John P. Gustin

Knockin of mutant PIK3CA activates multiple oncogenic pathways

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 GSK3β phosphorylation. Paradoxically, the GSK3β 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 GSK3β 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 GSK3β 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.

Mutation of a single allele of the cancer susceptibility gene BRCA1 leads to genomic instability in human breast epithelial cells

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 BRCA1s 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.

Tamoxifen-stimulated growth of breast cancer due to p21 loss

Tamoxifen is widely used for the treatment of hormonally responsive breast cancers. However, some resistant breast cancers develop a growth proliferative response to this drug, as evidenced by tumor regression upon its withdrawal. To elucidate the molecular mediators of this paradox, tissue samples from a patient with tamoxifen-stimulated breast cancer were analyzed. These studies revealed that loss of the cyclin-dependent kinase inhibitor p21 was associated with a tamoxifen growth-inducing phenotype. Immortalized human breast epithelial cells with somatic deletion of the p21 gene were then generated and displayed a growth proliferative response to tamoxifen, whereas p21 wild-type cells demonstrated growth inhibition upon tamoxifen exposure. Mutational and biochemical analyses revealed that loss of p21s cyclin-dependent kinase inhibitory property results in hyperphosphorylation of estrogen receptor-α, with subsequent increased gene expression of estrogen receptor-regulated genes. These data reveal a previously uncharacterized molecular mechanism of tamoxifen resistance and have potential clinical implications for the management of tamoxifen-resistant breast cancers.

Knock-in of Mutant K-ras in Nontumorigenic Human Epithelial Cells as a New Model for Studying K-ras–Mediated Transformation

The oncogenic function of mutant ras in mammalian cells has been extensively investigated using multiple human and animal models. These systems include overexpression of exogenous mutant ras transgenes, conditionally expressed knock-in mouse models, and somatic cell knockout of mutant and wild-type ras genes in human cancer cell lines. However, phenotypic discrepancies between knock-in mice and transgenic mutant ras overexpression prompted us to evaluate the consequences of targeted knock-in of an oncogenic K-ras mutation in the nontumorigenic human breast epithelial cell line MCF-10A and hTERT-immortalized human mammary epithelial cells. Our results show several significant differences between mutant K-ras knock-in cells versus their transgene counterparts, including limited phosphorylation of the downstream molecules extracellular signal-regulated kinase and AKT, minor proliferative capacity in the absence of an exogenous growth factor, and the inability to form colonies in semisolid medium. Analysis of 16 cancer cell lines carrying mutant K-ras genes indicated that 50% of cancer cells harbor nonoverexpressed heterozygous K-ras mutations similar to the expression seen in our knock-in cell lines. Thus, this system serves as a new model for elucidating the oncogenic contribution of mutant K-ras as expressed in a large fraction of human cancer cells.

The growth response to androgen receptor signaling in ERα-negative human breast cells is dependent on p21 and mediated by MAPK activation

IntroductionAlthough a high frequency of androgen receptor (AR) expression in human breast cancers has been described, exploiting this knowledge for therapy has been challenging. This is in part because androgens can either inhibit or stimulate cell proliferation in pre-clinical models of breast cancer. In addition, many breast cancers co-express other steroid hormone receptors that can affect AR signaling, further obfuscating the effects of androgens on breast cancer cells.MethodsTo create better-defined models of AR signaling in human breast epithelial cells, we took estrogen receptor (ER)-α-negative and progesterone receptor (PR)-negative human breast epithelial cell lines, both cancerous and non-cancerous, and engineered them to express AR, thus allowing the unambiguous study of AR signaling. We cloned a full-length cDNA of human AR, and expressed this transgene in MCF-10A non-tumorigenic human breast epithelial cells and MDA-MB-231 human breast-cancer cells. We characterized the responses to AR ligand binding using various assays, and used isogenic MCF-10A p21 knock-out cell lines expressing AR to demonstrate the requirement for p21 in mediating the proliferative responses to AR signaling in human breast epithelial cells.ResultsWe found that hyperactivation of the mitogen-activated protein kinase (MAPK) pathway from both AR and epidermal growth factor receptor (EGFR) signaling resulted in a growth-inhibitory response, whereas MAPK signaling from either AR or EGFR activation resulted in cellular proliferation. Additionally, p21 gene knock-out studies confirmed that AR signaling/activation of the MAPK pathway is dependent on p21.ConclusionsThese studies present a new model for the analysis of AR signaling in human breast epithelial cells lacking ERα/PR expression, providing an experimental system without the potential confounding effects of ERα/PR crosstalk. Using this system, we provide a mechanistic explanation for previous observations ascribing a dual role for AR signaling in human breast cancer cells. As previous reports have shown that approximately 40% of breast cancers can lack p21 expression, our data also identify potential new caveats for exploiting AR as a target for breast cancer therapy.

