Molecular classification reveals the sensitivity of lung adenocarcinoma to radiotherapy and immunotherapy: multi-omics clustering based on similarity network fusion.

BACKGROUND: Due to individual differences in tumors and immune systems, the response rate to immunotherapy is low in lung adenocarcinoma (LUAD) patients. Combinations with other therapeutic strategies improve the efficacy of immunotherapy in LUAD patients. Although radioimmunotherapy has been demonstrated to effectively suppress tumors, the underlying mechanisms still need to be investigated. METHODS: Total RNA from LUAD cells was sequenced before and after radiotherapy to identify differentially expressed radiation-associated genes. The similarity network fusion (SNF) algorithm was applied for molecular classification based on radiation-related genes, immune-related genes, methylation data, and somatic mutation data. The changes in gene expression, prognosis, immune cell infiltration, radiosensitivity, chemosensitivity, and sensitivity to immunotherapy were assessed for each subtype. RESULTS: We used the SNF algorithm and multi-omics data to divide TCGA-LUAD patients into three subtypes. Patients with the CS3 subtype had the best prognosis, while those with the CS1 and CS2 subtypes had poorer prognoses. Among the strains tested, CS2 exhibited the most elevated immune cell infiltration and expression of immune checkpoint genes, while CS1 exhibited the least. Patients in the CS2 subgroup were more likely to respond to PD-1 immunotherapy. The CS2 patients were most sensitive to docetaxel and cisplatin, while the CS1 patients were most sensitive to paclitaxel. Experimental validation of signature genes in the CS2 subtype showed that inhibiting the expression of RHCG and TRPA1 could enhance the sensitivity of lung cancer cells to radiation. CONCLUSIONS: In summary, this study identified a risk classifier based on multi-omics data that can guide treatment selection for LUAD patients.

Clinical Response of Advanced Lung Adenocarcinoma with Class III BRAF G466V Missense Mutation to Dabrafenib and Trametinib: A Case Report.

Aim: BRAF is a pivotal driver gene in cancer development. Based on this, the combination of dabrafenib and trametinib was approved for treating NSCLC patients with BRAFV600E mutations. However, the majority of BRAF mutations in lung cancer are non-V600E variants, particularly class III mutants, which currently lack targeted therapeutic options and result in unfavorable clinical outcomes. Case Presentation: We present a case of advanced lung adenocarcinoma with a class III BRAFG466V mutation. The patient experienced significant pleural and pericardial effusion, leading to chest tightness and an inability to lie flat. Severe pain and limited mobility from lumbar destruction seriously affected the patient's quality of life. Due to the patient's intolerance to chemotherapy, dabrafenib and trametinib combination therapy was chosen. After three months of targeted therapy, the patient's overall condition significantly improved, enabling self-care, and achieving partial response (PR) as an indicator of treatment efficacy. Conclusion: The combination therapy of dabrafenib and trametinib demonstrates remarkable clinical benefits for lung adenocarcinoma patients with the BRAFG466V mutation. Targeted therapy should be considered for patients with BRAF class III mutations, especially those in poor general condition and may not tolerate chemotherapy.

An advanced pulmonary sarcomatoid carcinoma patient harboring a BRAFV600E mutation responds to dabrafenib and trametinib: a case report and literature review.

Background: The pulmonary sarcomatoid carcinoma (PSC) is a rare and aggressive subtype of NSCLC with rapid progression and poor prognosis, and is resistant to conventional chemotherapy. Most PSC cases have potential targetable genomic alterations. Approximately 7% of PSC patients have BRAF mutations, and the efficacy of dabrafenib and trametinib in BRAFV600E mutated PSC is unclear. Case presentation: Our report describes a patient with mutated BRAFV600E PSC who underwent surgery and adjuvant chemotherapy early but quickly relapsed. Both chemotherapy and immunotherapy were ineffective for him, combined dabrafenib and trametinib produced a 6-month progression-free survival, and a partial response was observed in the tumor response evaluation. As a result of financial pressure, he stopped taking the targeted drugs, and his disease rapidly progressed. Conclusion: Dabrafenib combined with trametinib provides partial remission in patients with advanced PSC with BRAFV600E mutations, and large-scale NGS panels could offer more options for PSC treatment.

Abnormal lipid metabolism in cancer-associated cachexia and potential therapy strategy.

