p53 induces distinct epigenetic states at its direct target promoters

<p>Abstract</p> <p>Background</p> <p>The tumor suppressor protein p53 is a transcription factor that is mutated in many cancers. Regulation of gene expression by binding of wild-type p53 to its target sites is accompanied by changes in epigenetic marks like histone acetylation. We studied DNA binding and epigenetic changes induced by wild-type and mutant p53 in non-malignant hTERT-immortalized human mammary epithelial cells overexpressing either wild-type p53 or one of four p53 mutants (R175H, R249S, R273H and R280K) on a wild-type p53 background.</p> <p>Results</p> <p>Using chromatin immunoprecipitation coupled to a 13,000 human promoter microarray, we found that wild-type p53 bound 197 promoters on the microarray including known and novel p53 targets. Of these p53 targets only 20% showed a concomitant increase in histone acetylation, which was linked to increased gene expression, while 80% of targets showed no changes in histone acetylation. We did not observe any decreases in histone acetylation in genes directly bound by wild-type p53. DNA binding in samples expressing mutant p53 was reduced over 95% relative to wild-type p53 and very few changes in histone acetylation and no changes in DNA methylation were observed in mutant p53 expressing samples.</p> <p>Conclusion</p> <p>We conclude that wild-type p53 induces transcription of target genes by binding to DNA and differential induction of histone acetylation at target promoters. Several new wild-type p53 target genes, including <it>DGKZ</it>, <it>FBXO22 </it>and <it>GDF9</it>, were found. DNA binding of wild-type p53 is highly compromised if mutant p53 is present due to interaction of both p53 forms resulting in no direct effect on epigenetic marks.</p

Epigenetic silencing of DSC3 is a common event in human breast cancer

INTRODUCTION: Desmocollin 3 (DSC3) is a member of the cadherin superfamily of calcium-dependent cell adhesion molecules and a principle component of desmosomes. Desmosomal proteins such as DSC3 are integral to the maintenance of tissue architecture and the loss of these components leads to a lack of adhesion and a gain of cellular mobility. DSC3 expression is down-regulated in breast cancer cell lines and primary breast tumors; however, the loss of DSC3 is not due to gene deletion or gross rearrangement of the gene. In this study, we examined the prevalence of epigenetic silencing of DSC3 gene expression in primary breast tumor specimens. METHODS: We used bisulfite genomic sequencing to analyze the methylation state of the DSC3 promoter region from 32 primary breast tumor specimens. We also used a quantitative real-time RT-PCR approach, and analyzed all breast tumor specimens for DSC3 expression. Finally, in addition to bisulfite sequencing and RT-PCR, we used an in vivo nuclease accessibility assay to determine the chromatin architecture of the CpG island region from DSC3-negative breast cancer cells lines. RESULTS: DSC3 expression was downregulated in 23 of 32 (72%) breast cancer specimens comprising: 22 invasive ductal carcinomas, 7 invasive lobular breast carcinomas, 2 invasive ductal carcinomas that metastasized to the lymph node, and a mucoid ductal carcinoma. Of the 23 specimens showing a loss of DSC3 expression, 13 (56%) were associated with cytosine hypermethylation of the promoter region. Furthermore, DSC3 expression is limited to cells of epithelial origin and its expression of mRNA and protein is lost in a high proportion of breast tumor cell lines (79%). Lastly, DNA hypermethylation of the DSC3 promoter is highly correlated with a closed chromatin structure. CONCLUSION: These results indicate that the loss of DSC3 expression is a common event in primary breast tumor specimens, and that DSC3 gene silencing in breast tumors is frequently linked to aberrant cytosine methylation and concomitant changes in chromatin structure

Cancer Stem Cell Plasticity Drives Therapeutic Resistance

The connection between epithelial-mesenchymal (E-M) plasticity and cancer stem cell (CSC) properties has been paradigm-shifting, linking tumor cell invasion and metastasis with therapeutic recurrence. However, despite their importance, the molecular pathways involved in generating invasive, metastatic, and therapy-resistant CSCs remain poorly understood. The enrichment of cells with a mesenchymal/CSC phenotype following therapy has been interpreted in two different ways. The original interpretation posited that therapy kills non-CSCs while sparing pre-existing CSCs. However, evidence is emerging that suggests non-CSCs can be induced into a transient, drug-tolerant, CSC-like state by chemotherapy. The ability to transition between distinct cell states may be as critical for the survival of tumor cells following therapy as it is for metastatic progression. Therefore, inhibition of the pathways that promote E-M and CSC plasticity may suppress tumor recurrence following chemotherapy. Here, we review the emerging appreciation for how plasticity confers therapeutic resistance and tumor recurrence

Constitutive CCND1/CDK2 Activity Substitutes for p53 Loss, or MYC or Oncogenic RAS Expression in the Transformation of Human Mammary Epithelial Cells

<div><p>Cancer develops following the accumulation of genetic and epigenetic alterations that inactivate tumor suppressor genes and activate proto-oncogenes. Dysregulated cyclin-dependent kinase (CDK) activity has oncogenic potential in breast cancer due to its ability to inactivate key tumor suppressor networks and drive aberrant proliferation. Accumulation or over-expression of cyclin D1 (CCND1) occurs in a majority of breast cancers and over-expression of CCND1 leads to accumulation of activated CCND1/CDK2 complexes in breast cancer cells. We describe here the role of constitutively active CCND1/CDK2 complexes in human mammary epithelial cell (HMEC) transformation. A genetically-defined, stepwise HMEC transformation model was generated by inhibiting p16 and p53 with shRNA, and expressing exogenous MYC and mutant RAS. By replacing components of this model, we demonstrate that constitutive CCND1/CDK2 activity effectively confers anchorage independent growth by inhibiting p53 or replacing MYC or oncogenic RAS expression. These findings are consistent with several clinical observations of luminal breast cancer sub-types that show elevated CCND1 typically occurs in specimens that retain wild-type p53, do not amplify MYC, and contain no RAS mutations. Taken together, these data suggest that targeted inhibition of constitutive CCND1/CDK2 activity may enhance the effectiveness of current treatments for luminal breast cancer.</p> </div

