Novel treatment strategies for patients with HER2-positive breast cancer who do not benefit from current targeted therapy drugs.

Human epidermal growth factor receptor-2 positive breast cancer (HER2+ BC) is characterized by a high rate of metastasis and drug resistance. The advent of targeted therapy drugs greatly improves the prognosis of HER2+ BC patients. However, drug resistance or severe side effects have limited the application of targeted therapy drugs. To achieve more effective treatment, considerable research has concentrated on strategies to overcome drug resistance. Abemaciclib (CDK4/6 inhibitor), a new antibody-drug conjugate (ADC), src homology 2 (SH2) containing tyrosine phosphatase-1 (SHP-1) and fatty acid synthase (FASN) have been demonstrated to improve drug resistance. In addition, using an effective vector to accurately deliver drugs to tumors has shown good application prospects. Many studies have also found that natural anti-cancer substances produced effective results during in vitro and in vivo anti-HER2+ BC research. This review aimed to summarize the current status of potential clinical drugs that may benefit HER2+ BC patients in the future.

Efficacy and safety of endostar® combined with chemotherapy in patients with advanced soft tissue sarcomas.

BACKGROUND: Soft tissue sarcomas (STS) are a heterogeneous group of tumors, and approximately 40-50% of patients with STS develop metastatic disease. The median overall survival of those patients was 12 months and their 5-year survival rate was 8%. Therefore, study on more effective treatment, especially the targeting therapies, is urgently needed. OBJECTIVE: To evaluate the efficacy and safety of Endostar® combined with chemotherapy in patients with advanced STS. METHODS: A retrospective case-series study was conducted in Cancer Institute of PLA, Xinqiao Hospital. A total of 71 patients suffering from advanced STS (IIB - IV) were included, of whom 49 cases treated with chemotherapy alone were defined as the control group and the rest 22 cases treated with the traditional chemotherapy combined with Endostar® were defined as the test group. The short-term therapeutic effects including objective response rate (ORR), disease control rate (DCR) and safety were evaluated in the two groups. In the follow-up, progression-free survival (PFS) and overall survival (OS) were also observed. RESULTS: In the test and control groups, the ORR was 18.2% and 12.2%, respectively (P = 0.767), and the DCR was 86.4% and 61.2%, respectively (P=0.034). The median time to progression in the test and control groups was 120 days and 70 days with significant difference (P = 0.017), while the median overall survival was 452 days and 286 days without significant difference (P = 0.503). The one-year survival rate in the test group and control group was 56.2% and 35.4%, respectively, while the two-year survival rate was 30.2% and 26.5%, respectively. No significant difference in the side effects was found between the two groups. CONCLUSIONS: Endostar® combined with chemotherapy resulted in a higher DCR and longer PFS in the patients with advanced STS, and the toxicity was tolerable.

CCND1 G870A polymorphism interaction with cigarette smoking increases lung cancer risk: meta-analyses based on 5008 cases and 5214 controls.

Evidence indicates CCND1 G870A polymorphisms as a risk factor for a number of cancers. Increasing studies have been conducted on the association of CCND1 G870A polymorphism with lung cancer risk. However, the results were controversial. The aim of the present study was to derive a more precise estimation of the relationship. Meta-analyses examining the association between CCND1 G870A polymorphism and lung cancer were performed. Subgroup analyses regarding ethnicity, smoking status, histological types and source of controls were also implemented. All eligible studies for the period up to May 2012 were identified. The overall data from ten case-control studies including 5,008 cases and 5,214 controls indicated that variant A allele may have an association with increased lung cancer risk (AA vs GG: OR = 1.21; 95 % CI = 1.08-1.36, dominant model: OR = 1.09; 95 % CI = 1.00-1.19, recessive model: OR = 1.23; 95 % CI = 1.01-1.49). In the subgroup analysis by ethnicity, A allele may elevate lung cancer risk among Asians but not Caucasians or Mixed ethnicities. In smoking status subgroup, A allele was shown to associate with increased lung cancer risk among smokers but not non-smokers. In the subgroup analysis by histological types, increased cancer risks were shown in adenocarcinoma but not squamous cell carcinoma, under the homozygote comparison and recessive models. Collectively, the results of the present study suggest that CCND1 G870A polymorphism might be a low-penetrant risk factor for lung cancer, particularly among Asians and smokers. Moreover, homozygous AA alleles might have a correlation with increased lung adenocarcinoma susceptibility.