Cancer-associated cachexia (CAC) is a major characteristic of advanced cancer, associates with almost all types of cancer. Recent studies have found that lipopenia is an important feature of CAC, and it even occurs earlier than sarcopenia. Different types of adipose tissue are all important in the process of CAC. In CAC patients, the catabolism of white adipose tissue (WAT) is increased, leading to an increase in circulating free fatty acids (FFAs), resulting in " lipotoxic". At the same time, WAT also is induced by a variety of mechanisms, browning into brown adipose tissue (BAT). BAT is activated in CAC and greatly increases energy expenditure in patients. In addition, the production of lipid is reduced in CAC, and the cross-talk between adipose tissue and other systems, such as muscle tissue and immune system, also aggravates the progression of CAC. The treatment of CAC is still a vital clinical problem, and the abnormal lipid metabolism in CAC provides a new way for the treatment of CAC. In this article, we will review the mechanism of metabolic abnormalities of adipose tissue in CAC and its role in treatment.

Value of postoperative radiotherapy for stage IIIa-N2 non-small cell lung cancer: an analysis based on SEER database.

Background: The role of postoperative radiotherapy (PORT) in resected stage IIIa-N2 non-small cell lung cancer (NSCLC) patients who have received adjuvant chemotherapy remains controversial. This study aimed to explore the value of PORT and determine which patients could benefit from PORT. Methods: Stage IIIa-N2 NSCLC patients treated with surgery and adjuvant chemotherapy were identified from the Surveillance, Epidemiology and End Results (SEER) databases from 2004 to 2015. Eligible patients were divided into the following two groups: PORT group and non-PORT group. Overall survival (OS) was estimated by the Kaplan-Meier (KM) method, and differences in survival were evaluated with log-rank test. Long-term cause-specific mortality consisted of lung cancer-related mortality and non-lung cancer-related mortality was investigated through competing risk analysis. Cox regression analysis was performed to identify variables that significantly affected OS. Results: We identified 2,347 eligible patients, after propensity score matching (PSM), 877 pairs were selected. Overall, there was no significant difference in OS between two groups, but the patients who received PORT had a lower lung cancer-related mortality rate. Subgroup analysis showed that PORT was associated with a significantly better OS and lower lung cancer-related mortality rate in patients with T2, grade I-II and positive/resected lymph node ratio (LNR) ≥0.31. The non-lung cancer-related mortality of PORT group was higher in the patients with squamous cell carcinoma, although the difference was not significant. The independent prognostic factors for OS were age, sex, grade, histology, the American Joint Committee on Cancer (AJCC) T stage and LNR. Conclusions: Our results revealed that PORT appears to be the optimal treatment strategy in patients with AJCC T2, grade I-II and LNR ≥0.31. PORT may not be recommended for patients with squamous cell carcinoma.

Risk factors of acquired T790M mutation in patients with epidermal growth factor receptor-mutated advanced non-small cell lung cancer.

Background: It is still controversial to employ osimertinib as the first-line therapy for EGFR-mutated non-small cell lung cancer (NSCLC) patients in practice. The aim of the current study was to explore the risk factors of acquired T790M mutation during EGFR-TKIs therapy, and to identify the potential patients most likely to benefit from first-line osimertinib treatment. Methods: A total of 222 patients with EGFR-mutated (non-T790M) advanced NSCLC were analyzed. The progression-free survival (PFS), overall survival (OS), and cumulative incidence of acquired T790M mutation were calculated with the Kaplan-Meier method. The independent risk factors were investigated with the multivariate analysis. Results: A total of 70 patients acquired T790M mutation and were treated with osimertinib as a second-line treatment. These patients showed a significantly better OS (P=0.003) than those without T790M mutation. Multivariate analysis indicated that BMI ≤ 25 (P= 0.031), NSE > 17.9 ng/ml (P= 0.013) before treatment, and retroperitoneal lymph node (LN) metastasis (P= 0.002) were independent risk factors of acquired T790M mutation. At last, the actuarial risks of acquired T790M mutation at 1 year after EGFR-TKI treatment were 6.6% in patients with 0-1 risk factor and 31.5% in patients with 2-3 risk factors. Conclusions: Patients developing acquired T790M mutation during EGFR-TKI treatment had a better OS of osimertinib treatment. Lower BMI, higher NSE before treatment, and retroperitoneal LN metastasis are independent risk factors of acquired T790M mutation. Our study suggested that patients with 2-3 risk factors were highly recommended the first-line osimertinib treatment.

