Paulo Roberto Del Valle

Influence of the interaction between nodal fibroblast and breast cancer cells on gene expression.

Our aim was to evaluate the interaction between breast cancer cells and nodal fibroblasts, by means of their gene expression profile. Fibroblast primary cultures were established from negative and positive lymph nodes from breast cancer patients and a similar gene expression pattern was identified, following cell culture. Fibroblasts and breast cancer cells (MDA-MB231, MDA-MB435, and MCF7) were cultured alone or co-cultured separated by a porous membrane (which allows passage of soluble factors) for comparison. Each breast cancer lineage exerted a particular effect on fibroblasts viability and transcriptional profile. However, fibroblasts from positive and negative nodes had a parallel transcriptional behavior when co-cultured with a specific breast cancer cell line. The effects of nodal fibroblasts on breast cancer cells were also investigated. MDA MB-231 cells viability and migration were enhanced by the presence of fibroblasts and accordingly, MDA-MB435 and MCF7 cells viability followed a similar pattern. MDA-MB231 gene expression profile, as evaluated by cDNA microarray, was influenced by the fibroblasts presence, and HNMT, COMT, FN3K, and SOD2 were confirmed downregulated in MDA-MB231 co-cultured cells with fibroblasts from both negative and positive nodes, in a new series of RT-PCR assays. In summary, transcriptional changes induced in breast cancer cells by fibroblasts from positive as well as negative nodes are very much alike in a specific lineage. However, fibroblasts effects are distinct in each one of the breast cancer lineages, suggesting that the inter-relationships between stromal and malignant cells are dependent on the intrinsic subtype of the tumor.

Transcriptional effects of 1,25 dihydroxyvitamin D 3 physiological and supra-physiological concentrations in breast cancer organotypic culture

BackgroundVitamin D transcriptional effects were linked to tumor growth control, however, the hormone targets were determined in cell cultures exposed to supra physiological concentrations of 1,25(OH)2D3 (50-100nM). Our aim was to evaluate the transcriptional effects of 1,25(OH)2D3 in a more physiological model of breast cancer, consisting of fresh tumor slices exposed to 1,25(OH)2D3 at concentrations that can be attained in vivo.MethodsTumor samples from post-menopausal breast cancer patients were sliced and cultured for 24 hours with or without 1,25(OH)2D3 0.5nM or 100nM. Gene expression was analyzed by microarray (SAM paired analysis, FDR≤0.1) or RT-qPCR (p≤0.05, Friedman/Wilcoxon test). Expression of candidate genes was then evaluated in mammary epithelial/breast cancer lineages and cancer associated fibroblasts (CAFs), exposed or not to 1,25(OH)2D3 0.5nM, using RT-qPCR, western blot or immunocytochemistry.Results1,25(OH)2D3 0.5nM or 100nM effects were evaluated in five tumor samples by microarray and seven and 136 genes, respectively, were up-regulated. There was an enrichment of genes containing transcription factor binding sites for the vitamin D receptor (VDR) in samples exposed to 1,25(OH)2D3 near physiological concentration. Genes up-modulated by both 1,25(OH)2D3 concentrations were CYP24A1, DPP4, CA2, EFTUD1, TKTL1, KCNK3. Expression of candidate genes was subsequently evaluated in another 16 samples by RT-qPCR and up-regulation of CYP24A1, DPP4 and CA2 by 1,25(OH)2D3 was confirmed. To evaluate whether the transcripitonal targets of 1,25(OH)2D3 0.5nM were restricted to the epithelial or stromal compartments, gene expression was examined in HB4A, C5.4, SKBR3, MDA-MB231, MCF-7 lineages and CAFs, using RT-qPCR. In epithelial cells, there was a clear induction of CYP24A1, CA2, CD14 and IL1RL1. In fibroblasts, in addition to CYP24A1 induction, there was a trend towards up-regulation of CA2, IL1RL1, and DPP4. A higher protein expression of CD14 in epithelial cells and CA2 and DPP4 in CAFs exposed to 1,25(OH)2D3 0.5nM was detected.ConclusionsIn breast cancer specimens a short period of 1,25(OH)2D3 exposure at near physiological concentration modestly activates the hormone transcriptional pathway. Induction of CYP24A1, CA2, DPP4, IL1RL1 expression appears to reflect 1,25(OH)2D3 effects in epithelial as well as stromal cells, however, induction of CD14 expression is likely restricted to the epithelial compartment.

