10058-F4 – Carbetocin

The absence of PRDM2 involved the tumorigenesis of somatotroph adenomas through regulating c-Myc

Abstract

Somatotroph adenoma is the main cause of acromegaly which have peripheral signs with growth of soft tissues and multiple comorbidities. Surgery and adjuvant therapy with somatostatin analogs (SSA) fail in more than 25% of patients. PRDM2, a tumor suppressor, plays an important role in cancer and obesity, including pituitary adenomas. In this study, we analyze the correlation of PRDM2 and oncogene c-Myc in 70 somatotroph adenomas according immunohistochemical staining, furthermore, we probed that whether PRDM2 participates in c-Myc signaling pathway in vitro experiment. 70 somatotroph adenomas patients were divided into low patients and high patients according to median of H-score of PRDM2 or c-Myc. Low PRDM2 patients had higher risk of invasive behavior, larger tumor volume and recurrence chance than high PRDM2 group (P = 0.015, P = 0.031, P = 0.017). High c-Myc patients had higher risk of invasive behavior, larger tumor volume and recurrence chance than low c-Myc group (P = 0.012, P = 0.002, P = 0.015). It was a negative correlation between H-score of PRDM2 and c-Myc (PRDM2 = −0.163 × c-Myc + 67.11, r = −0.407). The ability of cell proliferation was declined in a time dependent manner after overexpression of PRDM2 (PRDM2 group) compared to that in control GH3 cells (P < 0.05). Through ﬂow cytometry assay, PRDM2 could induce the apoptosis and G2/M arrest in GH3 cell (both p < 0.05). Transwell experiment proved less trans-membrane cells in PRDM2 group than those in control group (415 ± 76 vs 145 ± 37, P < 0.01). RT-PCR and western blot both proved PRDM2 could inhibit the level c-Myc and elevate the levels of CDKN1A and CDKN1B. Combined with c-Myc inhibitor 10058-F4, PRDM2 further inhibited cell proliferation and induced more apoptosis in GH3 cell. Taken together, we found that PRDM2 negatively regulated the expression of c-Myc in somatotroph adenomas, and testiﬁed the synergism between PRDM2 gene therapy and c-Myc inhibitor in vitro experiment. 1. Background As second common intracranial tumor, pituitary adenomas may hypersecrete hormones or cause mass eﬀects and the prevalence ranges 1/865 to 1/2688 in adults, including somatotroph adenoma, lacto- toroph adenoma, thyrotroph adenoma, corticotroph adenoma, gona- dotroph adenoma and null cell adenoma (Melmed, 2011; Molitch, 2017). Somatotroph adenomas are the main reasons of acromegaly which have peripheral signs with growth of soft tissues and multiple comorbidities, such as metabolic, cardiovascular, oncological, etc (Gadelha et al., 2017). Surgery and adjuvant therapy with SSAs is ﬁrst therapy, and there are less than 1.0 ng/L serum growth hormone (GH) and normalization of insulin-like growth factor-1 (IGF-1) in 30% pa- tient although 45–50% have IGF-1 decreases by more than 50%. However, more than 25% of cases are fail to present medical strategy (Melmed, 2016). Increasing evidences have highlighted frequent alterations of oncogene, tumor suppressor gene and cell cycle in pituitary adenomas (Ezzat et al., 2018). As a vital oncogene, c-Myc comprises ubiquitous regulators of gene expression in over half human of cancers, and promotes tumorigenesis through several mechanisms, including gene ampliﬁcation, chromosomal translocation, mutation of signal transduction pathways and protein stability (Bayliss et al., 2017;Caforio et al., 2018). c-Myc is a promising targeting biomarker of tumor treatment based on the inhibition of cyclin-dependent kinase (CDKs), including antagonizing the activity of cell cycle inhibitors as CDKN1A and CDKN1B through diﬀerent mechanisms (Gartel and Shchors, 2003; Bretones et al., 2015). Fig. 1. Immunochemistry analysis of the expression of PRDM2 and c-Myc in PAs. A: MRI showed invasive and non-Invasive adenomas