A Common Role for Various Human Truncated Adenomatous Polyposis Coli Isoforms in the Control of Beta-Catenin Activity and Cell Proliferation

The tumour suppressor gene adenomatous polyposis coli (APC) is mutated in most colorectal cancer cases, leading to the synthesis of truncated APC products and the stabilization of β-catenin. Truncated APC is almost always retained in tumour cells, suggesting that it serves an essential function. Here, RNA interference has been used to down-regulate truncated APC in several colorectal cancer cell lines expressing truncated APCs of different lengths, thereby performing an analysis covering most of the mutation cluster region (MCR). The consequences on proliferation in vitro, tumour formation in vivo and the level and transcriptional activity of β-catenin have been investigated. Down-regulation of truncated APC results in an inhibition of tumour cell population expansion in vitro in 6 cell lines out of 6 and inhibition of tumour outgrowth in vivo as analysed in one of these cell lines, HT29. This provides a general rule explaining the retention of truncated APC in colorectal tumours and defines it as a suitable target for therapeutic intervention. Actually, we also show that it is possible to design a shRNA that targets a specific truncated isoform of APC without altering the expression of wild-type APC. Down-regulation of truncated APC is accompanied by an up-regulation of the transcriptional activity of β-catenin in 5 out of 6 cell lines. Surprisingly, the increased signalling is associated in most cases (4 out of 5) with an up-regulation of β-catenin levels, indicating that truncated APC can still modulate wnt signalling through controlling the level of β-catenin. This control can happen even when truncated APC lacks the β-catenin inhibiting domain (CiD) involved in targeting β-catenin for proteasomal degradation. Thus, truncated APC is an essential component of colorectal cancer cells, required for cell proliferation, possibly by adjusting β-catenin signalling to the “just right” level

Research of working area development parameters in conditions of deep steep deposit finalizing

Отримано формули розрахунку об’єму запасів корисних копалин в приконтурній та глибинній зоні. Встановлено характер впливу параметрів доробки глибоких крутоспадних родовищ відкритим способом на доцільне положення поточних та проектних контурів кар’єру. Встановлено, що найменший середній коефіцієнт розкриву досягається при мінімальному значенні суми обсягів корисної копалини приконтурної зони лежачого і висячого боків покладу в проектному положенні. Найменший поточний коефіцієнт розкриву досягається при мінімальному значенні суми обсягів корисної копалини приконтурної зони лежачого і висячого боків покладу, а також робочого борту кар'єру в поточному положенні

Combined changes in Wnt signalling response and contact inhibition induce altered proliferation in radiation treated intestinal crypts

Curative intervention is possible if colorectal cancer is identified early, underscoring the need to detect the earliest stages of malignant transformation. A candidate biomarker is the expanded proliferative zone observed in crypts before adenoma formation, also found in irradiated crypts. However, the underlying driving mechanism for this is not known. Wnt signaling is a key regulator of proliferation, and elevated Wnt signaling is implicated in cancer. Nonetheless, how cells differentiate Wnt signals of varying strengths is not understood. We use computational modeling to compare alternative hypotheses about how Wnt signaling and contact inhibition affect proliferation. Direct comparison of simulations with published experimental data revealed that the model that best reproduces proliferation patterns in normal crypts stipulates that proliferative fate and cell cycle duration are set by the Wnt stimulus experienced at birth. The model also showed that the broadened proliferation zone induced by tumorigenic radiation can be attributed to cells responding to lower Wnt concentrations and dividing at smaller volumes. Application of the model to data from irradiated crypts after an extended recovery period permitted deductions about the extent of the initial insult. Application of computational modeling to experimental data revealed how mechanisms that control cell dynamics are altered at the earliest stages of carcinogenesis

Myofibroblast-Derived SFRP1 as Potential Inhibitor of Colorectal Carcinoma Field Effect

Epigenetic changes of stromal-epithelial interactions are of key importance in the regulation of colorectal carcinoma (CRC) cells and morphologically normal, but genetically and epigenetically altered epithelium in normal adjacent tumor (NAT) areas. Here we demonstrated retained protein expression of well-known Wnt inhibitor, secreted frizzled-related protein 1 (SFRP1) in stromal myofibroblasts and decreasing epithelial expression from NAT tissues towards the tumor. SFRP1 was unmethylated in laser microdissected myofibroblasts and partially hypermethylated in epithelial cells in these areas. In contrast, we found epigenetically silenced myofibroblast-derived SFRP1 in CRC stroma. Our results suggest that the myofibroblast-derived SFRP1 protein might be a paracrine inhibitor of epithelial proliferation in NAT areas and loss of this signal may support tumor proliferation in CRC