Transcriptional profile of fibroblasts obtained from the primary site, lymph node and bone marrow of breast cancer patients

Cancer-associated fibroblasts (CAF) influence tumor development at primary as well as in metastatic sites, but there have been no direct comparisons of the transcriptional profiles of stromal cells from different tumor sites. In this study, we used customized cDNA microarrays to compare the gene expression profile of stromal cells from primary tumor (CAF, n = 4), lymph node metastasis (N+, n = 3) and bone marrow (BM, n = 4) obtained from breast cancer patients. Biological validation was done in another 16 samples by RT-qPCR. Differences between CAF vs N+, CAF vs BM and N+ vs BM were represented by 20, 235 and 245 genes, respectively (SAM test, FDR < 0.01). Functional analysis revealed that genes related to development and morphogenesis were overrepresented. In a biological validation set, NOTCH2 was confirmed to be more expressed in N+ (vs CAF) and ADCY2, HECTD1, HNMT, LOX, MACF1, SLC1A3 and USP16 more expressed in BM (vs CAF). Only small differences were observed in the transcriptional profiles of fibroblasts from the primary tumor and lymph node of breast cancer patients, whereas greater differences were observed between bone marrow stromal cells and the other two sites. These differences may reflect the activities of distinct differentiation programs.

Reestablishment of p53/Arf and interferon- β pathways mediated by a novel adenoviral vector potentiates antiviral response and immunogenic cell death

Late stage melanoma continues to be quite difficult to treat and new therapeutic approaches are needed. Since these tumors often retain wild-type p53 and have a strong immunogenic potential, we developed a gene transfer approach which targets these characteristics. Previously, we have shown that combined gene transfer of p19Arf and interferon-beta (IFNβ) results in higher levels of cell death and superior immune-mediated antitumor protection. However, these experiments were performed using B16 cells (p53wt) with forced expression of the adenovirus receptor and also the mechanism of death was largely unexplored. Here we take advantage of a novel adenoviral vector (AdRGD-PG), presenting an RGD-modified fiber as well as a p53-responsive promoter, in order to investigate further potential benefits and cell death mechanisms involved with the combined transfer of the p19Arf and IFNβ genes to the parental B16 cell line. Simultaneous p19Arf and IFNβ gene transfer is more effective for the induction of cell death than single gene treatment and we revealed that p19Arf can sensitize cells to the bystander effect mediated by secreted IFNβ. Strikingly, the levels of cell death induced upon activating the p53/p19Arf and interferon pathways were higher in the presence of the AdRGD-PG vectors as compared to approaches using pharmacological mimetics and this was accompanied by the upregulation of antiviral response genes. Only combined gene transfer conferred immunogenic cell death revealed by the detection of key markers both in vitro and in vivo. Finally, whole-genome transcriptome analysis revealed unique expression profiles depending on gene function, including immune activation, response to virus and p53 signaling. In this way, cooperation of p19Arf and IFNβ activates the p53 pathway in the presence of an antiviral response elicited by IFNβ, culminating in immunogenic cell death.

