Svein Carlsen

Enhanced HER-2/neu-specific antitumor immunity by cotransduction of mouse dendritic cells with two genes encoding HER-2/neu and alpha tumor necrosis factor

The present study uses an in vivo murine tumor model expressing the human HER-2/neu antigen to evaluate the potential vaccine using dendritic cells (DCs) infected with adenovirus AdVHER-2. We first investigated whether infected DCs (DCHER-2) engineered to express HER-2/neu could induce HER-2/neu-specific immune responses. Our data showed that (i) AdVHER2-infected DCHER-2 expressed HER-2/neu by Western blot and flow cytometric analysis, and (ii) vaccination of mice with DCHER-2 induced HER-2/neu-specific cytotoxic T-lymphocyte (CTL) responses, but protected only 25% of vaccinated mice from challenge of 3×105 MCA26/HER-2 tumor cells. Further, to enhance the efficacy of DCHER-2 vaccine, we coinfected DCs with both AdVHER-2 and AdVTNF-α. The infected DCs (DCHER-2/TNF-α) displayed the expression of both HER-2/neu and TNF-α by flow cytometric and ELISA analysis. We next investigated whether DCHER-2/TNF-α could induce stronger HER-2/neu-specific immune responses. We found that DCHER-2/TNF-α displayed up-regulation of immunologically important CD40, CD86, and ICAM-I molecules compared with DCHER-2, indicating that the former ones are more mature forms of DCs. Vaccination of DCHER-2/TNF-α induced stronger allogeneic T-cell proliferation and 36% enhanced HER-2/neu-specific T-cell responses in vitro than DCHER-2 cells. More importantly, it stimulated the significant anti–HER-2/neu immunity in vivo, which protected 8/8 mice from challenge of 3×105 MCA26/HER-2 tumor cells. Therefore, DCs genetically engineered to express both the tumor antigen and cytokines such as TNF-α as an immunoadjuvant are likely to represent a new direction in DC vaccine of cancer.

CREB3L1 is a metastasis suppressor that represses expression of genes regulating metastasis, invasion, and angiogenesis.

ABSTRACT The unfolded protein response (UPR) is activated in response to hypoxia-induced stress such as in the tumor microenvironment. This study examined the role of CREB3L1 (cyclic AMP [cAMP]-responsive element-binding protein 3-like protein 1), a member of the UPR, in breast cancer development and metastasis. Initial experiments identified the loss of CREB3L1 expression in metastatic breast cancer cell lines compared to low-metastasis or nonmetastatic cell lines. When metastatic cells were transfected with CREB3L1, they demonstrated reduced invasion and migration in vitro, as well as a significantly decreased ability to survive under nonadherent or hypoxic conditions. Interestingly, in an in vivo rat mammary tumor model, not only did CREB3L1-expressing cells fail to form metastases compared to CREB3L1 null cells but regression of the primary tumors was seen in 70% of the animals as a result of impaired angiogenesis. Microarray and chromatin immunoprecipitation with microarray technology (ChIP on Chip) analyses identified changes in the expression of many genes involved in cancer development and metastasis, including a decrease in those involved in angiogenesis. These data suggest that CREB3L1 plays an important role in suppressing tumorigenesis and that loss of expression is required for the development of a metastatic phenotype.

Adenovirus-mediated Transgene-engineered Dendritic Cell Vaccine of Cancer

Dendritic cells (DCs) are the most effective antigen presenting cells (APCs) to elicit both primary and secondary T-cell response that is critical for antitumor immunity and elimination of intracellular pathogens. Therefore, DCs pulsed ex vivo with antigens have the potential used as cell-based vaccines against tumors. Viral vectors derived from adenoviruses have been extensively used to pulse DCs ex vivo by delivering genes encoding immunomodulatory molecules and tumor antigens to DCs since these vectors are relatively safe, effective in inducing the maturation of DCs, and can accommodate large expression cassettes encoding antigens. One of the hurdles for gene delivery to DCs by adenovirus (Ad) vectors, however, is low transfection efficiency of DCs due to the paucity of Ad receptor on DCs. To overcome this obstacle, targeted Ad vectors have been made by modifying viral capsid proteins. These targeted Ad vectors not only enhance the gene delivery to DCs, but also allow in vivo gene delivery to DCs, thus avoiding ex vivo manipulation of DCs.