Down-regulation of frizzled-7 expression decreases survival, invasion and metastatic capabilities of colon cancer cells

BackgroundThe canonical Wnt signalling pathway is activated in most sporadic colorectal cancers (CRCs). We previously reported that FZD7 functions as a receptor for the canonical Wnt signalling pathway in colon cancer cells.Methods and resultsIn this study, we examined the function of FZD7 in survival, invasion and metastatic capabilities of colon cancer cells. FZD7_siRNA transfection decreased cell viability of HT-29 and HCT-116 colon cancer cells. Expression of c-Jun, phosphorylation of JNK and c-Jun, and activation of RhoA were suppressed after FZD7_siRNA transfection into HCT-116 cells. In vitro invasion activity and Wnt target gene expression were also reduced in HCT-116 cells transfected with FZD7_siRNA. Liver metastasis of stable FZD7_siRNA HCT-116 cell transfectants in scid mice was decreased to 40-50% compared to controls. The mRNA levels of FZD7 in 135 primary CRC tissues were examined by real-time PCR. FZD7 mRNA levels were significantly higher in stage II, III or IV tumours than in non-tumour tissues (P<0.005), and overall survival was shorter in those patients with higher FZD7 expression (P<0.001).ConclusionThese data suggest that FZD7 may be involved in enhancement of survival, invasion and metastatic capabilities of colon cancer cells through non-canonical Wnt signalling pathways as well as the canonical pathway

Androgen receptor expresion in breast cancer: Relationship with clinicopathological characteristics of the tumors, prognosis, and expression of metalloproteases and their inhibitors

<p>Abstract</p> <p>Background</p> <p>In the present study we analyze, in patients with breast cancer, the tumor expression of androgen receptors (AR), its relationship with clinicopathological characteristics and with the expression of several matrix metalloproteases (MMPs) and their inhibitors (TIMPs), as well as with prognosis.</p> <p>Methods</p> <p>An immunohistochemical study was performed using tissue microarrays and specific antibodies against AR, MMPs -1, -2, -7, -9, -11, -13, -14, and TIMPs -1, -2 and -3. More than 2,800 determinations on tumor specimens from 111 patients with primary invasive ductal carcinoma of the breast (52 with axillary lymph node metastases and 59 without them) and controls were performed. Staining results were categorized using a score based on the intensity of the staining and a specific software program calculated the percentage of immunostained cells automatically.</p> <p>Results</p> <p>A total of 83 cases (74.8%) showed a positive immunostaining for AR, but with a wide variation in the staining score values. There were no significant associations between the total immunostaining scores for AR and any clinicopathological parameters. However, score values for MMP-1, -7 and -13, were significantly higher in AR-positive tumors than in AR-negative tumors. Likewise, when we considered the cellular type expressing each factor, we found that AR-positive tumors had a higher percentage of cases positive for MMP-1, -7, -11, and TIMP-2 in their malignant cells, as well as for MMP-1 in intratumoral fibroblasts. On the other hand, multivariate analysis demonstrated that patients with AR-positive tumors have a significant longer overall survival than those with AR-negative breast carcinomas (<it>p </it>= 0.03).</p> <p>Conclusion</p> <p>Our results confirm that AR are commonly expressed in breast cancer, and are correlated with the expression of some MMPs and TIMP-2. Although we found a specific value of AR expression to be a prognostic indicator in breast cancer, the functional role of AR in these neoplasms is still unclear and further data are needed in order to clarify their biological signification in breast cancer.</p

Rho GTPase Cdc42 Is a Direct Interacting Partner of Adenomatous Polyposis Coli Protein and Can Alter Its Cellular Localization

Adenomatous Polyposis Coli (APC) is a tumor suppressor gene product involved in colon cancer. APC is a large multidomain molecule of 2843 amino acid residues and connects cell-cell adhesion, the F-actin/microtubule cytoskeleton and the nucleus. Here we show that Cdc42 interacts directly with the first three armadillo repeats of APC by yeast two-hybrid screens. We confirm the Cdc42-APC interaction using pulldown assays in vitro and FRET assays in vivo. Interestingly, Cdc42 interacts with APC at leading edge sites where F-actin is enriched. In contrast, Cdc42 interacts with the truncated mutant APC1–1638 in cellular puncta associated with the golgi-lysozome pathway in transfected CHO cells. In HCT116 and SW480 cells, Cdc42 induces the relocalization of endogenous APC and the mutant APC1–1338 to the plasma membrane and cellular puncta, respectively. Taken together, these data indicate that the Cdc42-APC interaction induces localization of both APC and mutant APC and may thus play a direct role in the functions of these proteins

Functional redundancy between Apc and Apc2 regulates tissue homeostasis and prevents tumorigenesis in murine mammary epithelium