according Knosp staging. B: Immunochemistry analysis of c-Myc and PRDM2. The PR/SET domain gene family (PRDM) have deletion, mutation, epigenetic changes in various tumor species (Mzoughi et al., 2016). For example, PRDM2/3/9/16 and ZFPM2/FOG2 were the most mutated genes with higher than 1% frequencies (Sorrentino et al., 2018a,b). PRDM2, coded protein RIZ1, is one of histone/protein methyltransferases (PRDMs), which regulates the methylation levels of serum H3K4/9/27 in pituitary adenomas patients (Xue et al., 2017). TAT-RIZ1-PR protein exerts histone methyltransferase activity and tumor-suppressive functions in human malignant meningiomas (Ding et al., 2015). PRDM2 relieved the level of Akt3 and increased the me- thylation of H3K9 in HEK293 cells (Liu et al., 2018). Our group re- ported that low PRDM2 expression is associated with dopamine-agonist resistance and tumor recurrence in lactotroph adenomas and the level of c-Myc is closely related to the invasive behavior of non-functioning pituitary adenomas (Gao et al., 2015; Liu et al., 2017). In addition, c-a High: According to the median H-score of PRDM2, more than 50%. Low: According to the median H-score of PRDM2, less than 50%. 2. Methods 2.1. Patient samples and cell lines All samples were obtained following transsphenoidal surgery per- formed at Beijing Tiantan Hospital from May 2012 through May 2017. Fresh tumor samples were stored in liquid nitrogen. 70 samples from the study population (age range, 17–69 years) were diagnosed as so- matotroph adenoma according to the 2017 World Health Organization classiﬁcation of tumors of endocrine organs (Mete et al., 2018). Five normal pituitary glands were obtained from a donation program. The study protocols were approved by the Internal Review Board of Beijing Tiantan Hospital, which was aﬃliated to Capital Medical University, and conformed to the ethical guidelines of the Declaration of Helsinki (no. KY-2013-02).GH3 cells were purchased from ATCC and cultured in a humidiﬁed incubator at 37 °C and 5% CO2 in F-12K medium (ATCC, Manassas, VA, USA) supplemented with 2.5% fetal bovine serum and 10% horse serum. PRDM2 plasmid (RC227715) was purchased (OriGene, Beijing, China). 2.2. Tissue microarray construction and immunochemistry staining A total of 75 formalin-ﬁXed paraﬃn-embedded tissue blocks were sectioned. Three core biopsies (2.0 mm in diameter) were selected from the paraﬃn-embedded tissue. The cores were transferred to tissue mi- croarrays using a semi-automated system (Aphelys MiniCore, Mitogen, UK). The microarrays were cut into 4-μm sections and incubated with PRDM2 (Rabbit polyclonal, 1:300, ab204110, Abcam), anti-CDKN1A (Mouse polyclonal, 1:400, ab80633, Abcam) and anti-CDKN1B (Rabbit monoclonal, 1:400, ab32034, Abcam) primary antibodies. The H-score was obtained by multiplying the staining intensity by a constant to adjust the mean to the strongest intensity [H-score = 3 × (percentage of strong staining)] (1.0%, weak; 2.0%, moderate; 3.0%, strong) to give Myc was associated with methyltransferase activity of H3K4 and sti- mulates demethylation and subsequently acetylation of H3K27 (Ullius et al., 2014). Targeting c-Myc sensitizes malignant mesothelioma cells to p21-activated kinase blockage-induced cytotoXicity (Tan et al., 2017). The imbalance of cell survive and cell death leads to tumorigenesis. Translational studies use precision gene editing to manipulate genes and proteins which lead to regulatory/functional/drug sensitivity dis- coveries as well as therapeutic approaches in cancer ﬁelds. In this study, we evaluated the levels and correlation of PRDM2 and c-Myc in 70 somatotroph adenoma patients in combination with follow-up data according tissue microarray. Furthermore, we probed that whether PRDM2 participates in the downstream signaling pathway of c-Myc through in vitro experiment, furthermore, we