Targeted RASSF1A expression inhibits proliferation of HER2‑positive breast cancer cells in vitro
- Authors:
- Published online on: April 11, 2023 https://doi.org/10.3892/etm.2023.11944
- Article Number: 245
Abstract
Introduction
Breast cancer is a heterogeneous malignancy that is categorized into clinically relevant molecular subtypes based on the expression of molecular markers, such as the estrogen receptor, progesterone receptor and human epidermal growth factor receptor 2 (HER-2, also called Neu) (1-4). HER2-positive (HER2+) cancers, which account for ~15-20% of all primary breast carcinomas, tend to grow more aggressively and have a worse prognosis (5,6). Despite the success of HER2-targeting therapies, HER2+ breast cancer remains a major challenge in clinical practice (7-9).
RAS association domain family protein 1 subtype A (RASSF1A), which belongs to the family of RAS effectors, is a tumor suppressor that is frequently silenced in a number of cancers through promoter hypermethylation (10). In fact, RASSF1A methylation might serve as a potential diagnostic biomarker for breast cancer (11). In addition, RASSF1A suppresses the growth of estrogen receptor α-positive (ERα+) breast cancer cells by keeping ERα expression and function under control via mechanisms involving the Hippo-Kinases LATS1 and 2 (12,13). However, the function of RASSF1A in HER2+ breast cancer remains to be elucidated.
Gene therapy is used to treat genetic and hereditary disorders by expressing a foreign gene in the host cells to produce desired therapeutic effects (14). The study of the molecular features of breast cancer has established gene therapy as a promising approach for this cancer (15). A key challenge of cancer gene therapy is the tumor-targeting efficiency, which means that the therapeutic gene should be specifically expressed in tumor cells, thereby reducing the damage to normal cells (16). The techniques to achieve tumor-specific gene expression include distinct delivery systems and expression driven by tumor-specific promoters (17). While tumor-targeting delivery systems have been extensively explored over the last two decades (18), the study of using a specific promoter to drive gene expression in tumor cells but not normal cells is relatively rare (19).
HER2 is a cell-specific oncogene that has low levels of expression in normal cells, but is highly expressed in a number of cancers, including HER2+ breast cancer (20). Thus, the HER2 promoter (HER2p) is exploited for targeted gene expression in HER2+ cancer cells (21).
Local hypoxia is a hallmark of a number of solid tumors and 25-40% of invasive breast cancers exhibit hypoxic regions (22). Hypoxia leads to increased activity of hypoxia-inducible factors, which bind hypoxia-response elements to promote the expression of genes involved in cell adaptations to hypoxia (23). The present study first investigated the clinical significance of RASSF1A in human HER2+ breast cancer. Next, an expression vector was constructed carrying RASSF1A under the control of HER2p and five copies of the HRE, aiming to selectively overexpress RASSF1A in HER2+ breast cancer cells, especially under hypoxic conditions. The effects of RASSF1A overexpression on the proliferation of HER2+ breast cancer cells was then assessed. The results of this study could help the future development of new targeted gene therapy strategies for HER2+ breast cancer.
Materials and methods
Patients and tissue samples
A total of 54 treatment-naïve patients with HER2+ breast cancer who underwent surgery at Shaanxi Provincial Cancer Hospital (Xi'an, Shaanxi, China) between January 2016 and December 2016 were included in this study. Patients who received any preoperatively adjuvant chemotherapy, radiotherapy or hormone therapy were excluded. The information on patient survival was obtained from a 5-year follow-up by telephone or outpatient examination. The overall survival rate was calculated as the percent of patients still alive at a specific time from the date of surgery during the 5-year follow-up period. The tumor stage, TNM stage, tumor status and nodal status were classified according to international standards for staging breast cancer (24) and the grouping in Table I was categorized according to clinical practice, which was in line with most papers (25,26). Tumor and adjacent non-tumor tissues were collected during surgery and stored at -80˚C until analysis. HER2 overexpression was confirmed by pathohistological examination of tumor tissues. The median relative RASSF1A mRNA level was used as the cutoff value for the definition of high and low RASSF1A expression in tumor tissues. The present study was approved by the Ethics Committee of Shaanxi Provincial Cancer Hospital. All participants provided written informed consent.
Table IRelationships between tumorous RASSF1A mRNA level and clinicopathologic characteristics of HER2+ breast cancer patients. |
Cell lines and culture
The AU565 (HER2+), SKBR-3 (HER2+), MCF-7 (HER2-) and BT474 (HER2+) human breast cancer cell lines and the MCF-10A human normal breast cell line were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). All cells were cultured in DMEM (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.) and 1% penicillin-streptomycin (Invitrogen; Thermo Fisher Scientific, Inc.) at 37˚C in a humidified atmosphere containing 5% CO2. To study the effects of hypoxia, cobalt (II) chloride (CoCl2), a well-known hypoxia mimic agent, was added to the medium to create hypoxia-like state in vitro.
