Over expression of PTEN induces apoptosis and prevents cell proliferation in breast cancer cells

1Department of Pathology, The First Affiliated Hospital of USTC, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, Anhui, 230036, China; 2Intelligent Pathology Institute, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, Anhui, 230036, China; 3Department of Pathology, The First Affiliated Hospital of USTC, Division of life Sciences and Medicine, University of Science and Technology of China, Hefei, Anhui, 230031, China; 4Department of Head And Neck Breast Surgery, The First Affiliated Hospital of USTC, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, Anhui, 230031, China


INTRODUCTION
In world wide, breast cancer is the most common cancer and one of the leading cause for cancer associated death rate among women (Becker, 2015). Breast cancer is heterogeneous whose molecular features include activation of hormone receptors (oestrogen and progesterone), epidermal growth factors (HER2) mutations in BRCA gens. Breast cancer is curable when it is diagnosed at early stage non-metastatic condition and it also depends on the patient's age because the main risk factor of the breast cancer is the women age (Harbeck et al., 2019). Advanced stages at distant organ metastasis are usually treated with different treatment strategies for instance surgery, radiation therapy and systemic therapy such as endocrine therapy for hormone receptors, chem-otherapy and anti-HER2 therapy (Ataollahi et al., 2015;Carbognin et al., 2019;Harbeck et al., 2019). The ultimate goals of such treatment approaches are prolonged survival and improved quality of patient's life. Although recent developments of molecular approaches in cancer treatment and plethora of drugs designed, the enhanced proliferation rate and cancer cell migration are the major implications in the diagnosis and treatment of cancers. Considering this fact, researchers should get more insights in to the molecular biology and immunology to unravel the molecular characteristics cancer metastasis. Therefore, detailed study of molecular and signaling pathways involved in cancer progression is urging one to design and develop a right anti-cancer drug.
Phosphatase and tensin (PTEN) is a lipid and protein phosphatase, acts as a tumor suppressor involved in the regulation of cell cycle, cell growth and survival Steck et al., 1997;Li et al., 2015). The key function of PTEN is inhibiting or down regulating the phosphatidylinositol-3-kinase (PI3K)/protein kinase B (Akt) signaling, a pathway involved in various cellular processes such as cell proliferation, motility, invasion and survival (Li et al., 2015). Therefore, PTEN exhibits anti-oncogenic effect by attenuating P13K/AKT pathway through dephosphorylation of PIP3 (phosphatidylinositol (3,4,5)-trisphosphate) to PIP2 (phosphatidylinositol (4,5)-bisphosphate), and thus terminating the signals from growth factors for PI3K/AKT signal transduction pathways and activation of downstream targets (Lu et al., 2016). However, it remains unclear whether PTEN can target any other cellular pathways other than P13K/ AKT. But it was reported that PTEN exert tumor suppressive effect by potentially inhibiting cell cycle progression, telomerase activity and as a consequence apoptosis was induced (Tian et al., 1999;Weng et al., 2001). Loss or mutated PTEN gene was frequently spotted at different stages of cancers such as several cancers such as breast, brain, prostate, lung, liver, colorectal and endometrium cancers Li et al., 2016). Further, loss of PTEN expression and its function has been associated with aggressive tumor phenotypes accompanied with reduced apoptosis and enhanced cell proliferation. Therefore, we have investigated the effect of over expression of PTEN on apoptosis induction, cell proliferation and tumor invasion in breast cancer cells (MCF-7). Anhui Provincial Cancer Hospital. Cells were cultured in DMEM with 10% FBS and antibiotics such as penicillin and streptomycin. Cells were usually maintained in 5% CO 2 with humidified air at 37°C. Cells were either cultured in Corning petri dished or T75 flask. Plasmid transfection. Cells were cultured in 6 well plate for 24 hours in 5% CO 2 at 37°C. For transfection, 8 μL of either of the plasmids pCMV-PTEN (Addgene) or GFP-PTEN (Addgene) are mixed in OPtiMEM (Thermofishers) containing 6 μL of transfection reagent Lipofectamine (Qiagen) and incubate at room temperature. After 20 minutes of incubation, the reaction mixture was added to the cells and incubated for either 24 or 48 hours.
Fluorescence Microscopy. Transiently transfected cells were collected after 48 hours by centrifugation for 5 minutes at 200 g and washed with 3 times to remove the cell debris. Subsequently cells were subjected to Hoechst 33344 staining by adding 10 μL of 10 mg/L of Hoechst 33344 dye to the cell suspension, incubated at dark for 30 minutes. Simultaneously, another portion of cells were subjected to 10 μL of acridine orange/ethidium bromide (AO/EtBr) solution. The stained cells were dripped (approximately 10 μL) on slides ad covered by coverslips. The morphological features of apoptotic cells were visualized by fluorescence microscope (400Å~), photographed and the acquired pictures were processed Adobe Photoshop software version 7.0.
Flow cytometry analysis. The apoptosis rate was measured by propidium iodide staining kit (Sigma-Aldrich). After 48 hours incubation of transient transfection with PTEN over expression cassette, cells were harvested and fixed in 70% ice-cold methanol. After overnight incubation with PI and RNase at a 37°C water bath in dark condition, cells were analyzed by flow cytometry and data were represented as a quantification graph showing percentage of apoptosis rate.
In vitro cell proliferation assay. Cells were seeded in a 96-well plate with a range of 1×10 6 cells/well (in triplicates) and the cell proliferation activity was measured every day for 7 days. 2-3 Hours before optical density measurement, cells were supplemented with CCK-8 solution (10 μL) and incubated in CO 2 incubator. Subsequently, optical density (OD) was determined at 490 nm was measured and the cell growth rate graph was made on the OD values and represented as a graph.
Chemoresistance assay. Cells were seeded in a 96well plate with a range of 1×10 6 cells/well (in triplicates). Followed by 24 hours incubation, docetaxel (10 μM) and doxorubicin and 5-fluorouracil (5-FU) of final concentra-tion of 10 μg/ml was added to the cell cultures. After 48 hours, chemoresistance assay and the formula was evaluated as described previously (He et al., 2014).
Statistical analysis. The t-test was used to compare between the groups and between multi-groups were analyzed by a multi-factor Q-test. The values defined were *P<0.05 and **P<0.01 significant.

