Single Copies of Mutant <i>KRAS</i> and Mutant <i>PIK3CA</i> Cooperate in Immortalized Human Epithelial Cells to Induce Tumor Formation

Wang, Grace M.; Wong, Hong Yuen; Konishi, Hiroyuki; Blair, Brian; Abukhdeir, Abde M.; Gustin, John P.; Rosen, David; Denmeade, Samuel R.; Rasheed, Zeshaan A.; Matsui, William; Garay, Joseph P.; Mohseni, Morassa; Higgins, Michaela J.; Cidado, Justin; Jelovac, Danijela; Croessmann, Sarah; Cochran, Rory L.; Karnan, Sivasundaram; Konishi, Yuko; Ota, Akinobu; Hosokawa, Yoshitaka; Argani, Pedram; Lauring, Josh; Park, Ben Ho

doi:10.1158/0008-5472.can-12-1578

Cited by 35 publications

(50 citation statements)

References 38 publications

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“…Additionally, the L755S and V777L DKI MCF-10A-derived cell lines did not form tumors in vivo (Fig. 7C), in contrast to our prior results with mutant KRAS and PIK3CA double knockin cell lines (14). We performed a tail vein injection assay to test the in vivo invasiveness of MCF7 HER2 mutant cell lines.…”

Section: Her2 Missense Mutations Do Not Lead To Increased Tumor Growtmentioning

confidence: 57%

“…It has been previously demonstrated that MCF-10A cells with knockin of PIK3CA E545K (referred to as MCF-10A + E545K) activated both the PI3 kinase and MAP kinase pathways compared with MCF-10A parental cells (14,17). MCF-10A HER2 V777L cells had increased levels of HER2 phosphorylation compared with MCF-10A + E545K cells but had similar levels of ERK phosphorylation (Fig.…”

Section: Acinar Morphology and Anchorage Independent Growth In Mcf7mentioning

confidence: 91%

“…HEK-293T cells were transfected using the Fugene 6 system (Promega). pCFG5 plasmids containing wild-type, G309A, L755S, or V777L HER2 cDNAs, and LXSN plasmids containing wild-type or E545K mutant PIK3CA were derived in previous studies (4,14). A wild-type HER3 expression vector was created by subcloning HER3 cDNA from the pCMV6-XL4-ERBB3 plasmid (SC118918; Origene) into a pIRES-neo3 backbone.…”

Section: Methodsmentioning

confidence: 99%

“…Additionally, we have shown KRAS G12V mutations, as single copies, have relatively little effect in breast epithelial cells, but have a profound effect on cancerous phenotypes and tumorigenicity when coupled with a PIK3CA oncogenic hotspot mutation (14). Given these caveats, we used two HER2 nonamplified human breast epithelial cell lines to create an isogenic panel of HER2 missense knockin mutants, to study the effects of single-copy HER2 mutations under gene expression levels comparable to what has been observed in HER2-mutant cancers.…”

mentioning

confidence: 88%

“…MCF-10A is a nontransformed human breast epithelial cell line with a mostly diploid karyotype that requires EGF supplementation for proliferation in culture (16). Using genome editing, we have demonstrated that MCF-10A cells can be transformed by the introduction of oncogenic mutations both in vitro and in vivo (14).…”

Section: Her2 V777l Mutation Increases Her2 Signaling Pathway Activatmentioning

confidence: 99%

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HER2 missense mutations have distinct effects on oncogenic signaling and migration

Zabransky

Yankaskas

Cochran

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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Recurrent human epidermal growth factor receptor 2 (HER2) missense mutations have been reported in human cancers. These mutations occur primarily in the absence of HER2 gene amplification such that most HER2-mutant tumors are classified as "negative" by FISH or immunohistochemistry assays. It remains unclear whether nonamplified HER2 missense mutations are oncogenic and whether they are targets for HER2-directed therapies that are currently approved for the treatment of HER2 gene-amplified breast cancers. Here we functionally characterize HER2 kinase and extracellular domain mutations through gene editing of the endogenous loci in HER2 nonamplified human breast epithelial cells. In in vitro and in vivo assays, the majority of HER2 missense mutations do not impart detectable oncogenic changes. However, the HER2 V777L mutation increased biochemical pathway activation and, in the context of a PIK3CA mutation, enhanced migratory features in vitro. However, the V777L mutation did not alter in vivo tumorigenicity or sensitivity to HER2-directed therapies in proliferation assays. Our results suggest the oncogenicity and potential targeting of HER2 missense mutations should be considered in the context of cooperating genetic alterations and provide previously unidentified insights into functional analysis of HER2 mutations and strategies to target them. A great success in the treatment of breast cancer has come from the identification of human epidermal growth factor receptor 2 (HER2)/Neu (ERBB2) amplification/overexpression as a targetable driver in ∼20% of breast cancers (1). HER2 is a member of the ErbB family of transmembrane receptor tyrosine kinases, which includes the epidermal growth factor receptor (EGFR/ErbB1), HER3 (ErbB3), and HER4 (ErbB4) (2). Activation of ErbB signaling causes receptor tyrosine autophosphorylation and induces interactions with cytoplasmic signal transduction partners that promote a wide variety of cellular processes including proliferation, motility, and escape from apoptosis. In addition to their key role in normal cellular growth and maintenance, the dysregulation of ErbB receptors has been extensively implicated in the development of numerous cancers (1).Whereas overexpression or amplification of HER2 has been well described to deregulate ErbB signaling, cancer genome sequencing studies have demonstrated that somatic point mutations in the HER2 gene occur in a number of cancers, including 2-4% of breast cancers (3-6). Importantly, these mutations are most often found in patients as single copies without amplification/overexpression of HER2 (HER2-"negative" breast cancers), though HER2 protein expression is often still present. Overexpression studies have implicated a number of these mutations as activating and oncogenic (4, 7-9). Additionally, one mutation, L755S, has been described to be associated with lapatinib resistance when overexpressed (4, 10).Past studies comparing overexpression of a mutant cDNA to single nucleotide knockin of mutant oncogenes have shown dramatic differences ...