Reverse transcription-quantitative (RT-q) PCR
Total RNA was extracted from 1x104 cells using TRIzol reagent (Thermo Fisher Scientific, Inc.) as previously described (27) and RNA was reverse-transcribed into the complementary DNA (cDNA) using a reverse transcription PCR (RT-PCR) kit (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocols. PCR amplification with the specific primers (Table II) was performed with the miScript SYBR Green PCR Kit (Qiagen GmbH) in triplicates at 98˚C for 2 min; 40 cycles of 95˚C for 15 sec, 60˚C for 30 sec, 72˚C for 1 min and a final 10 min extension at 72˚C. The mRNA levels were calculated using the 2-ΔΔCq method (28). Data were analyzed with the RealPlex analysis system (Eppendorf).
Western blot analysis
Western blot analysis was performed as described in our previous report (24). In brief, cells were lysed in RIPA lysis buffer (MilliporeSigma) in the presence of protease inhibitors and quantified using a BCA Protein Assay kit. Proteins (30 µg in each lane) were resolved with 10% SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes. Following blocking with 5% fat-free dry milk at room temperature for 1 h, the membranes were incubated with anti-RASSF1A (1:1,000; cat. no. ab126764; Abcam), anti-β-actin (1:1,000; cat. no. ab8226; Abcam), or anti-HER2 (1:1,000; cat. no. ab134182; Abcam) antibody at 4˚C overnight, followed by goat anti-rabbit IgG (1:3,000, cat. no. ab150077; Abcam) at room temperature for 1 h. Protein bands were detected using a chemiluminescence detection system (Beyotime Institute of Biotechnology) and densitometry analysis was performed using QuantityOne version 4.5.0 (Bio-Rad Laboratories, Inc.). Data were normalized to β-actin.
Lentivirus production
The full-length human RASSF1A cDNA (NM_007182) and HER2p were PCR-amplified from pcDNA-RASSF1A (Shanghai GeneChem Co., Ltd.) and genomic DNA from HER2+ breast cancer tissues, respectively, using specific primers shown in Table II. The HER2p product was digested with HindIII/PstI and subcloned into pLEGFP-N1-5HRE-CEAp (29) at the HindIII and PstI sites to replace CEAp, generating pLEGFP-N1-5HRE-HER2p. The 5HRE-HER2p fragment was subsequently subcloned into the pLVX-EGFP-3FLAG lentiviral vector to replace the CMV promoter, generating pLVX-5HRE-HER2p-EGFP-3FLAG. Next, the RASSF1A product was subcloned into pLVX-5HRE-HER2p-EGFP-3FLAG to replace EGFR, generating pLVX-5HRE-HER2p-RASSF1A-3FLAG (LV-5HH-RASSF1A). All primers were synthesized at Sangon Biotech Co., Ltd. The pLVX-5HRE-HER2p-3FLAG vector (LV-5HH) served as negative control. The recombinant lentiviral vectors were verified by restriction endonuclease digestion and DNA sequence analysis at Sangon Biotech Co., Ltd.
Lentiviral infection of breast cancer cells
Lentiviruses carrying LV-5HH-RASSF1A or LV-5HH (1x108 pfu) were custom-prepared by GeneChem. For lentiviral infection, breast cancer cells were seeded in 24-well plates (5x104 cells/well), cultured overnight and then infected for 72 h at 37˚C with corresponding lentiviruses at a MOI of 10 in the presence of polybrene. Following infection, the cells were selected with 10 µg/ml puromycin for two weeks. The surviving cells were used in subsequent experiments.
MTT assay
Cell viability was determined with the MTT assay. In brief, cells were seeded in 24-well plates (5x104 cells/well) and cultured for 7 days at 37˚C. To test the effects of hypoxia, cells were cultured for 7 days in the presence of 50 µmol/l CoCl2 (30). After repeated washing with serum-free DMEM to remove cell debris, the cells were incubated with 20 µl of 5 g/l 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; MilliporeSigma) for 4 h. The resulting formazan crystals were dissolved in 200 µl DMSO and the absorbance at 490 nm was recorded on a Victor 3 microplate reader (PerkinElmer, Inc.).
Colony formation assay
Cells were seeded in 60-mm cell culture dishes (200 cells/well) and incubated for 7 days. To test the effects of hypoxia, cells were incubated for 7 days at 37˚C in the presence of 50 µmol/l CoCl2. Then, the cells were fixed with methanol for 15 min at room temperature and stained with Giemsa for 30 min at room temperature. Colonies containing >50 cells were counted with an inverted microscope (Olympus Corporation; magnification, x10).