Predictive value of pancreatic dose-volume metrics on sarcopenia rate in gastric cancer patients treated with adjuvant chemoradiotherapy.

OBJECTIVE: To evaluate the relationship of sarcopenia with the pancreatic dose-volume histogram (DVH) in gastric cancer patients treated with adjuvant chemoradiotherapy (CRT) after radical gastrectomy. METHODS: A retrospective study was performed on the data in Zhongnan Hospital of Wuhan University from January 2008 to December 2016. Skeletal muscle index (SMI) was analyzed by cross-sectional areas of body composition at the level of third lumbar (L3) vertebrae, which was measured using single-slice computer tomograph (CT) prior to CRT, at 6 months and 12 months after CRT respectively. Logistic regression analysis was conducted to explore the potential clinical risk factors of sarcopenia in this patients cohort. Regarding the dosimetrics of pancreas, the sarcopenia rate was compared between the two groups divided according to the cut-off value determined by the receiver operating characteristic (ROC) curves. RESULTS: One hundred and fifty-three gastric cancer patients were eligible in this study. The median postoperative follow-up was 36 (7-115) months. The mean dose of pancreas was 4399.7 ± 396.0 cGy. The incidence of sarcopenia prior to CRT, at 6 months and 12 months later were 29.4% (45/153), 27.3% (35/128) and 37.0% (37/100). Both sarcopenia at 6 months (HR = 2.038, 95%CI = 1.084-3.833, P = 0.027) and sarcopenia at 12 months (HR = 2.216, 95%CI = 1.007-4.873, P = 0.048) were the independent prognostic factor of gastric cancer patients. V46 remained to be the only independent risk factor of sarcopenia at 6 months (OR = 3.889, 95%CI = 1.099-13.764, P = 0.035) and 12 months (OR = 6.067, 95%CI = 1.687-21.821, P = 0.006) in multivariate logistic regression analysis. Among the dosimetric parameters used for ROC analysis, the V46 showed the highest area under the curve (AUC = 0.707). Here is the relationship between sarcopenia rate and the cut-off value for V46. Higher sarcopenia rate at 6 months was noted in 42.6% patients with V46 ≥ 57% compared with 9% of patients with V46 < 57% (P < 0.001). The sarcopneia rate at 12 months was 52% with V46 ≥ 57% and 25% with V46 < 57% (P = 0.010). CONCLUSION: Gastric cancer with sarcopenia after adjuvant CRT had poorer survival. Higher dose and larger irradiated volume of pancreas correlated with higher risk of sarcopenia. Appropriated administration of pancreas dose-volume may be conducive to reduce the risk of sarcopenia and improve survival in gastric cancer patients treated with adjuvant CRT.

Value of radiotherapy in addition to esophagectomy for stage II and III thoracic esophageal squamous cell carcinoma: Analysis of surveillance, epidemiology, and end results database.

To determine the value of radiotherapy in addition to esophagectomy for stage II and III TESCC. We searched the Surveillance, Epidemiology, and End Results (SEER) database for all cases of stage II-III TESCC. Patients were grouped as those receiving pre- or postoperative radiotherapy plus esophagectomy and those receiving esophagectomy alone. Overall survival (OS) and cancer-specific survival (CSS) were compared between the groups. Among the 3292 patients, multimodality treatments (pre- or postoperative radiotherapy plus surgery) were more effective than surgery alone (5-year, OS: 17.3% vs 7.9%; P < 0.001; CSS: 51.8% vs 34.9%; P < 0.001). Among the patients receiving multimodality treatments, multivariate analyses revealed stage to be the most significant prognostic factor for OS (II vs III, HR, 0.726; P < 0.001), but the sequence of radiotherapy and surgery was only of the marginal significance (pre- vs postoperative, HR, 0.875; P = 0.093). Preoperative radiotherapy provided significantly better survival than postoperative radiotherapy in stage III disease (5-year, OS: 13.0% vs 11.0%, P < 0.04; CSS: 49.2% vs 31.7%, P < 0.003), but not in stage II disease (5-year OS: 23.5% vs 21.0%, P = 0.519; CSS: 62.0% vs 53.4%, P = 0.075). Radiotherapy in addition to esophagectomy provides better outcomes than esophagectomy alone for in stage II-III TESCC. Preoperative radiotherapy followed by surgery appears to be the optimal treatment strategy in stage III TESCC.

