The Epithelial-Mesenchymal Transition Generates Cells with Properties of Stem Cells

The transdifferentiation of epithelial cells into motile mesenchymal cells, a process known as epithelial-mesenchymal transition (EMT), is integral in development, wound healing and stem cell behaviour, and contributes pathologically to fibrosis and cancer progression. This switch in cell differentiation and behaviour is mediated by key transcription factors, including SNAIL, zinc-finger E-box-binding (ZEB) and basic helix-loop-helix transcription factors, the functions of which are finely regulated at the transcriptional, translational and post-translational levels. The reprogramming of gene expression during EMT, as well as non-transcriptional changes, are initiated and controlled by signalling pathways that respond to extracellular cues. Among these, transforming growth factor-β (TGFβ) family signalling has a predominant role; however, the convergence of signalling pathways is essential for EMT.

Molecular mechanisms of epithelial–mesenchymal transition

Cancers develop in complex tissue environments, which they depend on for sustained growth, invasion and metastasis. Unlike tumor cells, stromal cell types within the tumor microenvironment (TME) are genetically stable and thus represent an attractive therapeutic target with reduced risk of resistance and tumor recurrence. However, specifically disrupting the pro-tumorigenic TME is a challenging undertaking, as the TME has diverse capacities to induce both beneficial and adverse consequences for tumorigenesis. Furthermore, many studies have shown that the microenvironment is capable of normalizing tumor cells, suggesting that re-education of stromal cells, rather than targeted ablation per se, may be an effective strategy for treating cancer. Here we discuss the paradoxical roles of the TME during specific stages of cancer progression and metastasis, as well as recent therapeutic attempts to re-educate stromal cells within the TME to have anti-tumorigenic effects.

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Microenvironmental regulation of tumor progression and metastasis.

Mutations in codon 12, 13, or 61 of one of the three ras genes, H-ras, K-ras, and N-ras, convert these genes into active oncogenes. Rapid assays for the detection of these point mutations have been developed recently and used to investigate the role mutated ras genes play in the pathogenesis of human tumors. It appeared that ras gene mutations can be found in a variety of tumor types, although the incidence varies greatly. The highest incidences are found in adenocarcinomas of the pancreas (90%), the colon (50%), and the lung (30%); in thyroid tumors (50%); and in myeloid leukemia (30%). For some tumor types a relationship may exist between the presence of a ras mutation and clinical or histopathological features of the tumor. There is some evidence that environmental agents may be involved in the induction of the mutations.

https://cancerres.aacrjournals.org/content/canres/49/17/4682.full.pdf

ras Oncogenes in Human Cancer: A Review

Large intervening non-coding RNAs (lincRNAs) are pervasively transcribed in the genome yet their potential involvement in human disease is not well understood. Recent studies of dosage compensation, imprinting, and homeotic gene expression suggest that individual lincRNAs can function as the interface between DNA and specific chromatin remodelling activities. Here we show that lincRNAs in the HOX loci become systematically dysregulated during breast cancer progression. The lincRNA termed HOTAIR is increased in expression in primary breast tumours and metastases, and HOTAIR expression level in primary tumours is a powerful predictor of eventual metastasis and death. Enforced expression of HOTAIR in epithelial cancer cells induced genome-wide re-targeting of Polycomb repressive complex 2 (PRC2) to an occupancy pattern more resembling embryonic fibroblasts, leading to altered histone H3 lysine 27 methylation, gene expression, and increased cancer invasiveness and metastasis in a manner dependent on PRC2. Conversely, loss of HOTAIR can inhibit cancer invasiveness, particularly in cells that possess excessive PRC2 activity. These findings indicate that lincRNAs have active roles in modulating the cancer epigenome and may be important targets for cancer diagnosis and therapy.