Role of inhibitor of apoptosis protein Livin in radiation resistance in nonsmall cell lung cancer.

OBJECTIVE: The objective of the present study was to explore the role of the inhibitor of apoptosis protein (IAP) Livin in radioresistance in nonsmall cell lung cancer (NSCLC). METHODS: Lung adenocarcinoma cell lines A549 and SPC-A1 were used for this study. Using the technique of molecular cloning and gene transfection, two Livin isoforms, Livinα and ß, respectively, were expressed in A549 cells with the purpose of exploring the role of Livin in radiation resistance of A549 cells. Moreover, a Livin-specific gene-silencing system was developed using SPC-A1 cell line with the purpose of increasing radiosensitivity of SPC-A1 cells. RESULTS: A549 cells were induced by radiation to express Livin isoforms, Livinα and ß. A549 cells expressed Livin isoforms stably after gene transfection and the transfected cells demonstrated characteristics of antiradiation. However, Livin gene-silenced SPC-A1 cells exhibited remarkably enhanced radiation sensitivity. CONCLUSION: The IAP Livin is an important molecule in antiradiotherapy of NSCLC. Livin-specific gene silencing is likely to be an effective means to enhance radiation sensitivity of lung cancer.

Microarray-based analysis of microRNA expression in breast cancer stem cells.

BACKGROUND: This study aimed to determine the miRNA profile in breast cancer stem cells (BCSCs) and to explore the functions of characteristic BCSC miRNAs. METHODS: We isolated ESA+CD44+CD24-/low BCSCs from MCF-7 cells using fluorescence-activated cell sorting (FACS). A human breast cancer xenograft assay was performed to validate the stem cell properties of the isolated cells, and microarray analysis was performed to screen for BCSC-related miRNAs. These BCSC-related miRNAs were selected for bioinformatic analysis and target prediction using online software programs. RESULTS: The ESA+CD44+CD24-/low cells had up to 100- to 1000-fold greater tumor-initiating capability than the MCF-7 cells. Tumors initiated from the ESA+CD44+CD24-/low cells were included of luminal epithelial and myoepithelial cells, indicating stem cell properties. We also obtained miRNA profiles of ESA+CD44+CD24-/low BCSCs. Most of the possible targets of potential tumorigenesis-related miRNAs were oncogenes, anti-oncogenes or regulatory genes. CONCLUSIONS: We identified a subset of miRNAs that were differentially expressed in BCSCs, providing a starting point to explore the functions of these miRNAs. Evaluating characteristic BCSC miRNAs represents a new method for studying breast cancer-initiating cells and developing therapeutic strategies aimed at eradicating the tumorigenic subpopulation of cells in breast cancer.

Neutralization of complement regulatory proteins CD55 and CD59 augments therapeutic effect of herceptin against lung carcinoma cells.

Human monoclonal anti-Her2/neu antibody (herceptin, also named trastuzumab) failed in the treatment of lung cancer when in combination with two chemotherapy agents gemcitabine and cisplatin, despite of its clinical benefit in women with Her2 positive breast cancer. The capacity of herceptin to activate human complement and complement-dependent cytotoxicity against tumor cells was investigated in a study of tumor immunotherapy. We found that the expression of membrane complement regulatory proteins (mCRPs), CD55 and CD59 on non-small cell lung cancer (NSCLC) cells was closely correlated with histological types, prognosis and preoperational adjutant chemotherapy of the disease. Herceptin-mediated complement cytotoxicity to two human lung carcinoma cell lines exerted stronger killing effect on tumor cells after the neutralization of mCPRs via their antibodies. Furthermore, treatment of herceptin combined with chemo-agents had advantages over chemotherapy alone, while CD55 and CD59 expression levels both declined remarkably in A549 and H157 cell lines after incubation with IC50 cisplatin for 72 h. Our data indicated that overexpression of mCRPs on tumor cells contributes to herceptin's acquisition of resistance to NSCLC, and its anticancer efficacy was enhanced when mCRPs were neutralized or cisplatin could be used to down-regulate their expression.

MicroRNA expression profile of bronchioalveolar stem cells from mouse lung.