Salicylate •Phenanthroline copper (II) complex induces apoptosis in triple-negative breast cancer cells.

In this study, we investigated anti-tumor activity and associated molecular mechanism of action of Salicylate ●Phenanthroline Copper (II) Complex in triple-negative breast cancer. Salicylate ●Phenanthroline Copper (II) Complex inhibited the growth of four breast cancer cell lines (MCF-7, T47D, MDA-MB-231 and BT-20) and induced apoptosis in a concentration-dependent manner. The effect was more profound in MDA-MB-231 and BT-20 triple-negative breast cancer cell lines. Western blot showed that the expression of the apoptosis-related protein Bcl-2, Bcl-xl and survivin was significantly reduced in MDA-MB-231 after treatment with Salicylate ●Phenanthroline Copper (II) Complex. In vivo, Salicylate ●Phenanthroline Copper (II) Complex administration significantly attenuated tumor growth of MDA-MB-231 xenografts, and the expression levels of Bcl-2, Bcl-xL and survivin were reduced as measured by immunohistochemical staining. These data suggest that Salicylate ●Phenanthroline Copper (II) Complex is a promising novel therapeutic drug for triple-negative breast cancer and warrants further study.

Platinum-based therapy for triple-negative breast cancer treatment: A meta-analysis.

The aim of the present study was to evaluate the effect of platinum-based therapy on the short-term efficacy and survival rate in patients with triple-negative breast cancer (TNBC). A search of available databases was conducted, based on specific inclusion and exclusion criteria, for trials conducted between January 2006 and January 2014. The bibliographies of the included studies were examined with the same criteria. Included studies were evaluated using Grading of Recommendations Assessment, Development and Evaluation (GRADE), and extracted data were analyzed using RevMan 5.1 and GRADEprofiler 3.6. Eight studies with a total of 1,349 patients were included. The meta-analysis revealed that the pathological complete response rate and overall response rate in TNBC patients who were treated with a platinum-based regimen was significantly higher than that in those treated with a non-platinum-based regimen (49.2 and 64.3%, respectively). The disease-free survival rate and overall survival rate were not significantly different between TNBC patients treated with a platinum-based regimen and those treated with a non-platinum-based regiment (P>0.05). Platinum-based chemotherapy in TNBC patients resulted in improved short-term efficacy. Platinum-based regimens may therefore be more sensitive to TNBC patients. However, future multicenter randomized controlled trials are required to validate these findings and to determine whether platinum-based chemotherapy can extend the survival rate of TNBC patients.

Effect of neoadjuvant chemotherapy in patients with triple-negative breast cancer: A meta-analysis.

The present meta-analysis aimed to evaluate the effect of neoadjuvant chemotherapy on pathological complete response (pCR) and survival rate in patients with triple-negative breast cancer (TNBC). Specific inclusion and exclusion criteria were used to conduct a search of the available databases, in order to find studies performed between January 2006 and January 2014. The bibliographies of the included studies were examined with the same criteria. The Grading of Recommendations Assessment, Development and Evaluation (GRADE) working group framework was used to evaluate the included studies, and RevMan 5.1 and GRADEprofiler 3.6 were used to analyze the extracted data. A total of 19 studies with 6,180 patients were included. The meta-analysis revealed that the pCR rates in patients with TNBC were significantly higher than those in patients with non-TNBC. The 5-year disease-free survival (DFS) and overall survival (OS) rates were significantly lower in the patients with TNBC compared with those with non-TNBC. Furthermore, these survival rates were significantly higher in the patients with TNBC who achieved a pCR compared with those in the patients who did not achieve a pCR. pCR rates were higher among the patients with TNBC with high Ki-67 expression than among those with low Ki-67 expression. The patients with TNBC exhibited lower survival rates compared with those with non-TNBC, but achieved higher pCR rates. Moreover, those patients achieving a pCR exhibited improved 5-year survival rates, suggesting that the pCR rate could be predictive of survival in patients with TNBC. In addition, high Ki-67 expression may predict the likelihood of a pCR. However, future multicenter randomized controlled trials are required to enhance the quantity and quality of the clinical evidence.

Knockdown of telomeric repeat binding factor 2 enhances tumor radiosensitivity regardless of telomerase status.

