Combined 5-fluorouracil/systemic interferon-beta gene therapy results in long-term survival in mice with established colorectal liver metastases.

Preclinical in vitro and in vivo studies have demonstrated synergistic interactions between 5-fluorouracil (5-FU) and type I and II IFNs against human colorectal cancer cells. Despite these activities, randomized human trials have failed to identify a clinical benefit for this combination treatment. These limited clinical results may be secondary to the short half-life of recombinant IFN protein and the increased systemic toxicities of 5-FU/IFN combinations. We have previously reported an adenoviral-mediated IFN-beta gene therapy strategy, which may circumvent the pitfalls of recombinant IFN therapy. However, a dose-dependent toxicity and acute inflammatory response to systemically administered adenovirus vectors may limit the clinical application of this therapy. The combination of adenoviral-mediated IFN-beta gene therapy and 5-FU resulted in tumor regression, apoptosis, and improved survival in an established liver metastases model. These therapeutic effects were observed at a significantly lower vector dose than we had previously reported and with limited toxicity. This approach may allow for an effective clinical application of this therapy and warrants additional investigation.     

EpCAM- and EGFR-targeted selective gene therapy for biliary cancers using Z33-fiber-modified adenovirus

A critical issue in adenovirus (Ad)-based cancer gene therapy is to improve the specificity of gene delivery to cancer cells for better efficacy and safety. We explored methods of retargeting Ad vectors for selective gene therapy of human biliary cancers using the Ad incorporating an IgG Fc-binding motif (Z33) from the Staphylococcus protein A (Ad-FZ33) combined with tumor-specific antibodies. Flow cytometry analysis revealed high-expression levels of epithelial cell adhesion molecule (EpCAM) and epidermal growth factor receptor (EGFR) on human biliary cancer cells. Ad-FZ33 expressing LacZ combined with antibodies against EpCAM or EGFR, followed by β-gal assay, demonstrated highly efficient gene transduction in these biliary cancer cells, compared to the treatment with control antibody or without antibody. Ad-FZ33 expressing uracil phosphoribosyl transferase (UPRT), an enzyme which greatly enhances the toxicity of 5-fluorouracil (FU), combined with antibodies against EpCAM or EGFR, remarkably enhanced the sensitivity of biliary cancer cells to 5-FU. By contrast, the treatment did not affect the 5-FU sensitivity of the cells not expressing EpCAM or EGFR including normal hepatocytes. Finally, treatments with the UPRT-expressing Ad-FZ33 with antibodies against EpCAM or EGFR, followed by 5-FU administration, significantly suppressed the growth of biliary cancer xenografts in nude mice. These results indicate that the gene therapy mediated by the Z33 fiber modified Ad with anti-EpCAM or anti-EGFR antibodies offers a potentially effective therapeutic modality against biliary cancers.     

Advanced generation adenoviral vectors possess augmented gene transfer efficiency based upon coxsackie adenovirus receptor-independent cellular entry capacity

Adenoviral (Ad) vectors have been widely used in the context of cancer gene therapy approaches. Their utility in these contexts, however, has frequently been limited by tumor cell resistance to Ad infection. The basis of this resistance has been defined recently as resulting from a deficiency of the primary adenovirus receptor, coxsackie adenovirus receptor. As a means to circumvent this limitation, a variety of tropism modification strategies have allowed coxsackie adenovirus receptor-independent gene delivery via the Ad vector. These advanced generation adenovirus vectors exhibit enhanced infectivity, which can allow direct therapeutic gain. Such vectors may allow improvements in efficacy in the context of ongoing human clinical gene therapy approaches for cancer.     

Enhanced growth suppression in esophageal carcinoma cells using adenovirus-mediated fusion gene transfer (uracil phosphoribosyl transferase and herpes simplex virus thymidine kinase)

Advanced esophageal cancers are highly malignant and frequently resistant to 5-fluorouracil (5-FU). Escherichia coli uracil phosphoribosyltransferase (UP) is a pyrimidine salvage enzyme that alters 5-FU metabolism and sensitivity. A recombinant adenovirus encoding the UP gene (AxCA.UP) has been applied in gastric cancer gene therapy to sensitize cancer cells to lower concentrations of 5-FU. We have generated a recombinant adenovirus (AxCA.UT) encoding UP and herpes simplex virus thymidine kinase fusion protein (UT) to examine whether it would enhance the antitumor activity of AxCA.UP treatment. AxCA.UT treatment significantly enhanced the sensitivity of human esophageal cancer cells to and significantly enhanced the growth inhibition effects of UP gene therapy in vitro. Moreover, both 5-FU and ganciclovir showed bystander effects on growth inhibition. In an in vivo study, the therapeutic outcome of AxCA.UT treatment significantly enhanced the antitumor activity of AxCA.UP treatment. These observations suggest that AxCA.UT may be useful in esophageal cancer gene therapy.     