Combined alpha tumor necrosis factor gene therapy and engineered dendritic cell vaccine in combating well-established tumors

Although current immunotherapeutic strategies including adenovirus (AdV)‐mediated gene therapy and dendritic cell (DC) vaccine can all stimulate antitumor cytotoxic T lymphocyte (CLT) responses, their therapeutic efficiency has still been limited to generation of prophylactic antitumor immunity against re‐challenge with the parental tumor cells or growth inhibition of small tumors in vivo. However, it is the well‐established tumors in animal models that mimic clinical patients with existing tumor burdens. Alpha tumor necrosis factor (TNF‐α) is a multifunctional and immunoregulatory cytokine that induces antitumor activity and activates immune cells such as DCs and T cells. We hypothesized that a combined immunotherapy including gene therapy and DC vaccine would have some advantages over each modality administered as a monotherapy.

The identification of a neutral glycosphingolipid antigenic marker for metastatic cells in the R3230AC rat mammary adenocarcinoma.

The process of metastasis is a complex process involving numerous steps, and it is thought that cells able to complete all of these steps and form metastatic foci form a unique subpopulation of cells within the tumor. To study this metastatic subpopulation, a marker for the metastatic cells is required. We have previously described the enrichment of soybean agglutinin binding cells in tumor populations enriched for lymphatic metastasis [1]. In this study we provide evidence that the cell-surface structure binding soybean agglutinin is a neutral glycosphingolipid. Using monoclonal antibodies generated against this glycolipid, highly metastatic tumor populations were depleted of cells containing this glycolipid. These depleted cell populations were found to be equally tumorigenic to that of the untreated population but were much less metastatic. These results suggest that this glycolipid may be a useful marker for metastatic cells.

Epigenetic silencing of CREB3L1 by DNA methylation is associated with high-grade metastatic breast cancers with poor prognosis and is prevalent in triple negative breast cancers

BackgroundCREB3L1 (cAMP-responsive element-binding protein 3-like protein 1), a member of the unfolded protein response, has recently been identified as a metastasis suppressor in both breast and bladder cancer.MethodsQuantitative real time PCR (qPCR) and immunoblotting were used to determine the impact of histone deacetylation and DNA methylation inhibitors on CREB3L1 expression in breast cancer cell lines. Breast cancer cell lines and tumor samples were analyzed similarly, and CREB3L1 gene methylation was determined using sodium bisulfite conversion and DNA sequencing. Immunohistochemistry was used to determine nuclear versus cytoplasmic CREB3L1 protein. Large breast cancer database analyses were carried out to examine relationships between CREB3L1 gene methylation and mRNA expression in addition to CREB3L1 mRNA expression and prognosis.ResultsThis study demonstrates that the low CREB3L1 expression previously seen in highly metastatic breast cancer cell lines is caused in part by epigenetic silencing. Treatment of several highly metastatic breast cancer cell lines that had low CREB3L1 expression with DNA methyltransferase and histone deacetylase inhibitors induced expression of CREB3L1, both mRNA and protein. In human breast tumors, CREB3L1 mRNA expression was upregulated in low and medium-grade tumors, most frequently of the luminal and HER2 amplified subtypes. In contrast, CREB3L1 expression was repressed in high-grade tumors, and its loss was most frequently associated with triple negative breast cancers (TNBCs). Importantly, bioinformatics analyses of tumor databases support these findings, with methylation of the CREB3L1 gene associated with TNBCs, and strongly negatively correlated with CREB3L1 mRNA expression. Decreased CREB3L1 mRNA expression was associated with increased tumor grade and reduced progression-free survival. An immunohistochemistry analysis revealed that low-grade breast tumors frequently had nuclear CREB3L1 protein, in contrast to the high-grade breast tumors in which CREB3L1 was cytoplasmic, suggesting that differential localization may also regulate CREB3L1 effectiveness in metastasis suppression.ConclusionsOur data further strengthens the role for CREB3L1 as a metastasis suppressor in breast cancer and demonstrates that epigenetic silencing is a major regulator of the loss of CREB3L1 expression. We also highlight that CREB3L1 expression is frequently altered in many cancer types suggesting that it could have a broader role in cancer progression and metastasis.

