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.
Kim and colleagues reported a p53/p21 complex regulates cancer cell invasion and apoptosis by targeting the Bcl-2 family (1). Interaction of overexpressed p53 and p21 proteins in p53-null H1299 cells correlated with decreased cell invasion. No coimmunoprecipitation experiments documenting endogenous p53:p21 protein interaction were performed. There was no consideration of how the p53/p21/Bcl-w complex might relate to p53/MDM2 or p21/cyclin/CDK/PCNA complexes. The authors showed similar findings in IMR-32 cells that carry cytoplasmic wild-type (wt-p53). Although they demonstrated si-p53 or si-p21 affect Bcl-w:Bax interaction, they did not prove endogenous p53 interacts with p21.There was a modest effect of p53 on suppression of invasion (Fig. 1; ref. 1), and this phenotype was suppressed by si-p21. However, there was no effort to determine whether effects on invasion could be explained by altered cell-cycle phase or proliferation state. This is important as tumor cell growth and proliferation is required for invasiveness (2). On the other hand, some evidence suggests growth arrest may be required for invasion (3). Kim and colleagues state that cell invasion is regulated by p53/p21, but little insight is provided beyond cell-cycle and growth regulation. The authors suggest suppression of invasion is mediated by Bax. However, Bax is a direct target of p53, and its effects on apoptosis are well established (dead cells do not invade). The authors do not consider direct regulation of p53 toward Bax and whether this could explain their findings. Endogenous p53 decreases invasion by promoting MDM2:Slug interaction, resulting in Slug ubiquitin–mediated degradation (4). The authors did not experimentally consider these mechanisms.Kim and colleagues show results inconsistent with the paradigm in the p53 field. First, WT-p53 is stabilized following exposure of cells to DNA damage such as ionizing radiation (5). In Fig. 5B of ref. 1, lane 6 shows no increase in nuclear p53 expression following 20 Gy of radiation; rather, p53 remains cytoplasmic. Second, it is well known that overexpression of p53 suppresses cancer cell growth. Figure 6 of ref. 1 shows similar tumor growth of exogenously transfected wt-p53 versus vector control and greater than p53ΔC37 at 0 Gy. These unexpected results should be better documented, including WT-p53 localization by immunofluorescence and WT-p53 functionality in vivo, to validate whether these are cell type–specific phenotypes. Overexpression of p53ΔC37 was not characterized, and the mutant lacks one of two nuclear localization signal. Whether effects are due to failure of p21 interaction or inability of p53 nuclear import to activate transcription of p53 targets was not addressed.p21/CDKN1A is not infrequently mutated in bladder cancer (The Cancer Genome Atlas; ref. 6). We used p21-CRISPR in bladder cancer cells to directly test the hypothesis that p21 mutation might contribute to greater migration or invasiveness of tumor cells. CRISPR knockout of CDKN1A resulted in decreased cellular motility (Fig. 1A). This effect was muted when indels were targeted to the C-terminus of the protein, when a truncated peptide was still detectable and retaining the CDK-interacting domain (Fig. 1B). Importantly, SW780 cells carry WT-p53, and 50% of bladder cancers retain two intact copies of TP53. Thus, p21 ablation or mutation does not increase cell migration that is needed for invasion.p21 appears to protect from cell death through various mechanisms (6), including growth arrest and cytoplasmic effects of p21. The role of p21 in apoptosis following p53 overexpression in H1299 cells in Kim and colleagues should have been better substantiated as a cell type–specific phenotype.For multiple reasons, the evidence supporting the model put forth by Kim and colleagues is lacking in rigor and is not convincing due to alternative explanations. Kim and colleagues reported similar findings that have similar limitations (7, 8). It is important to document endogenous protein:protein interactions and to include critical controls to support novel paradigm-shifting results. It remains unclear whether a physiologic complex of p53/p21/Bcl-w regulates invasion or apoptosis.See the Response, p. 2772No potential conflicts of interest were disclosed.
Abstract 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
To improve the accuracy predictive models of response to neoadjuvante chemotherapy in breast cancer, cDNA microarray technology was used to study tumor transcriptional profile. Gene signatures associated with predicting the response to neoadjuvante chemotherapy are the subject of this review.The data base http://www.ncbi.nlm.nih.gov/pubmed/ search was conducted by using the words "breast cancer" AND "neoadjuvante/primary chemotherapy" AND "gene expression profile/microarray". After excluding the repeats and selecting the publications considered most relevant by the authors to be presented, 279 publications were retrieved.The number of publications regarding this subject has been increasing over the years, reaching over 50 in 2010, including the response to different chemotherapeutic drugs, such as anthracyclines and taxanes either alone or in combination. The first studies are from early last decade and used microarray platforms produced by the investigators. Recent studies have used commercial microarray platforms whose data have been stored in public databases, allowing for the analysis of a higher number of samples. Several transcriptional profiles associated with the complete pathological response were identified. Other authors used the clinical response to treatment as an endpoint, and, in this case, a predictive panel of resistance to the chemotherapeutic regimen at issue was determined. This is also a key issue, as it can contribute to individualize treatment, allowing patients resistant to a certain chemotherapeutic agent to be offered another therapeutic regimen.Identifying patients responsive to chemotherapy is of essential interest and despite major steps have been taken, the issue warrants further studies in view of its complexity.
