Mutant PIK3CA Induces EMT in a Cell Type Specific Manner

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Abstract

Breast cancer is characterized into different molecular subtypes, and each subtype is characterized by differential gene expression that are associated with distinct survival outcomes in patients. PIK3CA mutations are commonly associated with most breast cancer subtypes. More recently PIK3CA mutations have been shown to induce tumor heterogeneity and are associated with activation of EGFR-signaling and reduced relapse free survival in basal subtype of breast cancer. Thus, understanding what determines PIK3CA induced heterogeneity and oncogenesis, is an important area of investigation. In this study, we assessed the effect of mutant PIK3CA together with mutant Ras plus mutant p53 on oncogenic behavior of two distinct stem/progenitor breast cell lines, designated as K5+/K19- and K5+/K19+. Constructs were ectopically overexpressed in K5+/K19- and K5+/K19+ stem/progenitor cells, followed by various in-vitro and in-vivo analyses. Oncogene combination m-Ras/m-p53/m-PIK3CA efficiently transformed both K5+/K19- and K5+/K19+ cell lines in-vitro, as assessed by anchorage-independent soft agar colony formation assay. Significantly, while this oncogene combination induced a complete epithelial-to-mesenchymal transition (EMT) in K5+/K19- cell line, mostly epithelial phenotype with minor EMT component was seen in K5+/K19+ cell line. However, both K5+/K19- and K5+/K19+ transformed cells exhibited increased invasion and migration abilities. Analyses of CD44 and CD24 expression showed both cell lines had tumor-initiating CD44 ⁺/CD24 ^low cell population, however transformed K5+/K19- cells had more proportion of these cells. Significantly, both cell types exhibited in-vivo tumorigenesis, and maintained their EMT and epithelial nature in-vivo in mice tumors. Notably, while both cell types exhibited increase in tumor-initiating cell population, differential EMT phenotype was observed in these cell lines. These results suggest that EMT is a cell type dependent phenomenon and does not dictate oncogenesis.

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Morphogenesis and oncogenesis of MCF-10A mammary epithelial acini grown in three-dimensional basement membrane cultures.

Jayanta Debnath, Senthil Muthuswamy, Joan S Brugge (2003)

The three-dimensional culture of MCF-10A mammary epithelial cells on a reconstituted basement membrane results in formation of polarized, growth-arrested acini-like spheroids that recapitulate several aspects of glandular architecture in vivo. Oncogenes introduced into MCF-10A cells disrupt this morphogenetic process, and elicit distinct morphological phenotypes. Recent studies analyzing the mechanistic basis for phenotypic heterogeneity observed among different oncogenes (e.g., ErbB2, cyclin D1) have illustrated the utility of this three-dimensional culture system in modeling the biological activities of cancer genes, particularly with regard to their ability to disrupt epithelial architecture during the early aspects of carcinoma formation. Here we provide a collection of protocols to culture MCF-10A cells, to establish stable pools expressing a gene of interest via retroviral infection, as well as to grow and analyze MCF-10A cells in three-dimensional basement membrane culture.

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Multi-cancer computational analysis reveals invasion-associated variant of desmoplastic reaction involving INHBA, THBS2 and COL11A1

Tea Hoon Kim, John Watkinson, Vinay Varadan … (2010)

Background Despite extensive research, the details of the biological mechanisms by which cancer cells acquire motility and invasiveness are largely unknown. This study identifies an invasion associated gene signature shedding light on these mechanisms. Methods We analyze data from multiple cancers using a novel computational method identifying sets of genes whose coordinated overexpression indicates the presence of a particular phenotype, in this case high-stage cancer. Results We conclude that there is one shared "core" metastasis-associated gene expression signature corresponding to a specific variant of stromal desmoplastic reaction, present in a large subset of samples that have exceeded a threshold of invasive transition specific to each cancer, indicating that the corresponding biological mechanism is triggered at that point. For example this threshold is reached at stage IIIc in ovarian cancer and at stage II in colorectal cancer. Therefore, its presence indicates that the corresponding stage has been reached. It has several features, such as coordinated overexpression of particular collagens, mainly COL11A1 and other genes, mainly THBS2 and INHBA. The composition of the overexpressed genes indicates invasion-facilitating altered proteolysis in the extracellular matrix. The prominent presence in the signature of INHBA in all cancers strongly suggests a biological mechanism centered on activin A induced TGF-β signaling, because activin A is a member of the TGF-β superfamily consisting of an INHBA homodimer. Furthermore, we establish that the signature is predictive of neoadjuvant therapy response in at least one breast cancer data set. Conclusions Therefore, these results can be used for developing high specificity biomarkers sensing cancer invasion and predicting response to neoadjuvant therapy, as well as potential multi-cancer metastasis inhibiting therapeutics targeting the corresponding biological mechanism.

