FOXO3a shRNA and simultaneous induction of P27Kip1gene Inhibit the Breast Cancer Growth in Nude Mice

Background: FOXO proteins, which are overexpressed in multiple human tumors, belong to the Forkhead family of transcription factors that are involved in cell-cycle regulation, cell apoptosis, differentiation, stress response, and metabolism. The p27Kip1 gene leads to cell cycle arrest, cell apoptosis, tumor suppressor genes, and cell adhesion. The low expression level of the p27Kip1 gene is attributed to poor prognosis in patients with colorectal, gastric, pulmonary, and breast cancers. Accordingly, the present study aimed to investigate the possibility of tumor growth inhibition in a mouse model by targeting FOXO3a shRNA and the simultaneous induction of P27Kip1gene. Methods: The tumor model was generated by intratumoral inoculating with plasmids. When tumor size reached an average volume of 8 mm in diameter, the mice received injections of construct and control plasmids three times a week for two weeks, followed by tumor growth assessment. Results: Based on the obtained results, the delivery of construct plasmid signicantly inhibited tumor growth in nude mice, as compared to the control plasmid. Moreover, the immunohistochemical analysis indicated that the delivery of construct plasmid signicantly suppressed expression of FOXo3a and induced P27Kip1 in tumor samples. Conclusion: The ndings of the present study revealed that FOXO3a shRNA, along with simultaneous induction of P27Kip1gene using a useful in vivo gene delivery strategy, seems a practical therapeutic approach for breast cancer treatment and may provide profound insight into gene therapy of solid cancers.

Results: Based on the obtained results, the delivery of construct plasmid signi cantly inhibited tumor growth in nude mice, as compared to the control plasmid. Moreover, the immunohistochemical analysis indicated that the delivery of construct plasmid signi cantly suppressed expression of FOXo3a and induced P27Kip1 in tumor samples.
Conclusion: The ndings of the present study revealed that FOXO3a shRNA, along with simultaneous induction of P27Kip1gene using a useful in vivo gene delivery strategy, seems a practical therapeutic approach for breast cancer treatment and may provide profound insight into gene therapy of solid cancers.

Background
Breast cancer is the most prevalent cancer in women worldwide, which contributes to nearly 1.7 million new cases diagnosed accounts for than 520,000 related deaths just in 2012 (1). Triple-negative breast cancer (TNBC), which is highly aggressive, attributes to 15% of breast cancer incidence rates. This disease, which mostly affects younger patients, is characterized by tumors that lack expression of estrogen receptor(ER) progesterone receptor and HER2, as well as a poor clinical prognosis (2).
Expression of some genes, such as P27Kip1 and forkhead box O3 (FOXO3), changes in breast cancer. A low P27kip1 level can reduce the survival of cancer patients (3). The reduced p27Kip1, which is a crucial regulator of G1-to-S phase progression, is closely connected with high histopathologic tumor grade (4).
Loss of sensitivity to chemotherapy and a poor prognosis resulted in cancer recurrence in many patients with advanced breast cancer (1). Metastasis develops breast cancer into an incurable disease with a median survival time of around two years. In this stage, chemotherapy is the mainstay of treatment. A response rate of 35%-67% has been reported for combined chemotherapy with a short median response duration of about nine months. Accordingly, it seems essential to nd alternative therapies for patients who are in icted with chemotherapy or hormone-refractory cancers. Gene therapy can be regarded as an alternative therapy in this regard (7). Since in vivo studies of human breast cancer development in 1969 and the nude mice has been increasingly used in cancer research (8). In the current study, we designed a bidirectional construct that associated FOXO3a-shRNA and overexpression P27Kip1 simultaneously; after that, we investigated whether the constructed plasmid can suppress tumor growth in a nude mouse.

