting with the antibodies as indicated. The immunoblot results (repeated for at least three times) were quantified by Quantity One software and were shown in the lower panels. (F–H) The cells were transfected with the plasmids as indicated and then treated with or without MG132 (10 μM) for 6 h before immunoprecipitation (IP) and immunoblotting (IB) using the antibodies as indicated. The data are shown as mean ± SD. **P < 0.01 and ***P < 0.001. CRISPR/Cas9-mediated deletion of PAQR4 retarded growth and reduced CDK4 protein level in MCF7 cells In order to further investigate the effect of PAQR4 on human breast cancer cells, we established MCF7 cells with deletion of endogenous PAQR4 by CRISPR/Cas9 technology. The efficiency of PAQR4 deletion was confirmed by sequencing of the targeted genomic region. We found that the sequences following the sgRNA guide sequence were disrupted, confirming successful deletion of PAQR4 gene in these cells (Supplementary Figures S1 and S2, available at Carcinogenesis Online). We investigated the cell proliferation rate of these cells by MTT and colony formation assays. As shown in Figure 5A and B, the cell growth rate was significantly reduced by PAQR4 deletion in comparison with the parental cells. Next, we explored the effect of PAQR4 deletion on CDK4 protein in MCF7 cells. As shown in Figure 5C, deletion of PAQR4 reduced the steady state protein level of CDK4. We then performed CHX experiments to determine the half-life of CDK4 protein. Deletion of PAQR4 accelerated the degradation rate of CDK4 in MCF7 cells (Figure 5D). Furthermore, deletion of PAQR4 in MCF7 cells could reduce the growth of tumor xenografts in nude mice (Supplementary Figure S3, available at Carcinogenesis Online). These data, therefore, confirmed that PAQR4 is able to positively regulate the growth of breast cancer cells both in vitro and in vivo. Figure 5. View largeDownload slide CRISPR/Cas9-mediated disruption of PAQR4 gene affects cell growth and CDK4 protein in MCF7 cells (A) Effect of PAQR4 deletion on cell proliferation. The cell proliferation rate of parental or PAQR4-deleted MCF7 cells was determined by MTT assay at the indicated time point. (B) Effect of PAQR4 deletion on colony formation. Parental or PAQR4-deleted MCF7 cells were seeded into six-well plate with 300 cells per well and cultured for 11 days, followed by crystal violet staining and colony counting. (C) PAQR4 regulates the protein level of CDK4. Parental or PAQR4-deleted MCF7 cells were harvested for immunoblotting with the antibodies as indicated. (D) Effect of PAQR4 on the half-life of CDK4 protein. Parental or PAQR4-deleted MCF7 cells were plated in 12-well plate and treated with 100 μg/ml CHX for various times and then harvested for immunoblotting with the antibodies as indicated. The immunoblot results (repeated for three times) were quantified by Quantity One software, and the quantitation is shown in the lower panel. All data are shown as mean ± SD, **P < 0.01 and ***P < 0.001. Figure 5. View largeDownload slide CRISPR/Cas9-mediated disruption of PAQR4 gene affects cell growth and CDK4 protein in MCF7 cells (A) Effect of PAQR4 deletion on cell proliferation. The cell proliferation rate of parental or PAQR4-deleted MCF7 cells was determined by MTT assay at the indicated time point. (B) Effect of PAQR4 deletion on colony formation. Parental or PAQR4-deleted MCF7 cells were seeded into six-well plate with 300 cells per well and cultured for 11 days, followed by crystal violet staining and colony counting. (C) PAQR4 regulates the protein level of CDK4. Parental or PAQR4-deleted MCF7 cells were harvested for immunoblotting with the antibodies as indicated. (D) Effect of PAQR4 on the half-life of CDK4 protein. Parental or PAQR4-deleted MCF7 cells were plated in 12-well plate and treated with 100 μg/ml CHX for various times and then harvested for immunoblotting with the antibodies as indicated. The immunoblot results (repeated for three times) were quantified by Quantity One software, and the quantitation is shown in the lower panel. All data are shown as mean ± SD, **P < 0.01 and ***P < 0.001. Discussion Our studies have provided compelling evidence that PAQR4 has a tumorigenic function in human breast cancer cells via studies in vitro, in vivo and in human samples. At the cellular level, PAQR4 promotes the cell proliferation and tumorigenicity of human breast cancer cells. At the animal level, PAQR4 strongly enhances the growth of breast cancers in nude mice. Mechanistically, PAQR4 regulates the steady state level of CDK4, a critical protein that controls G1-to-S transition of the cell cycle. PAQR4 reduces ubiquitination and degradation rate of CDK4 in breast cancer cells and such regulation likely underlies its tumor-promoting activity in these cells. At the clinical level, PAQR4 expression level was robustly upregulated in human breast cancer samples as compared with the adjacent normal tissues. Furthermore, the expression status of PAQR4 was associated with the survival of the breast cancer patients. Due to potential significance of PAQR4 in regulating the tumorigenicity of breast cancers, it will be important to further explore the functions of PAQR4 in the context of breast cancer development. Firstly, it will be important to further elucidate the molecular mechanisms associated with the tumor-promoting activity of PAQR4 in breast cancer. Next, it will be important to investigate the functional interaction between PAQR4 and Her2. Her2 has stood out as an important target for the therapy of breast cancers in recent years. We found that the expression level of PAQR4 is closely correlated with HER2 expression in human breast cancer samples. High expression of PAQR4 is closely associated with positive HER2 expression (Table 1, P < 0.0001). It is currently unknown how PAQR4 expression is associated with HER2. In addition, the studies with the patient samples revealed that high expression of PAQR4 is associated with increased metastasis of breast cancer into the lymph nodes (Table 1, P = 0.001). It will be necessary to determine whether PAQR4 has a regulatory role on the migration and metastasis of breast cancer cells in the future. Finally, it will be of paramount significance to explore whether perturbation of PAQR4 function can be used as a new strategy to treat breast cancers. Discovery of chemicals or other means to suppress the tumorigenic activity of PAQR4 will have a beneficial effect on inhibiting tumor cell proliferation. Future studies at these directions will undoubtedly bring new hopes to breast cancer patients. Supplementary material Supplementary data are available at Carcinogenesis online. Funding This work was supported by research grants from National Natural Science Foundation of China (31630036 and 81390350 to Y.C.), Ministry of Science and Technology of China National Key R&D Program of China (2016YFA0500103 to Y.C.), Chinese Academy of Sciences (XDA12010102 and QYZDJ-SSW-SMC008 and ZDRW-ZS-2016–8 to Y.C.). Conflict of Interest Statement: The authors declare no conflicts of interest. 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CarcinogenesisOxford University Press

Published: Mar 1, 2018

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