PURPOSE: To investigate the effects of TRF2 depletion on radiosensitivity in both the telomerase-positive cell lines (A549) and alternative lengthening of telomere (ALT) cell lines (U2OS). METHODS: X-ray irradiation was used to establish two radioresistant cancer models (A549R and U2OSR) from A549 and U2OS. Colony formation assay was applied to examine the radiosensitivity of radioresistant A549R and U2OSR cells and TRF2 low-expression cells. Real-time PCR and TeloTAGGG Telomerase PCR ELISA Kit were performed to examine telomere length and telomerase activity separately. γ-H2AX was detected by immunofluorescence to assess the radiation-induced DSBs. RESULTS: Radioresistant cancer models were established, in which TRF2 was significantly over-expressed. Low expression of TRF2 protein could enhance the radiosensitivity and induce telomere length of A549 and U2OS cell shortening. In A549 cells with TRF2 down-regulated, the telomerase activity was inhibited, too. TRF2 deficiency increases γ-H2AX foci and fails to protect telomere from radiation. CONCLUSION: The data suggest that TRF2 is a radioresistant protein in A549 and U2OS cells, and could potentially be a target for radiosensitization of both telomerase-positive and ALT cells in radiotherapy.

RIG-I-like receptor LGP2 protects tumor cells from ionizing radiation.

An siRNA screen targeting 89 IFN stimulated genes in 14 different cancer cell lines pointed to the RIG-I (retinoic acid inducible gene I)-like receptor Laboratory of Genetics and Physiology 2 (LGP2) as playing a key role in conferring tumor cell survival following cytotoxic stress induced by ionizing radiation (IR). Studies on the role of LGP2 revealed the following: (i) Depletion of LGP2 in three cancer cell lines resulted in a significant increase in cell death following IR, (ii) ectopic expression of LGP2 in cells increased resistance to IR, and (iii) IR enhanced LGP2 expression in three cell lines tested. Studies designed to define the mechanism by which LGP2 acts point to its role in regulation of IFNß. Specifically (i) suppression of LGP2 leads to enhanced IFNß, (ii) cytotoxic effects following IR correlated with expression of IFNß inasmuch as inhibition of IFNß by neutralizing antibody conferred resistance to cell death, and (iii) mouse embryonic fibroblasts from IFN receptor 1 knockout mice are radioresistant compared with wild-type mouse embryonic fibroblasts. The role of LGP2 in cancer may be inferred from cumulative data showing elevated levels of LGP2 in cancer cells are associated with more adverse clinical outcomes. Our results indicate that cytotoxic stress exemplified by IR induces IFNß and enhances the expression of LGP2. Enhanced expression of LGP2 suppresses the IFN stimulated genes associated with cytotoxic stress by turning off the expression of IFNß.

Telomere-binding protein TPP1 modulates telomere homeostasis and confers radioresistance to human colorectal cancer cells.

BACKGROUND: Radiotherapy is one of the major therapeutic strategies in cancer treatment. The telomere-binding protein TPP1 is an important component of the shelterin complex at mammalian telomeres. Our previous reports showed that TPP1 expression was elevated in radioresistant cells, but the exact effects and mechanisms of TPP1 on radiosensitivity is unclear. PRINCIPAL FINDINGS: In this study, we found that elevated TPP1 expression significantly correlated with radioresistance and longer telomere length in human colorectal cancer cell lines. Moreover, TPP1 overexpression showed lengthened telomere length and a significant decrease of radiosensitivity to X-rays. TPP1 mediated radioresistance was correlated with a decreased apoptosis rate after IR exposure. Furthermore, TPP1 overexpression showed prolonged G2/M arrest mediated by ATM/ATR-Chk1 signal pathway after IR exposure. Moreover, TPP1 overexpression accelerated the repair kinetics of total DNA damage and telomere dysfunction induced by ionizing radiation. CONCLUSIONS: We demonstrated that elevated expressions of TPP1 in human colorectal cancer cells could protect telomere from DNA damage and confer radioresistance. These results suggested that TPP1 may be a potential target in the radiotherapy of colorectal cancer.

Inhibition of UBE2D3 expression attenuates radiosensitivity of MCF-7 human breast cancer cells by increasing hTERT expression and activity.