Increasing evidence has suggested that bronchioalveolar stem cell (BASC) is the progenitor cells of lung cancer stem cells. However, the mechanisms by which self-renewal of BSACs is controlled and how BASCs turn into cancer stem cells still remains to be unknown. In the present study, we successfully isolated bronchioalveolar stem cells (BASCs) from mouse lung using FACS. These BASCs were characterized by clonal growth, self-renewal and high capacity for differentiation, suggesting that these BASCs are indeed stem cells. We investigated the microRNA (miRNA) expression profile of these BASCs using miRNA array and quantitative RT-PCR. We discovered that BASCs possessed a unique miRNA profile, with altered expression of several microRNAs, such as miR-142-3p, miR-451, miR-106a, miR-142-5p, miR-15b, miR-20a, miR-106b, miR-25, miR-486, in BASCs compared to control cells. Our results suggest that microRNAs might play important roles in maintaining the self-renewal capacity of BASCs, and suggest the intriguing possibility that aberrant expression of microRNAs could involved in turning BASCs into lung cancer stem cells.

[Immunological efficiency of DNA vaccine targeting dendritic cells against tumor].

AIM: To construct the DNA vaccine containing ovalbumin (OVA) and Fc fusion gene targeting dentritic cells (DCs) and evaluate its anti-tumor efficiency in an E.G7-OVA-bearing tumor model. METHODS: Constructed OVA-Fc-pcDNA3.1 plasmids were transfected into CHO cells with lipofectamine and the in vitro expression of OVA-Fc was determined by flow cytometry and ELISA. (51)Cr-relaese assay was used to determine the anti-tumor activity of cytotoxic T lymphocytes (CTLs) from splenocytes of immunized mice. According to tumor volume and survival time of the mice, the therapeutic effect of this vaccine was evaluated in E.G7-OVA-bearing tumor model. RESULTS: DNA sequencing and restriction endonuclease digestion analysis indicated that the recombinant expression vector OVA-Fc-pcDNA3.1 had been constructed successfully. OVA-Fc expression could be detected in CHO cells transfected with OVA-Fc-pcDNA3.1 by ELISA and flow cytometry. The DNA vaccine containing OVA-Fc fusion gene inhibited tumor's growth and prolonged the time of tumor-bearing mice due to elicit the CTL-mediated anti-tumor immunity. CONCLUSION: OVA-Fc-pcDNA3.1 DNA vaccine targeting dendritic cells can elicit the CTL-mediated anti-tumor immunity, which lays the foundation for further clinical experiments.

Enhanced efficacy of radiation-induced gene therapy in mice bearing lung adenocarcinoma xenografts using hypoxia responsive elements.

The aim of the present study was to investigate whether the hypoxia responsive element (HRE) could be used to enhance suicide gene (HSV-tk) expression and tumoricidal activity in radiation-controlled gene therapy of human lung adenocarcinoma xenografts. A chimeric promoter, HRE-Egr, was generated by directly linking a 0.3-kb fragment of HRE to a 0.6-kb human Egr-1 promoter. Retroviral vectors containing luciferase or the HSV-tk gene driven by Egr-1 or HRE-Egr were constructed. A human adenocarcinoma cell line (A549) was stably transfected with the above vectors using the lipofectamine method. The sensitivity of transfected cells to prodrug ganciclovir (GCV) and cell survival rates were analyzed after exposure to a dose of 2 Gy radiation and hypoxia (1%). In vivo, tumor xenografts in BALB/c mice were transfected with the constructed retroviruses and irradiated to a total dose of 6 Gy, followed by GCV treatment (20 mg/kg for 14 days). When the HSV-tk gene controlled by the HRE-Egr promoter was introduced into A549 cells by a retroviral vector, the exposure to 1% O(2) and 2 Gy radiation induced significant enhancement of GCV cytotoxicity to the cells. Moreover, in nude mice bearing solid tumor xenografts, only the tumors infected with the hybrid promoter-containing virus gradually disappeared after GCV administration and radiation. These results indicate that HRE can enhance transgene expression and tumoricidal activity in HSV-tk gene therapy controlled by ionizing radiation in hypoxic human lung adenocarcinoma.

The co-transfection of p16(INK4a) and p14(ARF) genes into human lung cancer cell line A549 and the effects on cell growth and chemosensitivity.