testiﬁed the synergism of PRDM2 gene therapy combined with c-Myc inhibitor in GH3 cell line. 2.3. Cell proliferation, apoptosis, cell cycle and migration assays GH3 cells were adjusted to a density of 1 × 105 cells/ml. A total of 100 μL of the cell suspension was plated into each well of a 96-well plate and cultured overnight. After transient transfection for 24 h, 48 h, and 72 h, 20 μL of 3-(4,5-diethylthiazol-2-yl)-5-(3-carboXymethoXyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) solution was added to each well, and the cultures were further incubated for 4 h. Absorbance was measured at 490 nm using an ELISA plate reader (Thermo, USA). Apoptosis and cell cycle was determined by ﬂow cy- tometry using Detection Kit (Roche Diagnostics). Cell migration was measured using ﬁbronectin and Matrigel-coated polycarbonate ﬁlters, respectively, modiﬁed transwell chambers (Corning). GH3 cells (5 × 104 cells) were added into the upper chambers. Migrating cells that adhered to the lower membrane were ﬁXe in 4% paraformaldehyde and stained using hematoXyl in (ZSGB- BIO, China). EXperiments were performed in triplicate time. 2.4. ELISA assay The levels of GH in cell culture supernatant were measured by ELISA (Applygen, China) according to the manufacturer’s protocol. A total of 10 μL of supernatant was used per well. Absorbance was read at 450 nm using an ELISA plate reader (Thermo Fisher, USA). EXperiments were performed in triplicate time. 2.5. Polymerase chain reaction analysis (PCR) Microarray hybridization and qRT-PCR were performed as previously described (Feng et al., 2019). Total RNA of 30 samples was extracted and puriﬁed using the mirVana™ miRNA Isolation Kit (Am- bion, USA), following the manufacturer’s instructions. RT-qPCR was performed on an Applied Bio-systems 7500 Fast System (Life Technol- ogies). The fold-change in diﬀerential expression for each gene was calculated using the comparative CT method (2−ΔΔCT method) in R package with “pcr” functions (https://github.com/MahShaaban/pcr), a GAPDH reference gene, and the “somatroph adenoma” reference group (Pabinger et al., 2014). Fig. 2. PSF in 70 somatotroph adenomas patients. A: The correlation of c-Myc and PRDM2 according to H-score. r = −0.407, P < 0.001B: PSF according to the c- Myc levels. Hazard Ratio = 0.39 95% CI: 0.17–0.88. C: PSF according to the PRDM2 levels. Hazard Ratio = 2.82 95% CI: 1.26–6.290. 2.6. SDS-PAGE and western blot analyses Samples (10 mg) were lysed in lysis buﬀer containing 1% Nonidet P-40 (Calbiochem) and protease and phosphatase inhibitor cocktails (Roche) overnight at 4 °C overnight. Total extracts were centrifuged at 12000×g for 30 min at 4 °C, and protein concentration was determined with the BCA method (Pierce Biotechnology). A total of 40 μg of protein per lane was loaded onto 10% Bis-Tris SDS-PAGE gels, separated elec- trophoretically, and blotted onto polyvinylidene ﬂuoride (PVDF) membranes. The blots were incubated with antibodies against c-Myc (Rabbit monoclonal, 1:1000, ab32072, Abcam), anti-PRDM2 (Rabbit polyclonal, 1:1000, ab204110, Abcam), anti-CDKN1A (Mouse poly- clonal, 1:1000, ab80633, Abcam) and anti-CDKN1B (Rabbit mono- clonal, 1:2000, ab32034, Abcam) followed by a secondary antibody tagged with horseradish peroXidase (Santa Cruz Biotechnology). Blots were visualized by enhanced chemiluminescence, and densitometry was performed using a ﬂuorescence image analyzer (Amersham Imager 600). GAPDH was used as a loading control. 2.7. Statistical analysis All statistical analyses were conducted using SPSS Statistics Version22 (IBM Corporation, Armonk, New York, USA). An unpaired Student’s test and a chi-square (Fisher’s exact) test were used to compare quantitative and qualitative data. P value of less than 0.05 was considered signiﬁcant. 