PIK3CA and AKT1 Mutations Have Distinct Effects on Sensitivity to Targeted Pathway Inhibitors in an Isogenic Luminal Breast Cancer Model System

Purpose: Activating mutations in the phosphoinositide-3-kinase (PI3K)/AKT/mTOR pathway are present in the majority of breast cancers and therefore are a major focus of drug development and clinical trials. Pathway mutations have been proposed as predictive biomarkers for efficacy of PI3K-targeted therapies. However, the precise contribution of distinct PI3K pathway mutations to drug sensitivity is unknown. Experimental Design: We describe the creation of a physiologic human luminal breast cancer cell line model to study the phenotype of these mutations using the MCF-7 cell line. We used somatic cell gene targeting to “correct” PIK3CA E545K-mutant alleles in MCF-7 cells to wild-type sequence. The AKT1 E17K hotspot mutation was knocked in on this wild-type background. Results: Loss of mutant PIK3CA dramatically reduced phosphorylation of AKT proteins and several known AKT targets, but other AKT target proteins and downstream effectors of mTOR were not affected. PIK3CA wild-type cells exhibited reduced proliferation in vitro and in vivo. Knockin of the AKT1 E17K hotspot mutation on this PIK3CA wild-type background restored pathway signaling, proliferation, and tumor growth in vivo. PIK3CA, but not AKT1 mutation, increased sensitivity to the PI3K inhibitor GDC-0941 and the allosteric AKT inhibitor MK-2206. Conclusions: AKT1 E17K is a bona fide oncogene in a human luminal breast cancer context. Distinct PI3K pathway mutations confer differential sensitivity to drugs targeting the pathway at different points and by distinct mechanisms. These findings have implications for the use of tumor genome sequencing to assign patients to targeted therapies. Clin Cancer Res; 19(19); 5413–22. ©2013 AACR.

Knock in of the AKT1 E17K mutation in human breast epithelial cells does not recapitulate oncogenic PIK3CA mutations

An oncogenic mutation (G49A:E17K) in the AKT1 gene has been described recently in human breast, colon, and ovarian cancers. The low frequency of this mutation and perhaps other selective pressures have prevented the isolation of human cancer cell lines that harbor this mutation thereby limiting functional analysis. Here, we create a physiologic in vitro model to study the effects of this mutation by using somatic cell gene targeting using the nontumorigenic human breast epithelial cell line, MCF10A. Surprisingly, knock in of E17K into the AKT1 gene had minimal phenotypic consequences and importantly, did not recapitulate the biochemical and growth characteristics seen with somatic cell knock in of PIK3CA hotspot mutations. These results suggest that mutations in critical genes within the PI3-kinase (PI3K) pathway are not functionally equivalent, and that other cooperative genetic events may be necessary to achieve oncogenic PI3K pathway activation in cancers that contain the AKT1 E17K mutation.

The PIK3CA Gene as a Mutated Target for Cancer Therapy

The development of targeted therapies with true specificity for cancer relies upon exploiting differences between cancerous and normal cells. Genetic and genomic alterations including somatic mutations, translocations, and amplifications have served as recent examples of how such differences can be exploited as effective drug targets. Small molecule inhibitors and monoclonal antibodies directed against the protein products of these genetic anomalies have led to cancer therapies with high specificity and relatively low toxicity. Recently, our group and others have demonstrated that somatic mutations in the PIK3CA gene occur at high frequency in breast and other cancers. Moreover, the majority of mutations occur at three hotspots, making these ideal targets for therapeutic development. Here we review the literature on PIK3CA mutations in cancer, as well as existing data on PIK3CA inhibitors and inhibitors of downstream effectors for potential use as targeted cancer therapeutics.

Single copies of mutant KRAS and mutant PIK3CA cooperate in immortalized human epithelial cells to induce tumor formation

The selective pressures leading to cancers with mutations in both KRAS and PIK3CA are unclear. Here, we show that somatic cell knockin of both KRAS G12V and oncogenic PIK3CA mutations in human breast epithelial cells results in cooperative activation of the phosphoinositide 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) pathways in vitro, and leads to tumor formation in immunocompromised mice. Xenografts from double-knockin cells retain single copies of mutant KRAS and PIK3CA, suggesting that tumor formation does not require increased copy number of either oncogene, and these results were also observed in human colorectal cancer specimens. Mechanistically, the cooperativity between mutant KRAS and PIK3CA is mediated in part by Ras/p110α binding, as inactivating point mutations within the Ras-binding domain of PIK3CA significantly abates pathway signaling. In addition, Pdk1 activation of the downstream effector p90RSK is also increased by the combined presence of mutant KRAS and PIK3CA. These results provide new insights into mutant KRAS function and its role in carcinogenesis.

p21 Gene Knock Down Does Not Identify Genetic Effectors Seen with Gene Knock Out

RNA interference (RNAi) has become a popular tool for analyzing gene function in cancer research. The feasibility of using RNAi in cellular and animal models as an alternative to conventional gene knock out approaches has been demonstrated. Although these studies show that RNAi can recapitulate phenotypes seen in knock out animals and their derived cell lines, a systematic study rigorously comparing downstream effector genes between RNAi and gene knock out has not been performed. Here we present data contrasting the phenotypic and genotypic changes that occur with either stable knock down via RNAi of the cyclin dependent kinase inhibitor p21 versus its somatic cell knock out counterpart in the human mammary epithelial cell line MCF-10A. Our results demonstrate that p21 knock down clones display a growth proliferative response upon exposure to Transforming Growth Factor-Beta Type 1 (TGF- β) similar to p21 knock out clones. However, gene expression profiles were significantly different in p21 knock down cells versus p21 knock out clones. Importantly p21 knock down clones did not display increased gene expression of interleukin-1α (IL-1α), a critical effector of this growth response previously validated in p21 knock out cells. We conclude that gene knock out can yield additional vital information that may be missed with gene knock down strategies.