/pdf/long-non-coding-rna-hotair-reprograms-chromatin-state-to-5fx0t32kv1.pdf

Long non-coding RNA HOTAIR reprograms chromatin state to promote cancer metastasis

The inventors found that the gene, HOXB7, was frequently overexpressed in breast cancer, and is a major upstream regulator of events leading to tamoxifen resistance. The present invention provides double-stranded short interfering nucleic acid (siNA) molecules that targets the HOXB7 gene in cells, and also provides methods of use of this siNA molecule for methods of screening, diagnosis and prediction of treatment outcomes as well as treatment of cancer.

Compositions and methods for treatment of tamoxifen resistant breast cancer

Development of facile, sensitive, specific, and economical assays for the analysis of methylated alleles is crucial to the use of methylated biomarkers for cancer detection. We hereby report a novel method, MethySYBR, a SYBR green-based PCR assay for the dual analysis of DNA methylation and CpG methylation density. MethySYBR begins with multiplex PCR to enable the simultaneous amplification of many discrete target alleles in a single reaction using as little as 3 pg of bisulfite-converted DNA. In the second round of PCR, the specific methylated target is quantified from multiplex products using both nested methylation-independent and methylation-specific primer sets. Moreover, the use of SYBR green dye during quantitative PCR enables melting curve analysis of target amplicons to determine the methylation density of CpG sites on target alleles. To establish proof of principle, two cancer-specific methylated genes, RASSF1A and OGDHL , were assessed by MethySYBR. We demonstrated that MethySYBR sensitively detected methylated alleles in the presence of a 100,000fold excess of unmethylated allele. Furthermore, MethySYBR was shown to be capable of analyzing minute amounts of DNA from paraffin-embedded tissue. Therefore, the MethySYBR assay is a simple, highly specific, highly sensitive, high-throughput, and cost-effective method that is widely applicable to basic and clinical studies of DNA methylation. (J Mol

Technical Advance MethySYBR, a Novel Quantitative PCR Assay for the Dual Analysis of DNA Methylation and CpG Methylation Density

Proc Amer Assoc Cancer Res, Volume 45, 2004

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In recent years, several studies have demonstrated that the deregulation of Homeobox gene expression is associated with malignant transformation and tumorigenesis. Microarray analysis of microdissected epithelial cells from breast normal, lymph node positive primary carcinomas, and breast cancer metastasis to the bone revealed that compared to normal breast, HOXB7 was overexpressed 4.2 folds in the primary tumors and 18-folds in bone metastasis. To assess the role of HOXB7 in the transformation process, we generated stable cell lines of MCF-10A overexpressing HOXB7. Intriguingly, the MCF-10A HOXB7 transfectants presented significant morphological alterations from cobble-stone-like shape to spindle-like. The epithelial cell marker, E-cadherin, was lost in MCF-10A HOXB7 cells, and we also observed slightly upregulation of SNAIL in the transfectants. The over-expression of HOXB7 gene in MCF-10A cells also caused reorganization of the cytoskeletal protein, for instance the F-actin, into actin stress fibers. These data strongly suggest that high expression of HOXB7, even in normal breast epithelial cell line MCF10A, can induce epithelial-mesenchymal transition, which is the key phase for tumor cells acquiring metastatic potential. This model system also provides a means to study a de novo function for HOXB7 control of EMT.

Induction of epithelial-mesenchymal transition of MCF-10A cells with high expression of HOXB7.

Commentary Activation of CDK4 Gene Expression in Human Breast Cancer Cells by the Brn-3b POU Family Transcription Factor Laila Samady, Jonathan Dennis, Vishwanie Budhram-Mahadeo, and David S. Latchman

Saraswati Sukumar

Papers

Compositions and methods for treatment of tamoxifen resistant breast cancer

Technical Advance MethySYBR, a Novel Quantitative PCR Assay for the Dual Analysis of DNA Methylation and CpG Methylation Density

Induction of epithelial-mesenchymal transition of MCF-10A cells with high expression of HOXB7.

Coupling the transcriptional regulatory action of Brn-3b to the cell cycle clock.

Abstract 1526: HOXA5 inhibits cell plasticity and stemness in breast cancer cells by reinforcing epithelial traits