3. Results 3.1. Clinical and pathological features of 70 somatotroph adenomas patients Among these 70 patients, there were 39 males (55.7%) and 31 fe- males (44.3%) with average age (37.4 ± 7.6 year), average diameter (2.7 ± 0.84 cm, range 1.4–3.8 cm). The follow-up time was 27 ~ 69 months (47 ± 17 months on average). According to the Knosp/Hardy-Wilson classiﬁcation, we assured 32 invasive cases and 38 non-invasive cases. The average preoperative serum GH level was 35.6 ± 12.2 ng/ml and 34.3% (24/70) patients had headaches. According to the body mass index (BMI) of China standard, there are 15/70 (21.4%) normal, 24/70 (34.3%) overweight and 31/70 (44.3%) obesity. The recurrence patients of ﬁve years were 24/70 (34.3%). 3.2. Immunohistochemistry analysis of PRDM2 and c-Myc in 70 somatotroph adenomas patients Immunohistochemistry results showed that the H-score for PRDM2 and c-Myc was 38.4 ± 16.5 and 176.3 ± 42.3 respectively in Fig. 1. According to the median H-score of PRDM2 or c-Myc, 70 cases were divided into high group and low group. The low PRDM2 group (< 42.5) had more invasive behavior compare to the high PRDM2 group (≥42.5)(19/35 vs 9/35, P = 0.015) and had larger tumor size (7.67 ± 1.74 cm3 vs 5.23 ± 1.46 cm3, P = 0.031) in Table 1. The high c-Myc group (≥184.7) had more invasive behavior (20/35 vs 8/ 35, P = 0.003) and larger tumor size (8.32 ± 2.07 cm3 vs 4.58 ± 1.33 cm3, P = 0.002) than that in low c-Myc group in Table 2. There was a negative correlation between PRDM2 and c-myc (PRDM2 = −0.163 × c-Myc + 67.11, r = −0.407) in Fig. 2A. In additional, H-score of PRDM2 or c-Myc scores was independent of gender and age.Average progression free survival (PFS) time in the high PRDM2 group was longer than that in low PRDM2 group (9/35 vs 15/35, Hazard Ratio = 0.39, 95% CI: 0.17–0.88, P = 0.017), and 30.7 months vs 54.5 months p = 0.013 according to the level of c-Myc (17/35 vs7/ 35, Hazard Ratio = 2.82, 95% CI: 1.26–6.29, P = 0.015) in Fig. 2BC. 3.3. Eﬀect of PRDM2 on cell proliferation, apoptosis, cell cycle, GH secretion and migration of GH3 cell In present study, we constructed the plasmid of vector and PRDM2, and testiﬁed the mechanism of PRDM2 involved in cell proliferation, cell cycle, GH secretion, apoptosis and migration used rat pituitary GH3 cells. The cell proliferation test showed that the cell viability of PRDM2 group was (82.2 ± 6.3) %, (68.5 ± 5.2) % and (54.7 ± 3.6) % of that in control group after 24 h, 48 h and 72 h transfection in Fig. 3A, respectively (all P < 0.05). Annexin V-positive cells in control group was (3.35 ± 1.03) % and PI-positive cells was (1.34 ± 0.48) %, (5.69 ± 2.74%) and (2.59 ± 1.22) % in vector group (P > 0.05), and (14.66 ± 3.72) % and (6.21 ± 2.17) % in PRDM2 group after 72 h treatment in Fig. 3B (P < 0.05). Flow cytometry showed that PRDM2 induced G2/M phase arrest in Fig. 3C (P < 0.05). ELISA ex- periment proved that the level of GH in PRDM2 group was 35.7 ± 8.4% of that in control group in Fig. 3D (P < 0.05). Transwell chamber test showed that there were (415 ± 76) transmembrane cells in the control group, (382 ± 71) cells in the empty vector group, and (145 ± 37) cells in the PRDM2 group (P < 0.01) in Fig. 3E. Fig. 3. Anti-cell proliferation of PRDM2 in GH3 cell line. A: The inhibition of PRDM2 on cell viability in time manner and dose manner. B: PRDM2 induced the apoptosis in GH3 cell after 72 h transient transfection. C: PRDM2 induced G2/M phase arrest in GH3 cells. D: ELISA showed the declination of GH in supernatant of GH3 cell. E: Transwell experiment showed the impairment of migration ability of GH3 cell after overexpression of PRDM2. F: RT-PCR experiment showed that PRDM2 downregulated the mRNA level of c-Myc and upregulated the level of CDKN1A/CDKN1B after 72 h transient transfection. G:The bands of western blot. H: The statistic of western blot experiment showed that PRDM2 downregulated the protein level of c-Myc and upregulated