Intratumoral Immunization by p19Arf and Interferon-β Gene Transfer in a Heterotopic Mouse Model of Lung Carcinoma

Therapeutic strategies that act by eliciting and enhancing antitumor immunity have been clinically validated as an effective treatment modality but may benefit from the induction of both cell death and immune activation as primary stimuli. Using our AdRGD-PG adenovector platform, we show here for the first time that in situ gene transfer of p19Arf and interferon-β (IFNβ) in the LLC1 mouse model of lung carcinoma acts as an immunotherapy. Although p19Arf is sufficient to induce cell death, only its pairing with IFNβ significantly induced markers of immunogenic cell death. In situ gene therapy with IFNβ, either alone or in combination with p19Arf, could retard tumor progression, but only the combined treatment was associated with a protective immune response. Specifically in the case of combined intratumoral gene transfer, we identified 167 differentially expressed genes when using microarray to evaluate tumors that were treated in vivo and confirmed the activation of CCL3, CXCL3, IL1α, IL1β, CD274, and OSM, involved in immune response and chemotaxis. Histologic evaluation revealed significant tumor infiltration by neutrophils, whereas functional depletion of granulocytes ablated the antitumor effect of our approach. The association of in situ gene therapy with cisplatin resulted in synergistic elimination of tumor progression. In all, in situ gene transfer with p19Arf and IFNβ acts as an immunotherapy involving recruitment of neutrophils, a desirable but previously untested outcome, and this approach may be allied with chemotherapy, thus providing significant antitumor activity and warranting further development for the treatment of lung carcinoma.

Abstract 1236: Combined transfer of p19Arf and interferon-beta genes to mouse melanoma cells causes LC3B- and caspase-3-independent cell death and alters the expression of critical genes

Tumors cells are able to resist cell death and evade the immune system. This resistance can be caused by alterations in Trp53 itself or p19Arf loss and the reduced anti-tumor immune response may be caused by loss of interferon-β (IFN), an important immune-stimulatory cytokine. Here, we investigate the impact of p19Arf and IFN gene transfer in melanoma cells with regards to the expression profile of genes responsible for the induction of cell death. Recombinant adenoviral vectors with a p53-responsive promoter (PG) driving expression of p19Arf or IFN were applied alone or in combination in B16F10 cells in vitro (mouse melanoma, p53 wt). Cell death was evaluated by annexin/PI staining and MTT assays. LC3B expression was verified by western blot analysis of cells extracts. Caspase-3 activity was evaluated using a lentiviral vector which constitutively expressing luciferase protein linked to an ubiquitin site that can be cleaved by caspase-3. RNA expression profile was evaluated by microarray analysis of treated samples and validation of gene expression was performed by real-time PCR. We verified that combined gene transfer of p19Arf and IFN resulted in 74% hypodiploid cells whereas single gene transfer yielded only half of this number, as shown by cell cycle and MTT analysis. Although there was a high rate of cell death, only 25% of cells were stained for annexin/PI and activation of caspase-3 was reduced in cells treated with p19Arf and IFN combination compared to treatment of p19Arf alone. Also, there was no increase in LC3B expression when cells were treated with the p19Arf and IFN combination. These observations suggest that B16F10 cells are dying by other mechanisms rather than apoptosis or autophagy. Transduced cells were analyzed for gene expression profile and critical genes were selected for validation. Combination of p19Arf and IFN transduction in B16F10 cells revealed a synergistic effect modifying exclusively the expression of 1054 genes involved in cell death, immune system activation and cell cycle arrest. There was an increase of 4-20 fold in expression of MDM2, p53, p21Waf1, PUMA, MDM4, FOXO1, NR3C1, PHLDA3, RANBP9 and WDR46, and also an increase of 400-fold in p73 expression in cells treated with p19Arf and IFN combination. Conclusions: The use of p53-responsive vectors to express p19Arf and IFN promotes complementation of the p53/Arf and IFN pathways, resulting in a synergistic gene regulation of many essential functions in cancer cells, destabilizing cell cycle and cell organization in general, while promoting expression of genes related to cell death. As p73 is a transcription factor of caspase-1 expression, we hypothesize that this could be the major participant involved in death of these cells. Financial support: 2011/10656-5, 2011/50911-4 and 2013/25167-5 (FAPESP). Citation Format: Aline H. Ribeiro, Paulo R. Del Valle, Ruan F.V. Medrano, Daniel G. Ferrari, Daniela B. Zanatta, Bryan E. Strauss. Combined transfer of p19Arf and interferon-beta genes to mouse melanoma cells causes LC3B- and caspase-3-independent cell death and alters the expression of critical genes. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1236. doi:10.1158/1538-7445.AM2015-1236

