Cristina Peña

The Wnt antagonist DICKKOPF-1 gene is a downstream target of β -catenin/TCF and is downregulated in human colon cancer

Wnt glycoproteins regulate homeostasis and development by binding to membrane Frizzled-LRP5/6 receptor complexes. Wnt signaling includes a canonical pathway involving cytosolic β-catenin stabilization, nuclear translocation and gene regulation, acting as a co-activator of T-cell factor (TCF) proteins, and noncanonical pathways that activate Rho, Rac, JNK and PKC, or modulate Ca2+ levels. DICKKOPF-1 (DKK-1) encodes a secreted Wnt antagonist that binds to LRP5/6 and induces its endocytosis, leading to inhibition of the canonical pathway. We show that activation of canonical signaling by Wnt1 or ectopic expression of active β-catenin, TCF4 or LRP6 mutants induces transcription of the human DKK-1 gene. Multiple β-catenin/TCF4 sites in the DKK-1 gene promoter contribute to this activation. In contrast, Wnt5a, which signals through noncanonical pathways, does not activate DKK-1. Northern and Western blot studies show that activation of the Wnt/β-catenin pathway by treatment with lithium or Wnt3a-conditioned medium, or by stable expression of either Wnt1 or β-catenin, increases DKK-1 RNA and protein, thus initiating a negative feedback loop. However, we found that DKK-1 expression decreases in human colon tumors, which suggests that DKK-1 acts as a tumor suppressor gene in this neoplasia. Our data indicate that the Wnt/β-catenin pathway is downregulated by the induction of DKK-1 expression, a mechanism that is lost in colon cancer.

The transcription factor SNAIL represses vitamin D receptor expression and responsiveness in human colon cancer

Several non-hypercalcemic analogs of 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) show antitumor activity in a subset of cancer patients. High vitamin D receptor (VDR) expression, which is associated with good prognosis but is lost during tumor progression. We show that the SNAIL transcription factor represses VDR gene expression in human colon cancer cells and blocks the antitumor action of EB1089, a 1,25(OH)2D3 analog, in xenografted mice. In human colon cancers, elevated SNAIL expression correlates with downregulation of VDR.

Promoter methylation of the PTEN gene is a common molecular change in breast cancer.

About 25–50% of women with Cowden disease, a syndrome associated with germ‐line mutations of the PTEN gene (at 10q23), develop breast cancer (BC), but PTEN mutations have been found in only 5% of sporadic BCs. However, 29–48% of BCs display loss of heterozygosity in 10q23, and about 40% of BCs show a decrease or absence of PTEN protein levels at the time of diagnosis. Promoter hypermethylation has been identified as an alternative mechanism of tumor‐suppressor gene inactivation, but its importance in PTEN silencing in sporadic BC is unknown. We investigated PTEN promoter hypermethylation in 90 sporadic BCs and its correlations with 11 molecular and pathologic parameters, including mRNA levels of PTEN. The study, a methylation‐specific PCR assay, was carried out with methylated specific primers designed in a region with scarce homology with the psiPTEN pseudogene. Expression was analyzed by real‐time PCR. We found that the PTEN promoter was hypermethylated in 43 BCs (48%). PTEN hypermethylation was associated with ERBB2 overexpression, larger size, and higher histologic grade (P = 0.012, 0.03, and 0.009, respectively). We concluded that PTEN promoter hypermethylation is a common event in sporadic BC, correlating with other well‐established prognostic factors of this malignancy. Additionally, PTEN mRNA expression was lower in tumors with aberrant methylation.

Analysis of Exosome Release and Its Prognostic Value in Human Colorectal Cancer

A significant proportion of extracellular nucleic acids in plasma circulate highly protected in tumor‐specific exosomes, but it is unclear how the release of exosomes is modulated in carcinogenesis. We quantified by cytometry exosomes in plasma of 91 colorectal cancer patients to evaluate their potential as a tumor indicator and their repercussions on diagnosis and prognosis. We examined the involvement of TSAP6, a TP53‐regulated gene involved in the regulation of vesicular secretion, in levels of circulating exosomes in plasma of colorectal patients and in HCT116 TP53‐(wild‐type and null) human colorectal cancer cell lines. The fraction of exosomes in cancer patients was statistically higher than in healthy controls (mean rank = 53.93 vs. 24.35). High levels of exosomes in plasma of patients correlated with high levels of carcino‐embryonic antigen (P = 0.029) and with poorly differentiated tumors (P = 0.039) and tended to have shorter overall survival than patients with low levels (P = 0.056). Release of exosomes did not correlate with TSAP6 expression; and regulation of TSAP6 by TP53 was not shown either in tumor samples or in HCT116 cell lines. Although it was not suggested that the TP53/TSAP6 pathway regulates the release of exosomes into the plasma of colorectal cancer patients, the level of circulating exosomes may be used as a tumor indicator, because it correlates with poor prognosis parameters and shorter survival.