Effective gene therapy of biliary tract cancers by a conditionally replicative adenovirus expressing uracil phosphoribosyltransferase: significance of timing of 5-fluorouracil administration

In order to enhance the efficacy of conditionally replicating adenoviruses (CRAd) in the treatment of cancers of the biliary tract, we studied the efficacy in vitro and in vivo of AxE1CAUP, a CRAd vector that carries a gene for uracil phosphoribosyltransferase (UPRT), which converts 5-fluorouracil (5-FU) directly to 5-fluorouridine monophosphate and greatly enhances the cytotoxicity of 5-FU. AxE1CAUP replicated and induced an increased UPRT expression in biliary cancer cells more efficiently than AxCAUP, a nonreplicative adenovirus carrying the UPRT gene. Whereas AxCAUP and AxE1AdB, a CRAd without the UPRT gene, modestly increased the sensitivity of BC cells to 5-FU, AxE1CAUP markedly increased the sensitivity, especially when the timing of 5-FU administration was appropriately chosen. AxE1CAUP replicated much less efficiently in normal WI-38 fibroblasts without any change in the sensitivity to 5-FU. In nude mice with s.c. biliary cancer xenografts, i.t. AxE1CAUP/5-FU therapy inhibited tumor growth significantly more strongly than AxCAUP/5-FU or AxE1AdB/5-FU therapy. Furthermore, in mice with peritoneally disseminated biliary cancer, i.p. AxE1CAUP efficiently proliferated in the tumors, decreased the tumor burden, and prolonged the survival of the mice when 5-FU was started 10 or 15 days after the vector inoculation, whereas earlier initiation of 5-FU resulted in early eradication of the vector and no survival benefit. The present study shows that the CRAd expressing UPRT was a more potent sensitizer of biliary cancer to 5-FU, than was a nonreplicative UPRT-encoding vector or a CRAd without UPRT gene, even at a lower dose of the vector, and that timing of 5-FU administration was a key factor to maximize the efficacy. This gene therapy with appropriately timed administration of 5-FU should be useful in overcoming the resistance of biliary cancers to 5-FU.     

Carcinoembryonic antigen-targeted selective gene therapy for gastric cancer through FZ33 fiber-modified adenovirus vectors.

PURPOSE: A major problem when using the adenoviral vectors for gene therapy applications is thought to be related to low transduction efficiency in cancer cells or to side effects in normal cells. There is an urgent requirement to improve the specificity of gene delivery in the context of cancer gene therapy. EXPERIMENTAL DESIGN: We constructed a genetically modified adenovirus incorporating an IgG Fc-binding motif from the Staphylococcus protein A, Z33, within the HI loop (Adv-FZ33). A remarkable degree of targeted gene delivery to gastric cancer cells was obtained with Adv-FZ33 with the fully human anti-carcinoembryonic antigen (CEA) monoclonal antibody, C2-45. RESULTS: In vitro LacZ or EGFP gene expression after Adv-FZ33 infection via C2-45 was 20 times higher than control monoclonal antibody in MKN-45 at 1,000 viral particles/cell. We generated Ax3CAUP-FZ33 (UP-FZ33), which is an Adv-FZ33 derivative vector expressing a therapeutic gene (i.e., Escherichia coli uracil phosphoribosyltransferase), which converts 5-fluorouracil (5-FU) directly to 5-fluoro-UMP. UP-FZ33 with C2-45 enhanced the cytotoxicity of 5-FU by 10.5-fold in terms of IC(50) against MKN-45 compared with control IgG4. In a nude mouse peritoneal dissemination model, tumor growth in mice treated with UP-FZ33/C2-45/5-FU was significantly suppressed, and tumor volumes were less than one-fourth of those of the control IgG4 group (P < 0.05). The median survival time of the UP-FZ33/C2-45/5-FU group was significantly longer than those treated with PBS or 5-FU only (P < 0.01). CONCLUSIONS: These data suggest that CEA-targeted FZ33 mutant adenovirus-mediated gene delivery offers a strong and selective therapeutic modality against CEA-producing cancers.     