the level of CDKN1A/CDKN1B after 72 h transient transfection. (*P < 0.05 ** P < 0.01 *** P < 0.001, NS: nonsense). Promotion of the cell cycle is a major oncogenic mechanism of the oncogene c-Myc through antagonizes the activities and/or the expres- sion levels of CDKN1A, and CDKN1B (García-Gutiérrez et al., 2019). We found that the level of c-Myc was remarkably reduced by PRDM2 whether mRNA level Fig. 3F or protein level in GH3 cell in Fig. 3GH (both P < 0.05). The ascend tendency of CDKN1A and CDKN1B co- incident with the increased G1-phase arrest after overexpression of PRDM2. 3.4. PRDM2 combined and c-Myc inhibitor, 10058-F4, in GH3 cell Considering the role of c-Myc inhibitor in the growth arrest and chemosensitivity, we explored the eﬀect of PRDM2 combined c-Myc inhibitor, 10058-F4, in GH3 cell. We found that the inhibiting of PRDM2 and/or 10058-F4 on cell viability of GH3 cell was time manner in Fig. 4A (P < 0.01). There were more Annexin V-positive and PI- positive cells in PRDM2 combined with10058-F4 group than those in PRDM2 group or 10058-F4 group in Fig. 4B (P < 0.05). We also ob- served more G2/M phase arrest and lower GH level in combined group than other groups in Fig. 4CD (P < 0.05). Transwell experiment proved the relived trans-membrane positive cells after PRDM2 and/or 10058-F4 in Fig. 4E (P < 0.05). RT-PCR and western blot experiments both proved that there were the highest level of CDKN1A and CDKN1B in PRDM2 combined with 10058-F4 group in Fig. 4FGH (P < 0.05). Fig. 4. The synergism of PRDM2 and c-Myc inhibitor, 10058-F4, in GH3 cell line. A: The inhibition of PRDM2 and/or 10058-F4 on cell viability. B:The apoptosis in GH3 cell after PRDM2 and/or 10058-F4 treatment. C: G2/M phase arrest in GH3 cells after PRDM2 and/or 10058-F4 treatment. D: ELISA showed the declination of GH in supernatant of GH3 cell after PRDM2 and/or 10058-F4 treatment. E: Transwell experiment showed the impairment of migration ability of GH3 cell after overexpression of PRDM2 after PRDM2 and/or 10058-F4 treatment. F: The mRNA level of c-Myc and CDKN1A/CDKN1B after PRDM2 and/or 10058-F4 treatment. G: Bands of western blot after PRDM2 and/or 10058-F4 treatment. H:The protein level of c-Myc and CDKN1A/CDKN1B after PRDM2 and/or 10058-F4 treatment. Inhibitor: 10058-F4 group (40 µM), Combined group: PRDM2 + 10058-F4 group (40 µM). (* P < 0.05, ** P < 0.01 *** P < 0.001, NS: nonsense). 4. Discussion Recent advances in next-generation sequencing indicate that there is no high frequency somatic mutations in subtypes of pituitary ade- noma except adrenocorticotropic tumor (Chen et al., 2018). Imbalance of tumor suppressor genes would contribute more important role in the tumorigenesis of somatotroph adenomas. Pituitary tumor transforming gene 1 (PTTG1) increased c-Myc and proliferating cell nuclear antigen expression and inhibited cell proliferation. In this study, we explored the negative correlation of tumor suppressor gene, PRDM2, and c-Myc in somatotroph adenomas, furthermore discovered the possibility of PRDM2 combined c-Myc inhibitor as medical therapy of somatotroph adenomas. PRDM2/RIZ is a member of the histone/protein methyltransferase (PRDM) superfamily, which is characterized by conserved N-terminal domain, methyltransferase activity and C-terminal zinc ﬁnger array (Sorrentino et al., 2018a,b). PRDM2 was signiﬁcantly downregulated as the formation of meningiomas progressed, and PRDM2 inhibited cell proliferation and induced G2/M phase arrest in a meningioma cell line IOMM-Lee (Liu et al., 2013). Whether oncogene or tumor impress genes mainly involved into core cellular processes: cell fate, cell survival, and chromatin stability. PRDM2 participated to the formation of protein complexes involved in the DNA damage response and in genome maintenance (Schneider et al., 2002). In this study, we found larger BMI index, larger