Statistical analysis
All results are presented as the mean ± standard deviation (SD) from three independent experiments. Data analysis was performed with SPSS 22.0 (IBM Corp.) and GraphPad Prism 6.0 (Dotmatics). The RASSF1A mRNA levels in tumor and adjacent normal tissues from HER2+ breast cancer patients were compared using the paired t-test. Other data from different groups were compared using one-way ANOVA followed by Bonferroni multiple comparisons test. The data from groups that were separated by two independent variables (i.e. cell group and normoxia/hypoxia) were analyzed using two-way analysis of variance (ANOVA) followed by Bonferroni multiple comparisons test. The relationships between tumorous RASSF1A level and tumor grade, TNM stage, tumor size and lymph node metastasis were interpreted using the Fisher's exact test or chi-square test. Survival analysis was performed using the Kaplan-Meier method or log-rank test. P<0.05 was considered to indicate a statistically significant difference.
Results
Tumorous RASSF1A expression is negatively associated with disease progression and poor prognosis in HER+ breast cancer patients
The RASSF1A mRNA levels in tumor and adjacent normal tissues from 54 HER2+ breast cancer patients were determined by RT-PCR. The tumor tissues exhibited a significantly lower RASSF1A level than adjacent normal tissues (P<0.05, Fig. 1A). Kaplan-Meier survival analysis revealed a positive association between tumorous RASSF1A level and five-year overall survival (P<0.05, Fig. 1B). In addition, hierarchical cluster analysis showed that tumorous RASSF1A was negatively associated with tumor grade, TNM stage, tumor size and lymph node metastasis (P<0.05, Table I). Meanwhile, no significant correlation was detected between RASSF1A expression and age (P>0.05; Table I). These data supported the hypothesis of RASSF1A as a tumor suppressor in HER2+ breast cancer.
RASSF1A is downregulated in human breast cancer cells
The RASSF1A mRNA and protein levels in AU565, SKBR-3, MCF-7 and BT474 human breast cancer cells and MCF-10A human normal breast cells were determined by RT-PCR and western blot analysis, respectively. Similar to the clinical data, all four types of breast cancer cells exhibited lower RASSF1A mRNA and protein expression than MCF-10A normal breast cells (P<0.05, Fig. 1C and D). Among the four types of breast cancer cells, SKBR-3 showed the highest HER2 expression, while MCF-7 showed the lowest (Fig. 2A-C). Based on these results, SKBR-3 and MCF-7 cells were used as representative HER2+ and HER2-breast cancer cells, respectively, in subsequent experiments.
5HH drives RASSF1A expression in HER2+ but not HER2-breast cancer cells
For targeted RASSF1A expression in HER2+ breast cancer cells, especially under hypoxic conditions, a lentiviral expression system (LV-5HH-RASSF1A) was constructed that could express RASSF1A under the control of a promoter composed of five copies of HRE and one copy of HER2p (5HH; Fig. 3A and B). The 5HH-driven RASSF1A expression was confirmed by western blot analysis in the HER2+ SKBR-3 cells transfected with LV-5HH-RASSF1A (P<0.05; Fig. 3C and D). In keeping with hypoxia-induced activation of HRE-mediated transcription, more pronounced expression was detected under hypoxic conditions (P<0.01; Fig. 3C and D). By contrast, no 5HH-driven RASSF1A expression was detected in the HER2-MCF-7 cells transfected with LV-5HH-RASSF1A, either under normoxia or hypoxia (Fig. 3E and F). These results were in line with the low transcriptional activation activity of HER2p in HER2-breast cancer cells. Thus, the 5HH promoter only drives RASSF1A expression in HER2+ but not HER2-breast cancer cells.
5HH-driven RASSF1A expression in HER2+ breast cancer cells inhibits cell proliferation
MTT and colony formation assays were used to evaluate the effects of 5HH-driven RASSF1A expression on HER2+ breast cancer cell proliferation. After 7 days of cultivation, LV-5HH-RASSF1A-transfected SKBR-3 cells showed significantly reduced viability compared with LV-5HH-transfected cells (P<0.05; Fig. 4A) and the reduction in cell viability was even more pronounced under hypoxic conditions (P<0.01, Fig. 4A). In addition, LV-5HH-RASSF1A-transfected SKBR-3 cells exhibited decreased colony formation capacity compared with LV-5HH-transfected cells, especially under hypoxic conditions (P<0.05, Fig. 4B and C). Together, these results indicated that 5HH-driven RASSF1A expression in HER2+ breast cancer cells inhibited cell proliferation.