Abstract 3539: Modifications of adenoviral structure and genome improves transduction efficiency and transgene expression

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Innovative strategies are required for cancer treatment, especially evident given that many patients relapse or do not respond to current therapeutics. Adenoviral vectors are powerful tools for gene transfer and our laboratory employs a vector where the fiber was modified to present the RGD peptide, considerably improving cell transduction. We developed a system that controls transgene expression by the PG promoter, which is responsive to p53 transcriptional activity. We utilize this system to deliver the human p53 and interferon beta cDNAs. The present study evaluated the transduction efficiency, the dependence of transgene expression on p53 status in human colorectal cancer cell lines HCT116 (p53 wt), HCT116p53-/- and HT29 (p53 mut) and the influence of the transgenes on cell viability. The vectors efficiently transduced all of the tested cell lines. Even at MOIs as low as 10, it was possible to see 42% of cells HCT116 positive for GFP using the AdRGDPGeGFP vector. Higher MOIs (50 and 100) did not present major differences in transduction efficiency (approximately 85%), but the GFP expression intensity increased approximately 14 fold. Cells were treated with doxorubicin (1μM) in order to stimulate p53 activity and when a MOI of 10 was used, 54% of cells were GFP positive with a 16 fold increase in the expression level, yet expression intensity rose 16 fold (MOI 50) and two fold (MOI 100), when compared with non-stimulated with doxorubicin. Since p53 expression is absent in the HCT116p53-/- cell line, we would not expect to observe GFP expression when using the PG promoter. However, very low level GFP activity was detected and was slightly induced upon treatment with doxorubicin, suggesting a p53-independent mechanism for transgene expression. However, restoring p53 in this cell line led to higher expression levels of GFP. We observed an important benefit mediated by our vectors (MOI of 25) for the reduction of cell viability mediated by doxorubicin, cisplatin and 5-fluorouracil (5-FU). The effect of doxorubicin was especially interesting, even at a low dose (0.625μM) the co-transduction with both the p53 and IFN-β vectors conferred a benefit higher than that seen with isolated transductions. The dose of 10μM cisplatin and 6.25μM 5-FU also provided a major benefit in reducing cell viability upon co-transduction. Both HCT116 and HCT116p53-/-responded in a similar way to gene transfer combined with chemotherapeutics, but HT29 was more sensitive to IFN-β when associated with doxorubicin (0.625μM) and cisplatin (doses range 10 - 1.25 μM). Thus, our system reveals an opportunity to improve transduction efficiency and transgene expression and, as a consequence, increase sensitivity to genotoxic stress. Moreover, the co-transfection of p53 and IFN-β may improve the efficacy of chemotherapeutics in colorectal cancer. Financial support: Sao Paulo Research Foundation (FAPESP) 2013/16074-3; 2013/25167-5. Citation Format: Paulo Roberto Del Valle, Daniela B. Zanatta, Bryan E. Strauss. Modifications of adenoviral structure and genome improves transduction efficiency and transgene expression. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3539. doi:10.1158/1538-7445.AM2015-3539

Gene expression profile of stromal cells from potential metastatic sites in breast cancer patients.

Methods Fibroblasts primary culture was established from 11 breast cancer patients. Expression analysis was evaluated in PT (n=4), N+ (n=3) and BM (n=4) through a customized cDNA microarray platform (4,800 ORESTES) analyzed by SAM (TMEV; FDR 0%) and functional analysis was performed using DAVID. Technical validation was performed in 6 samples and biological validation was performed in fibroblasts obtained from others 25 patients as evaluated by RT-qPCR.