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PIK3CA cooperates with other phosphatidylinositol 3'-kinase pathway mutations to effect oncogenic transformation.

Grant A. Knight, Hiroyuki Kuramoto, Katsutoshi Oda … (2008)

Mutations in genes functioning in different pathways frequently occur together in the same cancer, whereas mutations in the same pathway tend to be mutually exclusive. However, the majority of colon, breast, and endometrial cancers that possess mutations in PIK3CA, the catalytic subunit p110alpha of phosphatidylinositol 3'-kinase (PI3K), also possess mutations or alterations in genes upstream of PI3K such as Ras, ERBB2/ERBB3, or PTEN. PIK3CA mutations occur almost exclusively in invasive tumors, whereas upstream mutations occur as frequently in early-stage and late-stage tumors, suggesting that PIK3CA mutation is a late-stage event that may augment earlier activation of the PI3K pathway. Consistent with this, we find that levels of p-AKT (Ser(473)) induced by mutant Ras or knockdown of PTEN were dramatically increased by addition of mutant PIK3CA. Soft agar assays revealed that anchorage-independent growth induced by mutant Ras was greatly increased in the presence of mutant PIK3CA. In breast, colon, and endometrial cancers in which the PI3K pathway is activated by a combination of mutant PIK3CA and alterations in Ras, ERBB2/3, or PTEN, signaling to downstream elements such as Akt was mediated exclusively by the p110alpha isoform, rather than a combination of different PI3K isoforms. Our data therefore suggest that in tumors with co-occurring mutations in multiple components of the PI3K pathway, selective inhibition of the alpha isoform of p110 is an attractive therapeutic strategy, especially for late-stage tumors.

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Author and article information

Contributors

Xinbin Chen: Role: Editor

Journal

Journal ID (nlm-ta): PLoS One

Journal ID (iso-abbrev): PLoS ONE

Journal ID (publisher-id): plos

Journal ID (pmc): plosone

Title: PLoS ONE

Publisher: Public Library of Science (San Francisco, CA USA )

ISSN (Electronic): 1932-6203

Publication date (Electronic): 12 December 2016

Publication date Collection: 2016

Volume: 11

Issue: 12

Electronic Location Identifier: e0167064

Affiliations

[1 ]Department of Genetics, Cell Biology and Anatomy, University of Nebraska Medical Center, Omaha, Nebraska, United States of America

[2 ]Pathology & Microbiology, College of Medicine, University of Nebraska Medical Center, Omaha, Nebraska, United States of America

[3 ]Eppley Institute for Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, Nebraska, United States of America

[4 ]Fred & Pamela Buffett Cancer Center, University of Nebraska Medical Center, Omaha, Nebraska, United States of America

University of California Davis, UNITED STATES

Author notes

Competing Interests: The authors have declared that no competing interests exist.

Conceptualization: DB XZ VB HB.
Data curation: DB XZ SM.
Formal analysis: DB XZ WW VB HB.
Funding acquisition: VB HB.
Investigation: DB XZ SM.
Methodology: DB XZ VB HB.
Project administration: VB HB.
Supervision: VB HB.
Writing – original draft: DB XZ VB HB.
Writing – review & editing: DB XZ SM VB HB.

* E-mail: vband@ 123456unmc.edu

Article

Publisher ID: PONE-D-16-25744

DOI: 10.1371/journal.pone.0167064

PMC ID: 5152840

PubMed ID: 27941987

SO-VID: c3d7b8f4-180a-493a-ba0e-4c18f61e6279

License:

This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

History

Date received : 11 July 2016

Date accepted : 8 November 2016