Discussion
Currently, HER2+ breast cancer is treated with HER2-targeting monoclonal antibodies such as trastuzumab and pertuzumab and tyrosine kinase inhibitors such as tucatinib and lapatinib (31,32). Although positive clinical outcomes are well documented, the continuous usage of these drugs may induce drug resistance in some patients. Patients who develop resistance to multiple HER2-targeting therapies often have limited treatment options and thus suffer poor clinical outcomes (33). Therefore, there exists a need for new targeted therapies for HER2+ breast cancer. The present study found that RASSF1A was downregulated in human HER2+ breast cancer and its expression was negatively associated with disease progression and mortality, which underscored the clinical significance of RASSF1A as a tumor suppressor in this specific subtype of breast cancer. Moreover, by using a lentiviral expression system under the control of a hypoxia-inducible, HER2p-driven promoter (5HH), the present study successfully introduced the RASSF1A gene into HER2+ breast cancer cells to express the RASSF1A protein. The same expression system failed to express RASSF1A in HER2-breast cancer cells, in which HER2p has a low transcriptional activation activity. This targeted RASSF1A expression inhibited the proliferation of HER2+ breast cancer cells and, to a greater degree, under hypoxia. Together, these clinical and in vitro findings supported 5HH-driven RASSF1A expression as a potential targeted gene therapy for HER2+ breast cancer.
Given the presence of a number of defective genes, breast cancer is an ideal candidate for gene therapy. At present, there are ~50 ongoing gene therapy clinical trials for breast cancer (34). These clinical trials target a variety of breast cancer susceptibility genes such as BRCA1, BRCA2, TP53 and PTEN and the genetic materials are transferred into cancer cells using both viral and non-viral vectors. The clinical trials have shown that gene therapy can be less toxic than conventional therapies, but this approach faces two key challenges to success: The persistent expression of anti-cancer gene products and a tumor-selective delivery system (35).
In recent years, universal tumor-specific promoters, such as the survivin and telomerase reverse transcriptase gene promoters and the hTERT promoter, have been exploited for cancer gene therapy (36-39). Hypoxia, caused by rapid tumor growth, has a key role in cancer progression and is the focus of a number of cancer treatment strategies (40). In 2000, Shibata et al (41) developed a hypoxia-responsive vector with five copies of HRE derived from the promoter region of the human VEGF (5HRE) for tumor-specific gene therapy. Since then, constructs with 5HRE have been successfully used to drive hypoxia-inducible gene expression in tumor cells (42-44). The present study identified RASSF1A as a new anti-tumor gene for HER2+ breast cancer and, more importantly, achieved selective delivery of this gene to HER2+ breast cancer cells using a lentiviral expression system under the control of HER2p and the 5HRE promoter. The results from the present study should encourage the development of new targeted gene therapy strategies for HER2+ breast cancer.
The present study is limited by the lack of investigations on the molecular mechanisms mediating the anti-tumor function of RASSF1A. RASSF1A has been shown to inhibit ERα+ breast cancer by suppressing ERα expression and function via Hippo-Kinases (12,13). The molecular pathways affected by RASSF1A in HER2+ breast cancer remain to be elucidated. In addition, further studies are required to confirm the efficacy of 5HH-driven RASSF1A expression for HER2+ breast cancer in vivo. HER2 amplification is not limited to breast cancer, but occurs in a number of other solid tumors such as bladder, cervical, uterine and testicular cancers, wherein it drives disease progression (45). Correspondingly, 5HH-driven gene therapy may hold promise as a general therapeutic approach for all types of HER2+ cancers.
In conclusion, the present study verified RASSF1A as a tumor suppressor in HER2+ breast cancer and achieved selective delivery of this gene to HER2+ breast cancer cells using a lentiviral expression system under the control of HER2p and the 5HRE promoter. The selective delivery of RASSF1A resulted in growth inhibition of HER2+ breast cancer cells. These findings encourage the development of new targeted gene therapy strategies for HER2+ breast cancer and possibly, all HER2+ cancers.
Acknowledgements
Not applicable.
Funding
Funding: The present study was funded by the Key Research and Development Program of Shaanxi Province (grant no: 2021SF-218), the Natural Science Foundation Research Program of Shaanxi Province (grant no: 2020JM-680), the Xi'an Science and Technology Plan Project [grant no: 20YXYJ0005(7)] and the National Natural Science Foundation Incubation Program of Shaanxi Provincial Cancer Hospital (grant no: SC211007).
Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
Authors' contributions
SH and JG contributed to the conceptualization and the design of the present study. SH, YH, LH, NC and XY performed the experiments and analyzed the data. HW and PH were responsible for the acquisition, analysis and interpretation of the data. YF, JZ and JYZ contributed to the drafting of the manuscript. SH and JG confirm the authenticity of all the raw data. All authors read and approved the final manuscript.
Ethics approval and consent to participate
Not applicable.
Patient consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
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