The known functions of telomerase in tumor cells include replenishing telomeric DNA and maintaining cell immortality. We have previously shown the existence of a negative correlation between human telomerase reverse transcriptase (hTERT) and radiosensitivity in tumor cells. Here we set out to elucidate the molecular mechanisms underlying regulation by telomerase of radiosensitivity in MCF-7 cells. Toward this aim, yeast two-hybrid (Y2H) screening of a human laryngeal squamous cell carcinoma radioresistant (Hep2R) cDNA library was first performed to search for potential hTERT interacting proteins. We identified ubiquitin-conjugating enzyme E2D3 (UBE2D3) as a principle hTERT-interacting protein and validated this association biochemically. ShRNA-mediated inhibition of UBE2D3 expression attenuated MCF-7 radiosensitivity, and induced the accumulation of hTERT and cyclin D1 in these cells. Moreover, down-regulation of UBE2D3 increased hTERT activity and cell proliferation, accelerating G1 to S phase transition in MCF-7 cells. Collectively these findings suggest that UBE2D3 participates in the process of hTERT-mediated radiosensitivity in human breast cancer MCF-7 cells by regulating hTERT and cyclin D1.

Novel, chimeric, cancer-specific, and radiation-inducible gene promoters for suicide gene therapy of cancer.

BACKGROUND: Although the promoter of the human telomerase reverse transcriptase (hTERT) gene has been widely used in gene therapy for targeted cancer cells, it has some limitations for clinical use because of its low activity and potential toxicity to certain normal cells. To overcome these defects, the authors generated novel chimeric hTERT promoters that contained the radiation-inducible sequence CC(A/T)(6) GG (known as CArG elements). METHODS: Chimeric hTERT promoters were synthesized that contained different numbers of CArG elements, and the activity of chimeric promoters was assessed in different cancer cell lines and normal cells. The potential of selected promoters to successfully control horseradish peroxidase (HRP) and prodrug indole-3-acetic acid (IAA) suicide gene therapy was tested in vitro and in vivo. RESULTS: The promoter activity assays indicated that the synthetic promoter that contained 6 repeating CArG units had the best radiation inducibility than any other promoters that contained different numbers of CArG units, and the chimeric promoters retained their cancer-specific characteristics. The chimeric promoter was better at driving radiation-inducible gene therapy than the control promoters. The sensitizer enhancement ratio of the chimeric promoter system determined by clonogenic assay was higher, and the chimeric promoter system resulted in a significantly higher apoptotic level compared with other promoter systems. The combination of chimeric/promoter-mediated gene therapy and radiotherapy significantly inhibited tumor volume in a xenograft mouse model and resulted in a significant prolongation of survival in mice. CONCLUSIONS: The current results indicated that a combinational cancer-specific promoter system that is responsive to irradiation has great potential for improving the efficacy of cancer treatment.

Co-expression of interleukin 12 enhances antitumor effects of a novel chimeric promoter-mediated suicide gene therapy in an immunocompetent mouse model.

The human telomerase reverse transcriptase (hTERT) promoter has been widely used in target gene therapy of cancer. However, low transcriptional activity limited its clinical application. Here, we designed a novel dual radiation-inducible and tumor-specific promoter system consisting of CArG elements and the hTERT promoter, resulting in increased expression of reporter genes after gamma-irradiation. Therapeutic and side effects of adenovirus-mediated horseradish peroxidase (HRP)/indole-3-acetic (IAA) system downstream of the chimeric promoter were evaluated in mice bearing Lewis lung carcinoma, combining with or without adenovirus-mediated interleukin 12 (IL12) gene driven by the cytomegalovirus promoter. The combination treatment showed more effective suppression of tumor growth than those with single agent alone, being associated with pronounced intratumoral T-lymphocyte infiltration and minor side effects. Our results suggest that the combination treatment with HRP/IAA system driven by the novel chimeric promoter and the co-expression of IL12 might be an effective and safe target gene therapy strategy of cancer.

[Anti-tumor effect of suicide gene therapy using chimeric promoter plus radiotherapy on cancer cell lines].