DICKKOPF-4 is induced by TCF/β-catenin and upregulated in human colon cancer, promotes tumour cell invasion and angiogenesis and is repressed by 1α,25-dihydroxyvitamin D3

Aberrant activation of the Wnt/β-catenin signaling pathway is a hallmark of colon cancer. We show that the Wnt antagonist DICKKOPF-4 (DKK-4) gene is repressed by 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) in human colon cancer cells. This effect correlated with the inhibition of the DKK-4 promoter. Chromatin immunoprecipitation assays revealed that 1,25(OH)2D3 induces early and transient binding of the vitamin D receptor (VDR) and the SMRT corepressor to the region adjacent to the transcription start site of DKK-4. Additionally, we demonstrate that the DKK-4 gene is a new downstream target of TCF/β-catenin. Remarkably, expression of DKK-4 messenger RNA (mRNA) was not detected in a series of 29 human normal (N) colon biopsies but expression was upregulated in all the matched tumour (T) tissues. An inverse correlation existed between the expression of DKK-4 and VDR RNA in the Ts. Ectopic DKK-4 expression increased the migration and invasion properties of colon cancer cells and conditioned media (CM) from DKK-4-expressing cells enhanced the capacity to migrate and form capillary-like tubules of human primary microvascular endothelial cells. In conclusion, DKK-4 is upregulated in colon cancer and is associated with the acquisition of malignant properties by neoplastic cells. DKK-4 downregulation is a novel effect of 1,25(OH)2D3 that may contribute to its anticancer action.

The expression levels of the transcriptional regulators p300 and CtBP modulate the correlations between SNAIL, ZEB1, E‐cadherin and vitamin D receptor in human colon carcinomas

ZEB1 and SNAIL repress CDH1 and induce epithelial–mesenchymal transition (EMT). However, SNAIL and ZEB1 also activate or regulate other target genes in different ways. For instance, vitamin D receptor (VDR), which activates CDH1 expression upon ligand binding, is repressed by SNAIL but induced by ZEB1. We examined whether the biological activity of SNAIL and ZEB1 in colon cancer is regulated by interacting cofactors. The mRNA expression levels of SNAIL and ZEB1, and of transcriptional regulators p300 and CtBP, were measured by RT‐PCR in tumor and normal tissue from 101 colon carcinoma patients. Overexpression of SNAIL was associated with down‐regulation of CDH1 and VDR (p = 0.004 and p < 0.001). CDH1 correlated with VDR (r = 0.49; p < 0.001). ZEB1 expression also correlated with VDR (r = 0.23; p = 0.019). However, when CtBP was strongly expressed, ZEB1 was inversely correlated with CDH1 (r = −0.39; p = 0.053). Furthermore, when there were elevated p300 expression levels, the correlation between expression of ZEB1 and VDR was stronger (r = 0.38; p = 0.070). Association between SNAIL expression and down‐regulation of CDH1 and VDR was lost in tumors in which p300 and CtBP were strongly expressed. These results indicate that the levels of expression of CtBP and p300 are critical for the action of SNAIL and ZEB1, which have a pivotal role in EMT, and show the importance of CtBP and p300 for tumor progression.

ΔTAp73 Upregulation Correlates With Poor Prognosis in Human Tumors: Putative In Vivo Network Involving p73 Isoforms, p53, and E2F-1

PURPOSE Although full-length TAp73 variants largely mimic p53 suppressor activities, the transactivation-deficient transcripts DeltaTAp73 exert an oncogenic effect by inactivating p53 and TAp73 suppressor properties. Additionally, DeltaTAp73 may cooperate with oncogenic RAS to induce cell transformation, confer drug resistance, and induce the phosphorylation of phosphorylated Rb. Here, we study the expression of TAp73 and DeltaTAp73 variants and assess possible associations with E2F-1, p53 and K-ras status. We address the possible clinical relevance of alterations in these genes. PATIENTS AND METHODS We determine in 113 colon and 60 breast cancer patients (a) the expression levels of TAp73, DeltaTAp73 (DeltaEx2p73, DeltaEx2/3p73, and DeltaNp73), and E2F-1 transcripts by quantitative real-time reverse transcriptase polymerase chain reaction (PCR); (b) mutations in the first exon of K-ras by PCR-single-stranded confirmational polymorphism; and (c) p53 status by immunohistochemistry. Tumor characteristics were examined in each patient. RESULTS Both suppressor and oncogenic isoforms of TP73 were significantly coupregulated in tumor tissues. Associations were observed between (a) p53 wild type status and upregulation of some TP73 variants; (b) overexpression of E2F-1 and some TP73 forms; and (c) upregulation of DeltaTAp73 variants and advanced pathologic stage, lymph node metastasis, vascular invasion, presence of polyps, and tumor localization. CONCLUSION Overexpression of TP73 variants in tumor tissues indicates that they may be involved in colon and breast carcinogenesis. The association between upregulation of DeltaTAp73 isoforms and poor prognosis features, specifically advanced tumor stage, suggests that they may be of practical clinical prognostic value. Interestingly, the in vivo associations identified here may indicate a functional network involving p73 variants, p53, and E2F-1.