tumor size, more invasive behavior and longer PSF time in low PRDM2 patients than those in high PRDM2 patients. The level of PRDM2 was negative correlated with high-grade endometrial carcinoma and positive expression of ERα, and estrogen could down- regulated the level of PRDM2 through in vitro experiments (Yang et al., 2017). Our group reported there were higher estrogen receptor α (ERα) in elder somatotroph adenomas and nonfunctioning adenomas. We also noticed that the opposite tendency between PRDM2 and ERα that came coincident with estrogen could down regulated the expression of PRDM2 in endometrial cancer cell (data no shown). Overexpression of PRDM2 played the antagonistic role in programs-myogenesis and the cell cycle in G0 myoblasts (Cheedipudi et al., 2015). Meanwhile, PRDM2 was associated with the downregulation of c-myc expression in GH3 cells. Our data shown that PRDM2 preserves key functions of the quiescent state through induce apoptosis and G2/M phase arrest in GH3 cells. PTTG1 promotes the tumorigenesis and metastasis of ovarian cancer regulating cellular metabolic reprogramming via c-myc pathway (Wang et al., 2015). c-Myc interacts with several central cell cycle regulators which participate into the balance of cell cycle progression and arrest (cellular senescence), including cyclin E/Cdk2 complex, CDKN1BKIP1 and the E3 ubiquitin ligase component S-phase kinase-associated pro- tein 2 (SKP2) (Hydbring et al., 2017). c-Myc plays an important role in the progression of cells through both the G1 and G2 phases of the cell cycle (Seth et al., 1993). Combined loss of pRB-E2F 30transcriptional regulation and p27KIP1 leads to defective proliferative control in re- sponse to various types of DNA damage (Thwaites et al., 2017). Genetic mouse models proved that the pituitary is a highly sensitive organ to genetic alteration of speciﬁc cell cycle regulators (Quereda and Malumbres, 2009). We reported that high cyclin E/Cdk2 and low CDKN1B in somatotroph adenomas had a poor prognosis (Dong et al., 2018). 10058-F4, a c-Myc inhibitor, could inhibit the cell proliferation in time manner and dose manner and induce G2/M phase arrest in GH3 cell line, furthermore, 10058-F4-induced apoptosis could also be observed. Promoter methylation and H3K9 modiﬁcations work together to silence the RIZ1 gene in hepatic carcinoma (Zhang et al., 2010). c-Myc and TXNIP, two putative downstream targets of H3K9 methylation, may be involved in regulating RIZ1 tumor-suppressive eﬀects (Ullius et al., 2014). Given the role of c-MYC in controling ribosome biogen- esis, it is reasonable that the c-Myc inhibitor contributes to the per- turbation of nuclear functions and RNA polymerase I activity (Chau et al., 2018). 10058-F4 showed enhanced therapeutic eﬃcacy combine with vincristine in acute lymphoblastic leukemia (Sheikh-Zeineddini et al., 2019). In this study, we measured the possibility of combined PRDM2 gene therapy and c-Myc inhibitor in GH3 cell line. In vitro experiments proved that more apoptosis and the lowest level of c-Myc in combined group compared to those in PRDM2 group or inhibitor group. The levels of CDKN1A and CDKN1B in combined group also testiﬁed the synergism of PRDM2 and 10058-F4. These changes re- ﬂected an early and persistent modulation of c-Myc and cyclin depen- dent kinase inhibitor CDKN1A/CDKN1B.

5. Conclusions

Recent evidence suggests that complex interactions among cell cycle control, cell proliferation, apoptosis, invasion, and angiogenic genetic and epigenetic changes ultimately lead to malignant transformation of malignant phenotypes. Considering the inactivation of PRDM2 and activation of c-Myc in somatotroph adenomas, restoring PRDM2 combined c-Myc inhibitor will provide new options for the treatment of somatotroph adenomas. Certainly, PRDM2 combined with radiotherapy would be another treatment strategy.