Acquired and innate resistance to the cancer-specific apoptosis-inducing cytokine, mda-7/IL-24: not insurmountable therapeutic problems

Despite significant progress in early cancer detection and aggressive therapies, effective treatments for metastatic disease frequently fall short of producing the desired effect of engendering a 'cure.' This can be attributed in part to inherent and acquired resistance of primary and evolving tumor cells to conventional therapeutic approaches. Agents that can interfere with critical aberrant cell signaling and survival pathways in tumor cells while displaying minimal or preferably no toxicity to normal cells represent potentially powerful tools for cancer therapy. A recently identified cancer gene therapeutic is melanoma differentiation associated gene-7/interleukin-24 (mda-7/IL-24) that has the unique ability of inducing apoptosis in diverse cancer cells without harming normal cells or tissues. As a secreted cytokine mda-7/IL-24 also exerts anti-angiogenic, radiosensitizing and immunomodulatory effects and the therapeutic benefits of an adenovirus expressing mda-7/IL-24 (Ad.mda-7) in human xenograft animal models has been successfully recapitulated in a Phase I clinical trial in patients with advanced cancers. However, as with most treatment modalities, particular subsets of tumor cells might be inherently resistant to mda-7/IL-24, as observed in pancreatic and specific colorectal cancer cells, or they might acquire resistance because of repeated exposure to this cytokine. Pataer et al. have developed Ad.mda-7-resistant lung cancer cells in vitro and demonstrated that the combination of Ad.mda-7 with bcl-2 siRNA or 17AAG could overcome this resistance. Indeed, previous studies have also demonstrated that combinatorial approaches employing Ad.mda-7 and diverse other modalities, such as chemo- or radiotherapy, small molecule inhibitors and monoclonal antibodies, can either augment the therapeutic effect of Ad.mda-7 or overcome resistance to this gene. In these contexts, should resistance to monotherapy with mda-7/IL-24 occur, combinatorial therapies with this cytokine might provide a viable path for potentially curing patients of primary and metastatic cancer.     

ABCB5 identifies a therapy-refractory tumor cell population in colorectal cancer patients

Identification and reversal of treatment resistance mechanisms of clinically refractory tumor cells is critical for successful cancer therapy. Here we show that ATP-binding cassette member B5 (ABCB5) identifies therapy-refractory tumor cells in colorectal cancer patients following fluorouracil (5-FU)-based chemoradiation therapy and provide evidence for a functional role of ABCB5 in colorectal cancer 5-FU resistance. Examination of human colon and colorectal cancer specimens revealed ABCB5 to be expressed only on rare cells within healthy intestinal tissue, whereas clinical colorectal cancers exhibited substantially increased levels of ABCB5 expression. Analysis of successive, patient-matched biopsy specimens obtained prior to and following neoadjuvant 5-FU-based chemoradiation therapy in a series of colorectal cancer patients revealed markedly enhanced abundance of ABCB5-positive tumor cells when residual disease was detected. Consistent with this finding, the ABCB5-expressing tumor cell population was also treatment refractory and exhibited resistance to 5-FU-induced apoptosis in a colorectal cancer xenograft model of 5-FU monotherapy. Mechanistically, short hairpin RNA-mediated ABCB5 knockdown significantly inhibited tumorigenic xenograft growth and sensitized colorectal cancer cells to 5-FU-induced cell killing. Our results identify ABCB5 as a novel molecular marker of therapy-refractory tumor cells in colorectal cancer patients and point to a need for consistent eradication of ABCB5-positive resistant tumor cell populations for more effective colorectal cancer therapy.     

溶瘤腺病毒联合化疗治疗肿瘤的研究

近年来用溶瘤腺病毒联合化疗治疗肿瘤的研究备受人们关注.肿瘤细胞对化疗药物的耐药性是目前临床上有效应用化疗方案的主要限制.溶瘤腺病毒能明显提高传统化疗对肿瘤细胞的杀伤效果,克服了传统肿瘤基因治疗中复制缺陷型腺病毒的转染效率低、靶向性差、抗癌基因表达量低的缺点.在肿瘤治疗中溶瘤腺病毒联合化疗将可能成为潜在有效的临床治疗方案.     