Abstract Intro. Glioblastoma is the most common and aggressive adult brain cancer, with a 5-year survival of less than 15%. Treatment decisions are guided by imaging, which at early timepoints cannot distinguish tumor pseudoprogression from non-response. Our work seeks to identify circulating biomarkers that can classify response as an alternative or adjunct to imaging. Here we present preliminary data exploring the potential of circulating cytokines and tumor cells collected before, during, and after treatment with the goal to identify potential makers for treatment adaptation. Methods. After consent, patient blood was collected the week before initiating chemoradiotherapy, weekly during treatment, and three weeks after treatment. Serum was tested by multiplex bead-based immunoassay (Legendplex Human Inflammation Panel 1, Biolegend inc.) targeting 13 cytokines and ELISA targeting soluble Human GFAP (Glial fibrillary acidic protein) (Biovendor R&D). The Circulogix filtration system was also used to capture cells of size consistent with circulating tumor cells (CTCs). Initial efforts focused on cells co-staining for GFAP and Olig2 and negative for CD45 (hematopoietic marker). All patient tumor specimens and cell lines were verified for GFAP and Olig2 expression by IHC. We selected 13 patients for the current analysis: 5 with growth due to non-response (NR), 6 stable disease (S) and two pseudo progression (PP). These classifications were evaluated radiologically at the first follow up after chemoradiotherapy (S or no) and based on whether changes observed stabilized or resolved spontaneously within six months or continued to progress (NR or PP). Results. The immunoassays detected approximately 70% of the analytes in our samples. Among those, IL10 and IL33 were higher before treatment on patients later determined to have stable disease (IL10 p=0.09; IL33 p=0.043 KW test; IL10 median S=22.93 ng/mL, NR=13.415ng/mL; IL33 median S= 164.10ng/mL, NR=55.57ng/mL). Interestingly, IL33 is reduced on stable patients during week 5 of treatment (PRE vs. W5 p=0.033; median PRE=164.1ng/mL, W5=57.57ng/mL). GFAP presented a slightly lower level after treatment in stable patients (S vs. NR p=0.014; median S=0.441 ng/ml, NR=0.473 ng/ml). As proof of concept, 50 GBM cells from each of U87 adherent cells, or 0913 and 0821 neurosphere cells (CD45 negative) glioblastoma cell lines were mixed in separate vials with 106 hematopoietic cells (RAJI; CD45 positive) and filtered per the procedure to identify CTCs in patient blood. We separated GBM cells from hematopoietic cells on the filtration system. Conclusion. Our preliminary data suggest that blood obtained during chemoradiotherapy can identify biomarkers associated with response. For example, IL10, IL33, GFAP, and CTCs might be used to classify treatment response at very early timepoints. We continue to recruit additional patients for additional analyses. Citation Format: Paulo Roberto Del Valle, Jonathan B. Bell, Scott M. Welford, Kaylie Kullison, Alessandro Valderrama, Gregory A. Azzam, Jessica J. Meshman, Macarena I. De La Fuente, Eric A. Mellon. Preliminary analysis of circulating biomarkers of glioblastoma response during chemoradiotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2162.
While treatments for colorectal cancer continue to improve, some 50% of patients succumb within 5 years, pointing to the need for additional therapeutic options. We have developed a modified non-replicating adenoviral vector for gene transfer, called AdRGD-PG, which offers improved levels of transduction and transgene expression. Here, we employ the p53-responsive PG promoter to drive expression of p53 or human interferon-β (hIFNβ) in human colorectal cancer cell lines HCT116wt (wtp53), HCT116−/- (p53 deficient) and HT29 (mutant p53). The HCT116 cell lines were both easily killed with p53 gene transfer, while combined p53 and hIFNβ cooperated for the induction of HT29 cell death and emission of immunogenic cell death (ICD) markers. Elevated annexinV staining and caspase 3/7 activity point to cell death by a mechanism consistent with apoptosis. P53 gene transfer alone or in combination with hIFNβ sensitized all cell lines to chemotherapy, permitting the application of low drug doses while still achieving significant loss of viability. While endogenous p53 status was not sufficient to predict response to treatment, combined p53 and hIFNβ provided an additive effect in HT29 cells. We propose that this approach may prove effective for the treatment of colorectal cancer, permitting the use of limited drug doses.
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.
Vitamin 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)(2)D(3) (50-100nM). Our aim was to evaluate the transcriptional effects of 1,25(OH)(2)D(3) in a more physiological model of breast cancer, consisting of fresh tumor slices exposed to 1,25(OH)(2)D(3) at concentrations that can be attained in vivo.Tumor samples from post-menopausal breast cancer patients were sliced and cultured for 24 hours with or without 1,25(OH)(2)D(3) 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)(2)D(3) 0.5nM, using RT-qPCR, western blot or immunocytochemistry.1,25(OH)(2)D(3) 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)(2)D(3) near physiological concentration. Genes up-modulated by both 1,25(OH)(2)D(3) 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)(2)D(3) was confirmed. To evaluate whether the transcripitonal targets of 1,25(OH)(2)D(3) 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)(2)D(3) 0.5nM was detected.In breast cancer specimens a short period of 1,25(OH)(2)D(3) exposure at near physiological concentration modestly activates the hormone transcriptional pathway. Induction of CYP24A1, CA2, DPP4, IL1RL1 expression appears to reflect 1,25(OH)(2)D(3) effects in epithelial as well as stromal cells, however, induction of CD14 expression is likely restricted to the epithelial compartment.
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.
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