Aberrant Wnt signaling within breast cancer is associated with poor prognosis, but regulation of this pathway in breast tissue remains poorly understood and the consequences of immediate or long-term dysregulation remain elusive. The exact contribution of the Wnt-regulating proteins adenomatous polyposis coli (APC) and APC2 in the pathogenesis of human breast cancer are ill-defined, but our analysis of publically available array data sets indicates that tumors with concomitant low expression of both proteins occurs more frequently in the ‘triple negative’ phenotype, which is a subtype of breast cancer with particularly poor prognosis. We have used mouse transgenics to delete Apc and/or Apc2 from mouse mammary epithelium to elucidate the significance of these proteins in mammary homeostasis and delineate their influences on Wnt signaling and tumorigenesis. Loss of either protein alone failed to affect Wnt signaling levels or tissue homeostasis. Strikingly, concomitant loss led to local disruption of β-catenin status, disruption in epithelial integrity, cohesion and polarity, increased cell division and a distinctive form of ductal hyperplasia with ‘squamoid’ ghost cell nodules in young animals. Upon aging, the development of Wnt activated mammary carcinomas with squamous differentiation was accompanied by a significantly reduced survival. This novel Wnt-driven mammary tumor model highlights the importance of functional redundancies existing between the Apc proteins both in normal homeostasis and in tumorigenesis

LGR5 Is a Negative Regulator of Tumourigenicity, Antagonizes Wnt Signalling and Regulates Cell Adhesion in Colorectal Cancer Cell Lines

BACKGROUND: LGR5 (Leucine-rich repeat-containing G-protein coupled receptor 5) is the most established marker for intestinal stem cells. Mouse models show that LGR5+ cells are the cells of origin of intestinal cancer, and LGR5 expression is elevated in human colorectal cancers, however very little is known about LGR5 function or its contribution to the stem cell phenotype and to colorectal cancer. PRINCIPAL FINDINGS: We have modulated the expression of LGR5 by RNAi (inhibitory RNAs) or overexpression in colorectal cancer cell lines. Paradoxically, ablation of LGR5 induces increased invasion and anchorage-independent growth, and enhances tumourigenicity in xenografts experiments. Conversely, overexpression of LGR5 augments cell adhesion, reduces clonogenicity and attenuates tumourigenicity. Expression profiling revealed enhanced wnt signalling and upregulation of EMT genes upon knockdown of LGR5, with opposite changes in LGR5 overexpressing cells. These findings suggest that LGR5 is important in restricting stem cells to their niche, and that loss of LGR5 concomitant with activated wnt signalling may contribute to the invasive phenotype of colorectal carcinomas

The endogenous retrovirus ENS-1 provides active binding sites for transcription factors in embryonic stem cells that specify extra embryonic tissue

<p>Abstract</p> <p>Background</p> <p>Long terminal repeats (LTR) from endogenous retroviruses (ERV) are source of binding sites for transcription factors which affect the host regulatory networks in different cell types, including pluripotent cells. The embryonic epiblast is made of pluripotent cells that are subjected to opposite transcriptional regulatory networks to give rise to distinct embryonic and extraembryonic lineages. To assess the transcriptional contribution of ERV to early developmental processes, we have characterized <it>in vitro </it>and <it>in vivo </it>the regulation of ENS-1, a host adopted and developmentally regulated ERV that is expressed in chick embryonic stem cells.</p> <p>Results</p> <p>We show that <it>Ens-1 </it>LTR activity is controlled by two transcriptional pathways that drive pluripotent cells to alternative developmental fates. Indeed, both Nanog that maintains pluripotency and Gata4 that induces differentiation toward extraembryonic endoderm independently activate the LTR. Ets coactivators are required to support Gata factors' activity thus preventing inappropriate activation before epigenetic silencing occurs during differentiation. Consistent with their expression patterns during chick embryonic development, Gata4, Nanog and Ets1 are recruited on the LTR in embryonic stem cells; in the epiblast the complementary expression of Nanog and Gata/Ets correlates with the <it>Ens-1 </it>gene expression pattern; and Ens-1 transcripts are also detected in the hypoblast, an extraembryonic tissue expressing Gata4 and Ets2, but not Nanog. Accordingly, over expression of Gata4 in embryos induces an ectopic expression of <it>Ens-1</it>.</p> <p>Conclusion</p> <p>Our results show that <it>Ens-1 </it>LTR have co-opted conditions required for the emergence of extraembryonic tissues from pluripotent epiblasts cells. By providing pluripotent cells with intact binding sites for Gata, Nanog, or both, <it>Ens-1 </it>LTR may promote distinct transcriptional networks in embryonic stem cells subpopulations and prime the separation between embryonic and extraembryonic fates.</p