Adenovirus-mediated N5 gene transfer inhibits tumor growth and metastasis of human carcinoma in nude mice

The therapeutic effectiveness of cancer therapy often relies on induction of apoptotic cell death. Gene-therapy-mediated induction of apoptosis, therefore, may provide an effective means to kill cancer cells. The N5 gene encodes a death-domain-containing protein (p84N5) that can trigger atypical apoptosis from within the nucleus, suggesting it may be a candidate for use as a gene therapy for cancer. In the present study, we test the potential utility of a recombinant adenovirus designed to express the N5 gene(AdN5) for the treatment of a variety of human cancers using in vitro and animal models. In vitro, adenoviral-mediated N5 gene transfer inhibits the growth of five different tumor cell lines, but not a normal diploid fibroblast cell line. Adenoviral-mediated N5 gene transfer also reduces the growth and metastasis of primary human tumors in subcutaneous and orthotopic xenograft mouse models. Reduction in tumor cell growth in vitro and in vivo correlates with increased expression of p84N5 and induction of apoptosis. The relative sensitivity of different human cancer cells to AdN5 or Adp53 varies, suggesting that AdN5 may be effective in tumors relatively resistant to p53 gene therapy. We conclude that N5 has potential utility for the gene therapy of cancer.     

Enzyme/prodrug gene therapy for human colon cancer cells using adenovirus-mediated transfer of the Escherichia coli cytosine deaminase gene driven by a CAG promoter associated with 5-fluorocytosine administration

Escherichia coli cytosine deaminase (CD), which is a prokaryotic enzyme, converts nontoxic prodrug 5-fluorocytosine (5-FC) into the toxic chemotherapeutic agent 5-fluorouracil (5-FU). To investigate an enzyme/prodrug gene therapy for colorectal cancer, using adenoviral gene transfer of the E. coli CD gene associated with administration of 5-FC, we constructed replication-defective adenovirus vectors expressing the E. coli CD gene or lacZ gene driven by a CAG promoter (composed of a cytomegalovirus immediate early enhancer and a chicken beta-actin promotor). The present study demonstrated that an adenoviral gene transfer system using a CAG promoter induced sufficient gene expression of CD to confer the cytotoxicity of 5-FC to HT29 human colon cancer cells by converting it into 5-FU even at an moi of one. Furthermore, experimental gene therapy using intratumoral injection of the CD-expressing adenovirus with systemical administration of 5'-FC successfully suppressed the growth of established HT29 subcutaneous tumors in nude mice. These results suggest that enzyme/prodrug gene therapy using the adenoviral gene transfer of the E. coli CD gene with concomitant administration of 5-FC may be an effective strategy in the local control of colorectal cancer.     

Gene therapy and molecular therapeutic for head and neck cancer

Despite recent progress in radiotherapy, chemotherapy and surgical techniques, the survival of patients with head and neck squamous cell carcinomas has not improved significantly over the past three decades. To conquer these malignant tumors, various new therapies have been under development, including gene therapies. In this paper, we have reviewed recent advances in gene therapies and molecular target therapy for head and neck cancers, with a special focus on the replication-selective adenovirus vector. At present, replication-defective adenovirus-based vectors have been widely used as convenient and safe vectors for transferring therapeutic genes into target cancer cells. However, these vectors have limited the efficacy of treatment by restricting the number of tumor cells to which the therapeutic gene can be delivered. Replication-selective adenoviral vectors (RSAV) are adenoviruses designed to have a limited ability to replicate themselves in the targeted tumor cells but not in other normal tissues. Tumor cell killing is achieved not by the genes delivered by the vectors but by the oncolysis induced by the replicated viruses by their original nature as adenovirus. Amplified viral vectors also spread to the adjacent tumor cells and kill these cells in the same manner. Recently, increased evidences of antitumor activity of RSAVs have been demonstrated preclinically, and several clinical trials have demonstrated the safety and clinical activity of replication-selective viruses.     