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Section: Her2 Missense Mutations Do Not Lead To Increased Tumor Growtmentioning

confidence: 57%

Section: Acinar Morphology and Anchorage Independent Growth In Mcf7mentioning

confidence: 91%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 88%

Section: Her2 V777l Mutation Increases Her2 Signaling Pathway Activatmentioning

confidence: 99%

See 3 more Smart Citations

HER2 missense mutations have distinct effects on oncogenic signaling and migration

Zabransky

Yankaskas

Cochran

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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Overexpression of lipid metabolism genes and PBX1 in the contralateral breasts of women with estrogen receptor-negative breast cancer

et al. 2017

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Risk biomarkers for estrogen receptor (ER)-negative breast cancer have clear value for breast cancer prevention. We previously reported a set of lipid metabolism (LiMe) genes with high expression in the contralateral unaffected breasts (CUBs) of ER-negative cancer cases. We now further examine LiMe gene expression in both tumor and CUB, and investigate the role of Pre-B-cell leukemia homeobox-1 (PBX1) as a candidate common transcription factor for LiMe gene expression. mRNA was extracted from laser-capture microdissected epithelium from tumor and CUB of 84 subjects (28 ER-positive cases, 28 ER-negative cases, 28 healthy controls). Gene expression was quantitated by qRT-PCR. Logistic regression models were generated to predict ER status of the contralateral cancer. Protein expression of HMGCS2 and PBX1 was measured using immunohistochemistry. The effect of PBX1 on LiMe gene expression was examined by overexpressing PBX1 in MCF10A cells with or without ER, and by suppressing PBX1 in MDA-MB-453 cells. The expression of DHRS2, HMGCS2, UGT2B7, UGT2B11, ALOX15B, HPGD, UGT2B28 and GLYATL1 was significantly higher in ER-negative versus ER-positive CUBs, and predicted ER status of the tumor in test and validation sets. In contrast, LiMe gene expression was significantly lower in ER-negative than ER-positive tumors. PBX1 overexpression in MCF10A cells up-regulated most LiMe genes, but not in MCF10A cells overexpressing ER. Suppressing PBX1 in MDA-MB-453 cells resulted in decrease of LiMe gene expression. Four binding sites of PBX1 and cofactor were identified in three lipid metabolism genes using ChIP-qPCR. These data suggest a novel role for PBX1 in the regulation of lipid metabolism genes in benign breast, which may contribute to ER-negative tumorigenesis.

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A phosphoproteomic screen demonstrates differential dependence on HER3 for MAP kinase pathway activation by distinct PIK3CA mutations

Blair

Zahari

et al. 2014

Proteomics

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The PIK3CA gene encodes for the p110 alpha isoform of PI3 kinase and is one of the most frequently mutated oncogenes in human cancers. However, the mechanisms by which PIK3CA mutations activate cell signaling are not fully understood. Here we used a phosphoproteomic approach to compare differential phosphorylation patterns between human breast epithelial cells and two isogenic somatic cell knock in derivatives, each harboring a distinct PIK3CA mutation. We demonstrated differential phosphorylation patterns between isogenic cell lines containing a PIK3CA helical domain mutation (E545K) compared to cells with a PIK3CA kinase domain mutation (H1047R). In particular, the receptor tyrosine kinase, HER3, showed increased phosphorylation at tyrosine 1328 in H1047R cells versus E545K cells. Genetic studies using shRNA demonstrated that H1047R cells have a profound decrease in growth factor independent proliferation upon HER3 knock down, but this effect was attenuated in E545K cells. In addition, HER3 knock down led to reductions in both PI3 kinase and MAP kinase pathway activation in H1047R cells, but in E545K cells only PI3 kinase pathway diminution was observed. These studies demonstrate the power of using paired isogenic cell lines for proteomic analysis to gain new insights into oncogenic signal transduction pathways.

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Single Copies of Mutant KRAS and Mutant PIK3CA Cooperate in Immortalized Human Epithelial Cells to Induce Tumor Formation

Cited by 35 publications

References 38 publications

HER2 missense mutations have distinct effects on oncogenic signaling and migration

HER2 missense mutations have distinct effects on oncogenic signaling and migration

Overexpression of lipid metabolism genes and PBX1 in the contralateral breasts of women with estrogen receptor-negative breast cancer

A phosphoproteomic screen demonstrates differential dependence on HER3 for MAP kinase pathway activation by distinct PIK3CA mutations

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