The phosphatidylinositol 3-kinase subunit PIK3CA is frequently mutated in human cancers. Here we used gene targeting to ''knock in'' PIK3CA mutations into human breast epithelial cells to identify new therapeutic targets associated with oncogenic PIK3CA. Mutant PIK3CA knockin cells were capable of epidermal growth factor and mTOR-independent cell proliferation that was associated with AKT, ERK, and GSK3␤ phosphorylation. Paradoxically, the GSK3␤ inhibitors lithium chloride and SB216763 selectively decreased the proliferation of human breast and colorectal cancer cell lines with oncogenic PIK3CA mutations and led to a decrease in the GSK3␤ target gene CYCLIN D1. Oral treatment with lithium preferentially inhibited the growth of nude mouse xenografts of HCT-116 colon cancer cells with mutant PIK3CA compared with isogenic HCT-116 knockout cells containing only wild-type PIK3CA. Our findings suggest GSK3␤ is an important effector of mutant PIK3CA, and that lithium, an FDA-approved therapy for bipolar disorders, has selective antineoplastic properties against cancers that harbor these mutations.GSK3␤ ͉ lithium ͉ mTOR ͉ phosphatidylinositol 3-kinase ͉ cancer

Biallelic inactivation of cancer susceptibility gene BRCA1 leads to breast and ovarian carcinogenesis. Paradoxically, BRCA1 deficiency in mice results in early embryonic lethality, and similarly, lack of BRCA1 in human cells is thought to result in cellular lethality in view of BRCA1's essential function. To survive homozygous BRCA1 inactivation during tumorigenesis, precancerous cells must accumulate additional genetic alterations, such as p53 mutations, but this requirement for an extra genetic "hit" contradicts the two-hit theory for the accelerated carcinogenesis associated with familial cancer syndromes. Here, we show that heterozygous BRCA1 inactivation results in genomic instability in nontumorigenic human breast epithelial cells in vitro and in vivo. Using somatic cell gene targeting, we demonstrated that a heterozygous BRCA1 185delAG mutation confers impaired homology-mediated DNA repair and hypersensitivity to genotoxic stress. Heterozygous mutant BRCA1 cell clones also showed a higher degree of gene copy number loss and loss of heterozygosity in SNP array analyses. In BRCA1 heterozygous clones and nontumorigenic breast epithelial tissues from BRCA mutation carriers, FISH revealed elevated genomic instability when compared with their respective controls. Thus, BRCA1 haploinsufficiency may accelerate hereditary breast carcinogenesis by facilitating additional genetic alterations.

Objective: To develop a microarray-based method for noninvasive prenatal testing (NIPT) and compare it with next-generation sequencing. Methods: Maternal plasma from 878 pregnant women, including 187 trisomy cases (18 trisomy 13, 37 trisomy 18, 132 trisomy 21), was evaluated for trisomy risk. Targeted chromosomes were analyzed using Digital Analysis of Selected Regions (DANSR™) assays. DANSR products were subsequently divided between two DNA quantification methods: microarrays and next-generation sequencing. For both microarray and sequencing methodologies, the Fetal-Fraction Optimized Risk of Trisomy Evaluation (FORTE™) algorithm was used to determine trisomy risk, assay variability across samples, and compute fetal fraction variability within samples. Results: NIPT using microarrays provided faster and more accurate cell-free DNA (cfDNA) measurements than sequencing. The assay variability, a measure of variance of chromosomal cfDNA counts, was lower for microarrays than for sequencing, 0.051 versus 0.099 (p < 0.0001). Analysis time using microarrays was faster, 7.5 versus 56 h for sequencing. Additionally, fetal fraction precision was improved 1.6-fold by assaying more polymorphic sites with microarrays (p < 0.0001). Microarrays correctly classified all trisomy and nontrisomy cases. Conclusions: NIPT using microarrays delivers more accurate cfDNA analysis than next-generation sequencing and can be performed in less time.

The PIK3CA gene is frequently mutated in human cancers. Here we carry out a SILAC-based quantitative phosphoproteomic analysis using isogenic knockin cell lines containing ‘driver’ oncogenic mutations of PIK3CA to dissect the signaling mechanisms responsible for oncogenic phenotypes induced by mutant PIK3CA. From 8,075 unique phosphopeptides identified, we observe that aberrant activation of PI3K pathway leads to increased phosphorylation of a surprisingly wide variety of kinases and downstream signaling networks. Here, by integrating phosphoproteomic data with human protein microarray-based AKT1 kinase assays, we discover and validate six novel AKT1 substrates, including cortactin. Through mutagenesis studies, we demonstrate that phosphorylation of cortactin by AKT1 is important for mutant PI3K enhanced cell migration and invasion. Our study describes a quantitative and global approach for identifying mutation-specific signaling events and for discovering novel signaling molecules as readouts of pathway activation or potential therapeutic targets.