Two functionally and structurally different proteins, p16(INK4a) and p14(ARF), encoded by the gene INK4a/ARF located at 9p21 are cyclin-dependent kinase (cdk) inhibitors and important cell cycle regulators. More and more evidences have been accumulated to show that the exogenous p16(INK4a) or p14(ARF) can inhibit the cell growth and/or induce the apoptosis. But it is still unclear if they can play positive role when combine with the conventional chemotherapy in cancer treatment. Here we show that cationic liposome-mediated gene transfection of INK4a/ARF into lung cancer cell line A549, in which the INK4a/ARF locus was lost, suppressed the growth and induced apoptosis. When treated with five different chemotherapy drugs with different mechanism after the transfection, A549 got an increased chemosensitivity for adriamycin and cisplatin and an unchanged result for topotecan, taxol or vinorelbine. The results indicated that cell cycle redistribution and increased apoptosis index after transfection might be the main explanation for the enhanced chemosensitivity. The combination of gene therapy with conventional chemotherapy is not always better than single chemotherapy. This trial will be of benefit to the treatment of lung cancer when combine the conventional chemotherapy and gene therapy in the future.

Cisplatin-controlled p53 gene therapy for human non-small cell lung cancer xenografts in athymic nude mice via the CArG elements.

Cisplatin, a commonly used chemotherapeutic agent, causes tumor cell death by producing DNA damage and generating reactive oxygen intermediates, which have been reported to activate the early growth response-1 (Egr-1) promoter through specific cis-acting sequences, termed CArG elements. The aim of this study was to construct an adenoviral vector containing CArG elements cloned upstream of the cDNA for human wt-p53, and to observe the effect of this vector on human non-small cell lung cancer (NSCLC) xenografts in athymic nude mice when combined with cisplatin treatment. The adenoviral vector AdEgr-p53 was generated by inserting CArG elements upstream of human wt-p53 cDNA. Two human NSCLC cell lines of varying p53 gene status, A549 (containing wild-type p53) and H358 (containing an internal homozygous deletion of the p53 gene) were used for in vitro and in vivo experiments. Wt-p53 production in cultured tumor cells and xenografts treated with the combination of AdEgr-p53 and cisplatin were detected by enzyme-linked immunosorbent assays. The antitumor responses in nude mice with the A549 or H358 xenografts following treatment with AdEgr-p53 and cisplatin were observed. We found that p53 was produced in tumor cells and xenografts treated with a combination of AdEgr-p53 and cisplatin. Furthermore, the Egr-1 promoter is induced by cisplatin, and this induction is mediated in part through the CArG elements. There was an enhanced antitumor response without an increase in toxicity following treatment with AdEgr-p53 and cisplatin, compared with either agent alone. Cisplatin-inducible p53 gene therapy may provide a means to control transgene expression while enhancing the effectiveness of commonly used chemotherapeutic agents. This is a novel treatment for human NSCLC.

[HSV-TK gene therapy of lung adenocarcinoma xenografts using a hypoxia/radiation dual-sensitive promoter].

BACKGROUND & OBJECTIVE: Radio-genetic therapy is a novel strategy for cancer treatment; however, a limited success was shown due to lower sensitivity of tumor cells to radiation under hypoxia, which is a unique feature for solid tumors. In order to improve the efficacy of radiogenetic therapy for lung cancer, a hypoxia/radiation dual-sensitive promoter was constructed to enhance the expression of HSV-TK in transfected cells exposed to radiation under hypoxia. METHODS: The chimeric promoter HRE-Egr was generated by insertion of hypoxia response elements (HREs) upstream of the Egr-1 (early growth response gene-1) promoter. HSV-TK expression vector was constructed by cloning HRE-Egr promoter upstream of HSV-TK gene, which was transfected into A549 cells via liposome. The expression of HSV-TK in transfected cells exposed to irradiation (6 Gy) and/or hypoxia (1% O2) were analyzed by Northern blot, and the relative survival rate of cells in presence of prodrug ganciclovir (GCV) was tested using MTT method. To examine the efficacy of this HRE-Egr-TK gene therapy in vivo, the A549 adenocarcinoma xenografts were planted in BALB/c nude mice. The tumor volumes and the suppression rates were assayed in nude mice bearing xenografts infected with plasmids and exposed to radiation. RESULTS: HSV-TK gene expression in transfected cells was markedly increased in both radiation (227 U) and hypoxia (94 U) groups compared with control group (21 U). The HSV-TK expression (769 U) in transfected cells exposed to radiation under hypoxia is much more higher than the former groups. The survival rate of transfected cells exposed to radiation under hypoxia in the presence of GCV was obviously decreased with comparison of cells under normoxia (7.2%+/-1.8 % vs 32.7%+/-4.6 %). HRE-Egr promoter transfected tumors regressed significantly after a combination therapy of irradiation and GCV in all mice (n=10), the tumor suppression rates was 91.2%. CONCLUSIONS: The hypoxia/radiation dual-sensitive promoter HRE-Egr can enhance the HSV-TK gene expression in solid tumors under hypoxia. Enhanced tumor suppression effect was observed in A549 xenografts infected with HRE-Egr promoter exposed to radiation.