OBJECTIVE: To explore the synergistic anti-tumor effect of radiotherapy and horseradish peroxidase/prodrug indole-3-acetic acid (HRP/IAA) gene therapy system using chimeric hTERT promoter responsive to ionizing radiation. METHODS: The synthetic hTERT promoters containing four tandem-repeat copies of radio-inducible CArG elements, and the chimeric promoter containing cytomegalovirus (CMV) early promoter were both constructed. The activities of the chimeric promoters in cancer cell lines (HeLa, A549, and MHCC97) and normal cell line (MRC-5) were detected using luciferase reporter gene expression analysis after a (60)Co γ-irradiation treatment at a series of doses(a single dose of 0 to 10 Gy). The anti-tumor effect of combining irradiation with HRP/IAA gene-directed enzyme prodrug therapy system controlled by the chimeric promoter was tested by colony formation assay, cell counting and apoptosis analysis. RESULTS: The chimeric promoters were ineffective in normal human cells, even after irradiation, but the expression of luciferase gene in tumor cells was significantly higher. The activity of the chimeric promoter in MRC-5 cells was 22.3%, 12.9% and 13.6% of that in HeLa, A549 and MHCC97 cells, respectively. After irradiation, the ratios were 11.7%, 8.7% and 8.8%, respectively. Furthermore, the chimeric promoters could successfully induce the expression of luciferase gene following different doses of radiation, with maximal inducible activity seen after 6 Gy irradiation. The chimeric promoter containing four tandem-repeat copies of radio-inducible CArG elements and CMV early promoter showed the highest activity with 6 Gy irradiation. The relative luciferase activities in HeLa, A549 and MHCC97 cells were 1.7 ± 0.2, 2.3 ± 0.2 and 2.3 ± 0.1, respectively. The chimeric promoter mediated suicide gene therapy system could increase radio-sensitivity in different cancer cells. Compared with the control system, it plus irradiation showed stronger cell proliferation inhibition, 67.3% vs. 26.1% in HeLa, 69.0% vs. 28.3% in A549, 64.6% vs. 20.8% in MHCC97 cells, and also higher apoptosis-inducing effect, 39.6% vs. 14.2% in HeLa, 33.0% vs. 12.4% in A549, and 33.2% vs. 14.2% in MHCC97 cells. CONCLUSIONS: Chimeric promoter containing hTERT promoter, CArG elements and CMV promoter preserve the tumor-specificity in telomerase-positive tumor cells, and irradiation-responsive to low dose of radiation. The suicide gene therapy using this promoter plus radiotherapy show a strong anti-tumor effect in vitro. It is expected to have a good potential for future application in gene radiotherapy.

Gene therapy with tumor-specific promoter mediated suicide gene plus IL-12 gene enhanced tumor inhibition and prolonged host survival in a murine model of Lewis lung carcinoma.

BACKGROUND: Gene therapy is a promising therapeutic approach for cancer. Targeted expression of desired therapeutic proteins within the tumor is the best approach to reduce toxicity and improve survival. This study is to establish a more effective and less toxic gene therapy of cancer. METHODS: Combined gene therapy strategy with recombinant adenovirus expressing horseradish peroxidase (HRP) mediated by human telomerase reverse transcriptase (hTERT) promoter (AdhTERTHRP) and murine interleukin-12 (mIL-12) under the control of Cytomegalovirus (CMV) promoter (AdCMVmIL-12) was developed and evaluated against Lewis lung carcinoma (LLC) both in vivo and in vitro. The mechanism of action and systemic toxicities were also investigated. RESULTS: The combination of AdhTERTHRP/indole-3-acetic acid (IAA) treatment and AdCMVmIL-12 resulted in significant tumor growth inhibition and survival improvement compared with AdhTERTHRP/IAA alone (tumor volume, 427.4 ± 48.7 mm3 vs 581.9 ± 46.9 mm3, p = 0.005 on day 15; median overall survival (OS), 51 d vs 33 d) or AdCMVmIL-12 alone (tumor volume, 362.2 ± 33.8 mm3 vs 494.4 ± 70.2 mm3, p = 0.046 on day 12; median OS, 51 d vs 36 d). The combination treatment stimulated more CD4+ and CD8+ T lymphocyte infiltration in tumors, compared with either AdCMVmIL-12 alone (1.3-fold increase for CD4+ T cells and 1.2-fold increase for CD8+ T cells, P < 0.01) or AdhTERTHRP alone (2.1-fold increase for CD4+ T cells and 2.2-fold increase for CD8+ T cells, P < 0.01). The apoptotic cells in combination group were significantly increased in comparison with AdCMVmIL-12 alone group (2.8-fold increase, P < 0.01) or AdhTERTHRP alone group (1.6-fold increase, P < 0.01). No significant systematic toxicities were observed. CONCLUSIONS: Combination gene therapy with AdhTERTHRP/IAA and AdCMVmIL-12 could significantly inhibit tumor growth and improve host survival in LLC model, without significant systemic adverse effects.