PTEN over expression inhibits cell proliferation via downregulating P13K/AKT signaling pathway
It has been previously reported that over expression of PTEN contributes to cell cycle arrest by and induce apoptosis by suppressing the P13K/AKT pathway in hepatocellular carcinoma, renal carcinoma, breast cancer cells and glioma cells Lu et al., 2016). Therefore, we have investigated the protein expression levels of PTEN, P13K and AKT in MCF-7 breast cancer cells over expressing PTEN. As shown in the Fig. 1A, the reduced PTEN protein was observed in control cells and thus indicating the tumor suppressor PTEN protein level is modulated in cancer cells and thus leads to aberrant activation of P13K and AKT proteins. However, when we over express PTEN in breast cancer cells, the expression level of P13K/AKT pathways are significantly downregulated.
Next we have analyzed the cell proliferation rate between the control and PTEN over expressed breast cancer cells. The cell growth curve (Fig. 1B) showing the logarithmic growth phase for the first four days after that the cell growth was gradually deceased. In these four days, the PTEN over expressed cells showed significantly compromised cell growth (P<0.05) when compared to the controls. These results suggest that PTEN over expression suppressing the cell proliferation efficiently and that might be due to the cell cycle arrest (G1 phase) by inhibiting P13K/AKT pathways as reported in the previous studies (Tamura et al., 1998;Weng et al., 1999;Ding et al., 2009;Gao et al., 2009;Lu et al., 2016).

PTEN over expression induces apoptotic cell death
We have examined the PTEN over expressed cells for apoptosis induction. Activation of caspases are the crucial event for the mitochondrial mediated intrinsic apoptotic pathways. Hence, we have analyzed the expression levels of caspases 9, caspases 3 and BCL- 2. By western blot analysis, we found that caspases 9 and caspase 3 are significantly enhanced in PTEN over expressed breast cancer cells. Simultaneously, the anti-apoptotic or pro-survival protein BCL-2 expression level is reduced ( Fig. 2A). We have quantified the rate of apoptosis between the control and PTEN over expressed cells by flow cytometry. We found that significantly enhanced rate of apoptosis (P<0.01) in the group over expressing PTEN (Fig. 2B). This data suggest that apoptosis is initiated in the PTEN over expressed cells. Accordingly, the post transfected cells were subjected to apoptosis evaluation by AO-EtBr staining and Hoechst 33344 staining. By fluorescence microscopic analysis, we observed significantly more dead cells in PTEN over expressed groups, which emits red fluorescence confirming the apoptotic cells (Fig. 3A); whereas the live cells extrapolate green fluorescence. Further, the morphology of apoptotic cells is visualized as chromatin condensation and presence of bright blue pyknotic nucleus, which are not observed in control cells (Fig. 3B). Finally, our flow cytometry analysis revealed All these findings suggest that PTEN over expression efficiently induce apoptotic cell death in breast cancer cells and these data were consistent with previous findings in different cancers.

PTEN over expression contributes increased sensitivity to chemotherapy drugs
Major hurdles of the current chemotherapy strategies are the multi-drug resistance and tumor regrowth of cancer cells owing to the over expression of ABC transporter proteins. We hypothesize that reduced expression of tumor suppressor gene PTEN might also contribute to chemotherapy resistance. Therefore, we have performed chemotherapy resistance assay to determine the effect of PTEN over expression on cell viability for 5-FU, docetaxel and doxorubicin. Figure 4 showing that PTEN over expressed cells showed significantly increased sensitivity towards the above-mentioned drugs, when compared to controls. Therefore, the increased sensitivity towards chemotherapeutic drugs leads to apoptosis induction and cell death.