Adenoviral transfer of xenogeneic MHC class I gene results in loss of tumorigenicity and inhibition of tumor growth

The immune system confers protection against a variety of pathogens and contributes to the destruction of neoplastic cells. Foreign major histocompatibility complex (MHC) protein serves as a potent stimulus to the immune system. In this report, a mouse H-2Kb gene was introduced into two poorly immunogenic tumor cell lines, a mouse colonic carcinoma cell line, MCA-26 (H-2Kd), and a rat mammalian carcinoma cell line, LN-4, in an effort to stimulate tumor rejection. Our results showed that the expression of xenogeneic MHC class I antigen completely abolished the LN-4 tumorigenicity in rats, whereas the expression of allogeneic MHC class I antigen only partially reduced the MCA-26 tumorigenicity in mice. Rats with tumor regression of LN-4/H-2Kb developed a T helper type 1-dominant response, whereas rats with LN-4 tumor growth developed a T helper type 2-dominant response. The immunized rats that experienced LN-4/H-2Kb tumor regression further developed protective immunity against a subsequent challenge of LN-4 cells. This protective immunity was mediated by the LN-4 tumor-specific cellular immune response against both the transduced and the parental LN-4 cells. Recombinant adenoviral vectors are highly efficient at in vitro and in vivo gene delivery. The LN4 cells transfected with the recombinant adenovirus AdV-H-2Kb in vitro expressed the cell surface H-2Kb molecule by fluorescence-activated cell sorter analysis. Adenovirus-mediated H-2Kb gene transfer in vivo can further significantly inhibit pre-established LN-4 tumors. Those rats with complete tumor regression further developed protective immunity against the subsequent challenge of a parental LN-4 tumor. Therefore, our study indicates that the adenovirus-mediated transfer of xenogeneic MHC class I gene may be an effective alternative to the current protocol of cancer gene therapy in which the allogeneic MHC class I gene is used.

Abstract 1450: Loss of the UPR member CREB3L1 is vital for the survival of breast cancer cells in the tumor microenvironment and development of the metastatic phenotype

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL The unfolded protein response (UPR) is required for proper protein folding in the endoplasmic reticulum under conditions of stress such as those encountered in the tumor microenvironment. Recently, a number of studies have suggested a role for the unfolded protein response in the development of cancer, more specifically in regulating the balance between cell death, dormancy and the growth of cancer cells in the tumor microenvironment. This study examined the role of the transcription factor CREB3L1, a member of the UPR machinery, in breast cancer development and metastasis. Northern blotting analysis identified a loss of CREB3L1 expression in metastatic breast cancer cell lines compared to low- or non-metastatic cell lines, which was subsequently shown, through bisulphate sequencing, to be the result of enhanced promoter methylation. When metastatic cells were transfected with CREB3L1 they demonstrated a significant decrease in survival in hypoxic and non-adherent growth conditions, and reduced in vitro invasion and migration in chemotaxis assays. Interestingly, in an in vivo rat mammary tumor model where rats were injected with cells in the hind footpad, the CREB3L1 expressing rat cells not only failed to form metastases but tumor regression was seen in 70% of the animals. In contrast, metastases were seen in the popliteal lymph node of 90% of rats injected with wild-type and empty vector-transfected cells. Microarray and ChIP on Chip analyses identified changes in the expression of many genes involved in cancer development and metastasis, including a decrease in those involved in angiogenesis when CREB3L1 was expressed. We also observed a significant decrease in the number of migrating endothelial cells in scratch and chemotaxis assays in media conditioned by CREB3L1 expressing cells, compared to conditioned media from untransfected cells. To determine if these findings translated to human breast cancer, real-time PCR analysis of CREB3L1 expression in human breast cancer tissues of differing grade (n=30) was performed. Expression of CREB3L1 was elevated in low and medium grade tissue but substantially reduced in high grade tissue, compared to normal. The high grade breast cancer tissues demonstrated a 6.6 fold decrease in the expression of CREB3L1 compared to the low grade tissues (p<0.001), with about a 2 fold decrease compared to normal tissue. In conclusion, CREB3L1 expression is elevated in low and medium grade breast cancer tissue, perhaps as the cancer cells respond to various stresses. However, loss of CREB3L1 expression is required for the long-term survival of breast cancer cells in the tumor microenvironment and the progression to a highly metastatic phenotype. CREB3L1 expression may be a useful marker in predicting tumor grade and patient prognosis, in addition to providing a therapeutic target. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1450. doi:10.1158/1538-7445.AM2011-1450