Constructed plasmid
A short hairpin RNA (shRNA) targeting FOXO3a along with simultaneous induction of P27Kip1 gene ( Figure 1) were designed, synthesized (BioMatic, Canada), and inserted into the blank expression plasmid (PcDNA3.1 + expression vector with EGFP). Restriction enzymes digestion and sequencing were used to validate the recombinant plasmid (6).
Cell culture MDA-MB-231 cell line, which is a human breast cancer cell line, was provided from Tehran Pasteur Institute (Tehran, Iran). Cells were preserved in a DMEM-Hi glucose medium (Gibco 31966-047), and 10% fetal bovine serum was used as the growth supplement(FBS Gibco BI102-100) with penicillinstreptomycin (100 units/mL) at 37°C in a humidi ed incubator with 5% CO2.

Animal model
Athymic female nude mice (6 weeks old and weighed 18-22g) were provided by Pasteur Amol Institute (Amol, Iran) Laboratory Animal Center. MDA-MB-231 cells were injected into 6-8 week old mice using Matrigel (50:50), which were kept in the Animal Pasteur Amol Institute. Throughout the study, the standard condition was maintained for the mice in the following way, the temperature of 25±2° C, humidity within the range of 40%-60%, and12L/12D light cycle in speci c pathogen free housing. Initially, the nude mice skin was sanitized with 75% ethanol (9), 1x10 6 cells in 0.1 ml of DMEM were then injected subcutaneously into the lower right hind ank of nude mice using a sterile syringe (10). After that, any sign of disease, including subcutaneous tumors or weight loss due to potential tumor growth in internal sites, were tracked in mice. Moreover, during the study, the mice were monitored for the growth of tumors, and tumor volume measurement was performed using calipers every three days. In this regard, tumor volume was obtained as πls 2 /6, where l represents the long side and "s" demonstrates the short side (9). All nude mice were sacri ced by spinal cord injury.

Plasmid treatment
Based on previous studies we used 15 nude mice. When tumor diameters reached a size of about 8mm. (9), mice were assigned into three groups (n=5 for each): 1) vector containing the genes group which received a subcutaneous injection of plasmid construct and jet polyethyleneimine (jetPEI; Polyplus Co., France); 2) control group which received subcutaneous injection with empty vector and jetPEI 3) reagent group which were injected with jetPEI, according to the protocol. The plasmids were administered three times a week for two weeks (6 total injections), and Caliper measurements of tumor size were performed. At 28d following injection, the experiments were terminated, and all nude mice were sacri ced by spinal cord injury, and tumors were excised and weighed. It is worthy to note all animal experiments were performed following the 'Guide for the Care and Use of Laboratory Animals' published by the National Institutes of Health and were approved by the "Animal Care and Use Committee "of our university.

Immunohistochemistry assessment of FOXO3a and P27kip1 expression
In an attempt to determine the expression of FOXO3a and P27kip1, tumor samples were exposed to immunohistochemical examination. Tissue xation was performed at 4°C using 4% paraformaldehyde before para n embedding. After that, they were cut into 5μm sections and transferred to silicon-coated slides, which were then stained with a monoclonal antibody against FOXO3a (Santa Cruz Biotechnology) at a dilution of 1:30 and use monoclonal antibody against P27kip1 (Santa Cruz Biotechnology) at a dilution of 1:100. The 3, 3'-diaminobenzidine tetrahydrochloride (DAB) was utilized for visualization, and Mayer's hematoxylin was used for counterstaining. Light microscopy was utilized for the evaluation of FOXO3a and P27kip1immunostained slides with a total magni cation of 400x and a 10x10 square grid placed in the ocular. When tumor cells showed a distinct cytoplasmic and nuclear reaction, they were regarded as positive. The positive tumor cells were counted in 500 tumor cells in continuous high power.
FOXO3a and P27kip1 were determined by counting 500 tumor cells and were calculated as the percentage of positively labeled cells (8).

Statistical analysis
The data were analyzed in GraphPad Prism software (version 5.03, GraphPad, San Diego, CA, USA) using a 2-sided Student's paired t-test for single comparisons and one-way ANOVA with LSD posthoc test for multiple comparisons. All data were expressed as Mean±SEM. Moreover, Bonferroni's correction was used to adjust for multiple comparisons. A P value less than 0.05 was considered statistically signi cant.