DISCUSSION
Studies have been demonstrated that PTEN can exert tumor suppressive effect by inducing apoptosis, inhibiting tumor invasion and maintaining genome integrity (Zhao et al., 2013). The PTEN mediated tumor suppression can be achieved by attenuation of the PI3K/ AKT signaling pathway and its downstream targets such as FRAP/mTOR, FAK and APK (Ohta et al., 2015). Therefore, for normal physiological processes such as regulated cell cycle, cell proliferation, differentiation, apoptosis and metabolism PTEN gene function is essential. However, PTEN was found to be mutated or loss its function frequently and as a consequence P13K/ AKT pathway is aberrantly activated in cancer cells (Carbognin et al., 2019).
In the present study, the breast cancer cells showed reduced expression of PTEN and enhanced regulation of P13K/AKT signaling pathways, which are crucial for increased cell proliferation, malignant transformation and migration. In the mean time, when we over express the PTEN in breast cancer cells, the P13K/ AKT pathway is down regulated, which is confirmed by our western blot analysis. This finding is consistent with the previous reports where they showed mutated or deleted PTEN results in excessive activation of P13K signal leads to hyper cell proliferation (Yan et al., 2015). However, when we complement the PTEN function in breast cancer cells, the rate of proliferation was significantly reduced when compared to the control cells. PTEN inhibits P13K signaling and its downstream targets by dephosphorylation of PIP3 to PIP2 so that AKT binding is interrupted. Consequently, the PTEN induce cell cycle arrest at G1 phase and ultimately the induction of apoptosis takes place (Tamura et al., 1998;Ding Gao et al., 2009). Studies in MCF-7 cell line observed stimulation of apoptosis and cell cycle arrest in G1 phase upon over expression of PTEN under serum free conditions (Lu et al., 1999;Zhu et al., 2001;Li et al., 2007;Yan et al., 2015). In addition, the focal adhesion kinase phosphorylation also regulated by PTEN which in turn prevent cell adhesion, migration and apoptosis (Lu et al., 2016).
In our study, we have also examined the impact of PTEN over expression in apoptosis induction in breast cancer cells. We have shown the correlation between the number of apoptotic cells by flow cytometry/staining (PI, Hoechst 33344 and AO-EtBr) and expression of caspases involved in mitochondria dependent apoptosis. Our study further revealed that PTEN over expression cause down regulation of pro-survival protein BCL-2. As a result, mitochondrial membrane potential is distorted and releases the cytochrome c. Several studies has been reported that released cytochrome c involved in the activation of caspases 9, which binds to the Apoptotic protease activating factor 1 (APAF-1). In turn, caspases 3 gets activated and thus cause apoptotic cell death (Weng et al., 2009;McIlwain et al., 2013;Campbell et al., 2018). Consequently, in our study we have also estimated the expression level of cytochrome c by western blot analysis. We found that cytochrome c level is enhanced in the PTEN over expressed MCF-7 cells (not shown). Therefore, PTEN over expressed breast cancer cells showed enhanced mitochondrial dependent intrinsic apoptosis, which is confirmed by caspases over expression and the persistence apoptotic features. Our findings are in agreed with previously reports, which stated that over expression of PTEN is responsible for the induction of apoptosis (Lu et al., 1999;Weng et al., 2001;Li et al., 2007;Hemmings et al., 2012;Carbognin et al., 2019).
The interesting finding in our study is PTEN over expression breast cancer cells are more susceptible towards chemotherapeutic drugs such as 5-FU, docetaxel and doxorubicin, which killed cells effectively. This suggests that PTEN over expressed can efficiently uptake the drugs leads to induction of apoptosis and thus cell viability is deceased. However, findings from our present study are preliminary and the lack of animal models and in vivo experiments are the limitations. In future, detailed research work will be carried by performing more in vivo experiments by using animal models and different cancer cell lines as well.
Meanwhile, Liu and others (Liu et al., 2012) showed that PTEN over expression combined with the docetaxel are effective adjuvant therapy for liver cancer.
Similarly, it has been demonstrated in MCF-7 cells that PTEN plays an essential role in the etoposide induced check point activation and cell cycle arrest . It is well known that chemoresistance property of the cancer cell is due to the over expression of ATPase binding cassette (ABC) transporter proteins which efflux the therapeutic drugs efficiently out of the cells (He et al., 2014;Wang et al., 2015). Therefore, further detailed investigation is requited to elucidate the role of PTEN in inhibiting ABC proteins expression and efficient drug uptake will provide more molecular insights and that would be clinically significant for the effective cancer treatment.
In summary, our finding suggests that over expression of PTEN attenuate the cell growth and induce apoptosis in breast cancer cells. Therefore, the tumor suppression activity exerted by PTEN is crucial for the prevention of cancer and therefore PTEN might be potential target for anti-cancer therapy.