Proteome Profiling of Cancer-Associated Fibroblasts Identifies Novel Proinflammatory Signatures and Prognostic Markers for Colorectal Cancer

Purpose: Cancer-associated fibroblasts (CAF) are essential components of the stroma that play a critical role in cancer progression. This study aimed to identify novel CAFs markers that might contribute to the invasion and the prognosis of colorectal cancer. Experimental Design: The azoxymethane/dextran sodium sulfate mouse model of sporadic colon cancer represents an adequate source for the isolation of CAFs and normal fibroblasts. By using the explants technique, we purified CAFs and normal fibroblasts from colon tissues. Whole-cell extracts and supernatants were subjected to in-depth quantitative proteomic analysis by tandem mass spectrometry. Further validations of upregulated proteins in CAFs were carried out by chemokine microarray and immunohistochemical analyses of mouse and human tissues. Results: Using a fold-change of 1.4 or more, we found 132 and 125 differentially expressed proteins in whole-cell extracts and supernatants, respectively. We found CAFs-associated proinflammatory and desmoplastic signatures. The proinflammatory signature was composed of several cytokines. Among them, CCL2 and CCL8 caused an increase in migration and invasion of colorectal cancer KM12 cells. The desmoplastic signature was composed of 30 secreted proteins. In mouse and human samples, expression of LTBP2, CDH11, OLFML3, and, particularly, FSTL1 was significantly increased in the tumoral stroma, without significant expression in the cancer epithelial cells. The combination of CALU and CDH11 stromal expression showed a significant association with disease-free survival and poor prognosis. Conclusion: We have identified LTBP2, CDH11, OLFML3, and FSTL1 as selective biomarkers of cancer stroma, and CALU and CDH11 as candidate stromal biomarkers of prognostic significance in colon cancer. Clin Cancer Res; 19(21); 6006–19. ©2013 AACR.

Cancer-associated fibroblast and M2 macrophage markers together predict outcome in colorectal cancer patients.

Tumor epithelial cells within a tumor coexist with a complex microenvironment in which a variety of interactions between its various components determine the behavior of the primary tumors. Cancer‐associated fibroblasts (CAF) and M2 macrophages, characterized by high expression of different markers, including α‐SMA, FSP1 and FAP, or CD163 and DCSIGN, respectively, are involved in the malignancy of different tumors. In the present study, expression of the above markers in CAF and M2 macrophages was analyzed using RT‐PCR and immunohistochemistry in the normal mucosa and tumor tissue from a cohort of 289 colorectal cancer patients. Expression of CAF and M2 markers is associated with the clinical outcome of colorectal cancer patients. Moreover, the combination of CAF and M2 markers identifies three groups of patients with clear differences in the progression of the disease. This combined variable could be a decisive factor in the survival of advanced‐stage patients. Taken together, these analyses demonstrate the prognostic involvement of interrelationships between DCSIGN, CD163, α‐SMA, FSP1 and FAP markers in the survival of colon cancer patients.

Extracellular plasma RNA from colon cancer patients is confined in a vesicle-like structure and is mRNA-enriched

Little is yet known about the origin and protective mechanism of free nucleic acids in plasma. We investigated the possibility of these free nucleic acids being particle associated. Plasma samples from colon cancer patients and cell culture media were subjected to various antibody incubations, ultracentrifugation, and RNA extraction protocols for total RNA, epithelial RNA, and mRNA. Flow cytometry using a Ber-EP4 antibody and confocal laser microscopy after staining with propidium iodide were also performed. mRNA levels of the LISCH7 and SDHA genes were determined in cells and in culture media. Ber-EP4 antibody and polystyrene beads coated with oligo dT sequences were employed. We observed that, after incubation, total RNA and mRNA were always detected after membrane digestion, and that epithelial RNA was detected before this procedure. In ultracentrifugation, mRNA was caught in the supernatant only if a former lysis mediated or in the pellet if there was no previous digestion. Flow cytometry determinations showed that antibody-coated microbeads keep acellular structures bearing epithelial antigens apart. Confocal laser microscopy made 1- to 2-microm-diameter particles perceptible in the vicinity of magnetic polystyrene beads. Relevant differences were observed between mRNA of cells and culture media, as there was a considerable difference in LISCH7 mRNA levels between HT29 and IMR90 cell co-cultures and their culture media. Our results support the view that extracellular RNA found in plasma from cancer patients circulates in association with or is protected in a multiparticle complex, and that an active release mechanism by tumor cells may be a possible origin.