Tumor Microenvironmental Signaling Elicits Epithelial-Mesenchymal Plasticity through Cooperation with Transforming Genetic Events

Epithelial-to-mesenchymal transition (EMT) facilitates the escape of epithelial cancer cells from the primary tumor site, which is a key event early in metastasis. Here, we explore how extrinsic, tumor microenvironmental cytokines cooperate with intrinsic, genetic changes to promote EMT in human mammary epithelial cells (HMECs). Viral transduction of transforming genetic events into HMECs routinely generated two distinct cell populations. One population retained epithelial characteristics, while an emergent population spontaneously acquired a mesenchymal morphology and properties associated with cancer stem cells (CSCs). Interestingly, the spontaneous mesenchymal/CSCs were unable to differentiate and lacked epithelial-mesenchymal plasticity. In contrast, exposure of the transformed HMECs retaining epithelial characteristics to exogenous transforming growth factor-β (TGF-β) generated a mesenchymal/CSC population with remarkable plasticity. The TGF-β-induced mesenchymal/CSC population was dependent on the continued presence of TGF-β. Removal of TGF-β or pharmacologic or genetic inhibition of TGF-β/SMAD signaling led to the reversion of mesenchymal/CSC to epithelial/non-CSC. Our results demonstrate that targeting exogenous cytokine signaling disrupts epithelial-mesenchymal plasticity and may be an effective strategy to inhibit the emergence of circulating tumor cells. The model of epithelial-mesenchymal plasticity we describe here can be used to identify novel tumor microenvironmental factors and downstream signaling that cooperate with intrinsic genetic changes to drive metastasis. Understanding the interaction between extrinsic and intrinsic factors that regulate epithelial-mesenchymal plasticity will allow the development of new therapies that target tumor microenvironmental signals to reduce metastasis

Constitutive CCND1/CDK2 activity replaces either MYC or RAS in the transformation of HMEC.

<p>48R-shp16-shp53 cells were infected with control virus (Vec), virus encoding RAS alone (RAS), MYC alone (MYC), or MYC and RAS together (M/R). Additionally, 48R-shp16-shp53 cells were infected with viruses encoding CCND1/CDK2 (D1/K2) followed by RAS, or viruses encoding MYC followed by CCND1/CDK2 (D1/K2). (A) Western blot analysis comparing parental 48R passage 11 to derivative cells. (B) Each derivative was plated in soft agar to assess AIG. The bar graph represents the average colony number per plate of quadruplicates. Error bars represent the standard deviation.</p

Constitutive CCND1/CDK2 activity enhances the growth of 48R-shp16 cells despite elevated p53 and p21.

<p>48R-shp16 cells were infected with viruses encoding CCND1/CDK2 (D1/K2), a shRNA targeting p53 (shp53), or control virus (Vec). (A) Western blot analysis comparing parental 48R passage 11 to derivative cells. (B) Population doublings of the parental 48R (diamonds), 48R-shp16 (squares), 48R-shp16-D1/K2 (crosses), 48R-shp16-shp53 (triangles), and 48R-shp16-shp53-D1/K2 (circles) cells. Cells were grown from passage 6 at the origin and infected with shp16 at passage 11 indicated by the arrow.</p

Generation of a genetically-defined, stepwise model of HMEC transformation.

<p>48R expressing shRNA targeting p16 (shp16), p53 (shp53), or both (shp16-shp53) were further infected with control virus (Vec), or viruses encoding RAS alone (RAS), MYC alone (MYC), or MYC and RAS together (M/R) and assessed at passage 19 for AIG. (A) The bar graph represents the average colony number per plate of quadruplicates. Error bars represent the standard deviation. (B) Pictorial representation of the HMEC transformation model and growth in soft agar. Normal cells (left) are transformed (right) by sequential inactivation of p16 and p53 using shRNA, and over-expression of MYC and oncogenic RAS.</p

Constitutive CCND1/CDK2 activity replaces p53 inactivation in the transformation of HMEC.

<p>48R-shp16 cells were infected with control virus (Vec), or viruses encoding CCND1/CDK2 (D1/K2), or shRNA targeting p53 (shp53), followed by virus encoding MYC and RAS (M/R). (A) Western blot analysis comparing parental 48R passage 11 to derivative cells. (B) Derivative cells from A were assessed for AIG. The bar graph represents the average colony number per plate of quadruplicates. Error bars represent the standard deviation.</p

Constitutive CCND1/CDK2 activity does not replace multiple components of transformation.

<p>48R-shp16-D1/K2 and 48R-shp16-shp53-D1/K2 cells were infected with control virus (Vec), virus encoding RAS alone (RAS), MYC alone (MYC), or MYC and RAS together (M/R). (A) Western blot analyses comparing parental 48R passage 11 to derivative cells. (B) Each derivative was plated in soft agar to assess AIG. The bar graph represents the average colony number per plate of quadruplicates. Error bars represent the standard deviation.</p