[Oncostatin M gene therapy in mice bearing lung adenocarcinoma xenograft using a hypoxia/radiation dual-sensitive promoter].

OBJECTIVE: To improve the efficacy of radiogenetic therapy for lung cancer, a hypoxia/radiation dual-sensitive promoter was constructed to enhance the expression of oncostatin M (OSM) in transfected cells exposed to radiation under hypoxia. METHODS: The chimeric promoter HRE-Egr was generated by insertion of hypoxia response elements (HREs) upstream of the Early growth response gene-1 (Egr-1) promoter. OSM expression vector was constructed by cloning HRE-Egr promoter upstream of OSM gene, which was transfected into A549 cells. The expression of OSM in transfected cells exposed to irradiation and(or) hypoxia was analyzed, and the relative survival rate of transfected cells exposed to the above conditions was tested. To examine the efficacy of this HRE-Egr-OSM gene therapy in vivo, the tumor suppression effects were investigated in 40 nude mice bearing lung adenocarcinoma xenograft. RESULTS: Expression of OSM gene in transfected cells exposed 6 Gy irradiation was markedly increased under hypoxia. A gene therapy experiment in vitro showed that the survival rate of transfected cells exposed to radiation under hypoxia was obviously decreased with comparison of cells under normoxia. HRE-Egr promoter transfected tumors regressed significantly after a combination therapy of irradiation and HRE-Egr transfection in all mice (n = 10), and six tumors disappeared in 3 weeks without any side effects. CONCLUSION: The data indicate that tumor targeted expression of OSM gene under the control of a hypoxia/radiation dual-sensitive promoter represents a novel strategy for safe and effective gene therapy of lung carcinoma and might have clinical application in the future.

[Experimental study on lung carcinoma-targeted suicide gene therapy induced by irradiation].

OBJECTIVE: To improve the efficacy and selectivity of gene therapy for lung carcinoma, a strategy was designed for suicide gene therapy in conjunction with irradiation therapy, by constructing a plasmid tgEgr-HyTK in which HSV-TK gene was driven by the early growth responsive gene-1 (Egr-1) promoter. METHODS: The radio-inducible suicide gene was constructed by insertion of Egr-1 promoter upstream of the HSV-TK gene. The expressions of HSV-TK in lung carcinoma cell lines A549 which were infected with tgEgr-HyTK and exposed to different doses of gamma-ray irradiation were analyzed, and the relative survival rates of cells in presence of prodrug ganciclovir (GCV) were tested. The tumor suppression effects were investigated in 40 nude mice bearing lung tumors to examine the efficacy of this Egr-TK gene therapy in vivo. RESULTS: Expression of HSV-TK gene in lung carcinoma cells infected with tgEgr-HyTK plasmids was markedly increased in a radiation dose-dependent manner. A gene therapy experiment in vitro showed that tgEgr-HyTK transduced lung carcinoma cells became highly sensitive to GCV after irradiation, but not without irradiation. tgEgr-HyTK transfected tumors regressed significantly after a combination therapy of irradiation and GCV in all mice (n = 10), and five tumors disappeared in 3 weeks without any side effect. CONCLUSION: The data indicate that tumor targeted expression of HSV-TK gene under the control of a radio-inducible promoter represents a novel strategy for safe and effective gene therapy of lung carcinoma, which may have clinical application in the future.