Adenovirus-mediated HCCS1 overexpression elicits a potent antitumor efficacy on human colorectal cancer and hepatoma cells both in vitro and in vivo

We had characterized earlier the novel tumor suppressor gene hepatocellular carcinoma suppressor 1 (HCCS1), and demonstrated that expression of exogenous HCCS1 gene in human hepatocarcinoma cells could remarkably suppress their abilities to develop tumors in nude mice and to form colonies in soft agar. In this study, we provide further experimental evidence to confirm the role of HCCS1 as a tumor suppressor gene and investigate its potential in therapeutic applications by using adenovirus vectors. We show that HCCS1 overexpression, mediated by replication-deficient adenovirus, significantly suppressed the growth of human colorectal cancer cells, as well as hepatocellular carcinoma cells in vitro and in vivo. To further improve its antitumor efficacy, we inserted the HCCS1 gene into an oncolytic adenovirus. This HCCS1-armed oncolytic adenovirus exhibited a dramatic inhibitory effect on cancer cells in vitro and in vivo, and led to a complete regression of 50% of established tumor xenografts in nude mice. Taken together, our data suggest that HCCS1 is a promising therapeutic gene for the treatment of human cancers.     

Target for cancer therapy: proliferating cells or stem cells.

Tumor stem cells are quiescent and, therefore, resistant to therapy, yet harbor the capacity to replenish a tumor after therapy. Therefore, it is tempting to explain all therapeutic failures by the persistence of tumor stem cells. Yet, this explanation is relevant only to initial stages of stem-cell-dependent tumors (such as chronic myeloid leukemia) that, actually, are well controlled by therapy. In advanced cancers that poorly respond to therapy, quiescent tumor stem cells play a negligible role. Instead, proliferating cells determine disease progression, prognosis, therapeutic failures, and resistance to therapy. And therapy fails not because it eliminates only proliferating tumor cells, but because it does not eliminate them. With noticeable exceptions, it is the proliferating cell that should be targeted, whereas resting cancer cells including stem and dormant cells need to be targeted only when they 'wake up'. Finally, I discuss a strategy of selectively killing dominant proliferating clones, including proliferating stem-like and drug-resistant cancer cells, while sparing normal cells.     

Survivin promoter-regulated oncolytic adenovirus with Hsp70 gene exerts effective antitumor efficacy in gastric cancer immunotherapy

Gene therapy is a promising adjuvant therapeutic strategy for cancer treatment. To overcome the limitations of current gene therapy, such as poor transfection efficiency of vectors, low levels of transgene expression and lack of tumor targeting, the Survivin promoter was used to regulate the selective replication of oncolytic adenovirus in tumor cells, and the heat shock protein 70 (Hsp70) gene was loaded as the anticancer transgene to generate an AdSurp-Hsp70 viral therapy system. The efficacy of this targeted immunotherapy was examined in gastric cancer. The experiments showed that the oncolytic adenovirus can selectively replicate in and lyse the Survivin-positive gastric cancer cells, without significant toxicity to normal cells. AdSurp-Hsp70 reduced viability of cancer cells and inhibited tumor growth of gastric cancer xenografts in immuno-deficient and immuno-reconstruction mouse models. AdSurp-Hsp70 produced dual antitumor effects due to viral replication and high Hsp70 expression. This therapeutic system used the Survivin promoter-regulated oncolytic adenovirus vector to mediate targeted expression of the Hsp70 gene and ensure safety and efficacy for subsequent gene therapy programs against a variety of cancers.     

PUMA mediates the combinational therapy of 5-FU and NVP-BEZ235 in colon cancer

Colon cancer is the third most common cancer in humans which has a high mortality rate, and 5-Fluorouracil (5-FU) is one of the most widely used drugs in colon cancer therapy. However, acquired chemoresistance is becoming the major challenges for patients, and the molecular mechanism underlying the development of 5-FU resistance is still poorly understood. In this study, a newly designed therapy in combination with 5-FU and NVP-BEZ235 in colon cancer cells (HCT-116 and RKO) was established, to investigate the mechanism of 5-FU resistance and optimize drug therapy to improve outcome for patients. Our results show 5-FU induced cell apoptosis through p53/PUMA pathway, with aberrant Akt activation, which may well explain the mechanism of 5-FU resistance. NVP-BEZ235 effectively up-regulated PUMA expression, mainly through inactivation of PI3K/Akt and activation of FOXO3a, leading to cell apoptosis even in the p53^−/− HCT-116 cells. Combination treatment of 5-FU and NVP-BEZ235 further increased cell apoptosis in a PUMA/Bax dependent manner. Moreover, significantly enhanced anti-tumor effects were observed in combination treatment in vivo. Together, these results demonstrated that the combination treatment of 5-FU and NVP-BEZ235 caused PUMA-dependent tumor suppression both in vitro and in vivo, which may promise a more effective strategy for colon cancer therapy.