Antitumor Activity of shFOXO3a and induced P27
Based on the obtained data, the constructed plasmid resulted in tumor growth inhibition in vivo. The nude mouse tumor models were established by subcutaneous inoculation of 1×10 6 cells. Before the administration of plasmids or vehicle, the tumors were allowed to reach 8 mm in diameter. As depicted in Figure2, a signi cant reduction in tumor growth was observed in mice treated with construct plasmid, as compared to treatment with the negative control or the reagent (Figure 2). Besides, signi cant differences in tumor volumes were detected between the treated group (5/15) and controls group (10/15:empty vector and reagent), 14 days after the injection, 447±17.72 mm3, and (662±36.1, 608±19.3) mm3, respectively (P<0.05, Figure 1).
Nonetheless, no signi cant difference was reported between the empty vector control group and the reagent group. Accordingly, the treatment of construct plasmid halted tumor growth of cancer cells in nude mice. H&E staining was used to prove the tumor was cancerous. It includes mitotic changes, severe eosinophilic cytoplasm, and increase the ratio of the nucleus to the cytoplasm (Figure 3). Percentage cancer cells of nude mouse tumor tissues that were staining with anti-P27 presented that it was 32.6% of construct compared with empty vector and reagent respectively 65.8% and 63.5%, p<0.05 and with anti-FOXO3 monoclonal antibody it was 22.1% of construct compared with empty vector and reagent 58.5% and 56.2% respectively (Figure 4,5), p<0.05.

Discussion
Triple-negative breast cancer (TNBC) is a challenging and aggressive type of breast cancer associated with a poorer prognosis, along with a higher risk of recurrence and metastasis (11). The present study aimed to examine the possibility of using a construct as a therapeutic agent against breast cancer. RNA interference (RNAi) is an evolutionarily conserved mechanism for speci c gene silencing (12). Studies revealed that the transfection of the construct into mammalian cells could e ciently inhibit cancer cells. The tumor was generated in nude mouse models through subcutaneous inoculation of breast cancer cell lines. The ndings of the experiment and immunohistochemical results were indicative of signi cant attenuation of tumor growth by construct was. Moreover, the results suggested that shRNA against the FOXO3a gene and overexpression of P27 could signi cantly suppress the proliferation of breast cancer cell lines. Also, the results of a study conducted by Spratt et al. indicated that the decrease of P27 in a mouse model led to lung cancer (13). Storz et al. revealed that knockdown of FOXO3a resulted in decreased tumor size; moreover, they noted that any therapies involving the inactivation of FOXO3a might effectively block tumor expansion and metastasis (14). Besides, the obtained results of the present study data suggested that delivery of construct inhibits proliferation of breast cancer cell line leading to a decreased number of breast cancer and suppression of tumor cell growth in nude mice. Therefore, the current study suggested that silencing FOXO3a and simultaneous induction of P27 signi cantly contribute to the regulation of breast cancer cell line, growth antitumor activity against breast cancer.
In conclusion, the results were suggestive of the enormous impact of construct delivery on the ability to grow in vivo, suppression of breast cancer proliferation in vivo, and its effectiveness in breast cancer treatment. It is suggested that future studies focus on the e ciency of FOXO3a silencing and induced P27 as a novel biotherapy strategy for breast cancer patients.

Conclusions
In summary, the present study described that the construct we designed can suppress tumor growth in a nude mouse. Consequently gene delivery strategy, seems a practical therapeutic approach for breast cancer treatment. It is worthy to note all animal experiments were performed following the 'Guide for the Care and Use of Laboratory Animals' published by the National Institutes of Health and were approved by the "Animal Care and Use Committee "of our university. Consent for publication: Not applicable' for that section.
Availability of data and materials: Not applicable' for that section.
Competing interests: The authors declare that they have no competing interests.