Michaela J. Higgins

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.

CYP2D6 Polymorphisms and Tamoxifen Metabolism: Clinical Relevance

The selective estrogen receptor modulator tamoxifen has been used for more than three decades to treat metastatic and early-stage receptor-positive breast cancer and, more recently, to prevent the disease. Biotransformation of tamoxifen to the potent antiestrogen endoxifen is performed by cytochrome P450 (CYP) enzymes, in particular the CYP2D6 isoform. Genetic variants in the CYP2D6 gene may result in CYP2D6 enzymes with reduced or null activity. Strong and intermediate inhibitors of CYP2D6, which may be used to treat hot flashes or psychiatric conditions in breast cancer patients, can also negatively impact enzyme function. Prospective data are lacking, but the balance of current evidence strongly suggests that, compared with women with two wild-type alleles, the presence of two null alleles, and possibly one null allele, predicts reduced tamoxifen metabolism and an inferior outcome in postmenopausal women with early breast cancer who receive adjuvant treatment with the drug. Unfortunately, studies to date have been largely retrospective and the interpretation of their results is limited by examination of archival tissue samples and the inclusion of heterogeneous populations. Although we do not currently recommend routine CYP2D6 testing for women who do not have alternative standard therapies, the use of concomitant strong or intermediate inhibitors of CYP2D6 should be avoided if feasible. This review summarizes the literature to date with a focus on clinically relevant recent studies that examined the association between CYP2D6 polymorphisms and tamoxifen-associated outcomes.

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.

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.

Chemotherapy for lung cancer: the state of the art in 2009

Lung cancer remains the most common cause of cancer-related death among men and women worldwide. Incremental and significant advances in available systemic treatments, however, have taken place in the last decade to provide improved survival rates and better palliation for patients with non-small-cell and small-cell lung cancer. Superior imaging techniques have enabled the detection of early-stage disease and adjuvant chemotherapy has earned a place for select patients following resection of their tumors. Perhaps the largest growth has been in the area of advanced non-small-cell lung cancer, in which multiple new combination and single-agent systemic therapies have become standard where previously only ‘best supportive care’ was thought appropriate. In concert with broader applicability of chemotherapy, translational studies have provided the rationale for using molecular markers to identify the patients most likely to benefit from biological and targeted therapies. This review will discuss the current role of chemotherapy in both early and advanced non-small-cell and small-cell lung cancer. Novel targeted systemic therapies and the appropriate selection of treatments for patients based on their tumors’ molecular phenotypes and histologies will also be reviewed.

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.

Understanding Resistance to Tamoxifen in Hormone Receptor–Positive Breast Cancer

The selective estrogen receptor (ER)1 modulator tamoxifen has been used for more than 30 years to treat and, more recently, to prevent breast cancer. Unfortunately, even among patients with ER-positive tumors, the response achieved with tamoxifen treatment is variable. Recent years have seen a sharp increase in the number of studies suggesting that the efficacy and safety of anticancer therapies such as tamoxifen depend not only on tumor characteristics but also on characteristics of the host. It is probable that future prescribing of truly “personalized” treatment approaches for our patients will be determined after analysis of both sets of traits. This Perspective summarizes recent basic and translational studies of putative mechanisms of tamoxifen resistance. Resistance to tamoxifen therapy may be intrinsic or acquired. Breast cancers that produce either ER or progesterone receptor (PR) have the potential to respond to tamoxifen; however, a proportion of ER-positive tumors are intrinsically resistant to the drug. Historically, metastatic breast cancer that is both ER and PR positive (suggesting that the ER is functional) has an approximately 80% rate of response to antihormonal therapy, whereas tumors that are ER positive but PR negative have lower response rates, approximately 40%. Tumors that do not produce ER or PR are not expected to respond to tamoxifen. Investigators have proposed several mechanisms of intrinsic resistance, including cross talk with growth factor–signaling pathways and a balance of ER coregulators. In addition, alterations in epigenetic regulation may cause a lack of ER production or lead to the acquisition of resistance to hormone therapy. Recently, breast tumor phenotypes have been further characterized with DNA microarrays, and such studies have identified 5 clinically distinct subtypes: luminal A, luminal B, ERBB2 -amplified, basal-like, and normal breast-like. So-called luminal B breast cancers that are classically associated with amplification of ERBB2 2 [v-erb-b2 erythroblastic …

The androgen receptor in breast cancer: learning from the past

Care of the cancer patient is becoming increasingly individualized. At the fulcrum of such therapeutic decisionmaking lies a relevant and accurate understanding of unique patient characteristics and preferences, and of tumor biology. In a pure sense, prognostic factors reflect tumor biology that may be used to estimate outcome independent of systemic treatment, whereas predictive factors reflect a relative resistance or sensitivity to specific therapy(ies) and are increasingly used to select optimal treatments for an individual patient. Perhaps the best and oldest example of a biomarker with proven clinical utility is the estrogen receptor alpha (ER), the expression of which on breast tumors has served both as a prognostic and predictive biomarker for the last three decades [1]. The Early Breast Cancer Trialists’ Collaborative Group (EBCTCG) 2005 overview clearly demonstrated that tamoxifen substantially reduces the risk for breast cancer recurrence and death across all age groups in patients with ER-positive earlystage breast cancer, whereas patients with ER-negative disease do not benefit from tamoxifen [2, 3]. ER status also predicts response to endocrine therapy in the metastatic setting [4]. In this issue of Breast Cancer Research and Treatment, a well designed and thorough study by Castellano et al. [5] explores the concept of androgen receptor (AR) as a novel prognostic factor in ER-positive breast cancer, and suggests that it may have additional clinical utility in identifying a better prognosis subgroup of luminal B breast cancers.

Expanding role of bisphosphonates in the management of early breast cancer

Evaluation of: Gnant M, Mlineritsch B, Schippinger W et al. Endocrine therapy plus zoledronic acid in premenopausal breast cancer. N. Engl. J. Med. 360(7), 679–691 (2009) Current expert guidelines support tamoxifen alone or tamoxifen plus ovarian suppression as adjuvant endocrine therapy for premenopausal women with hormone receptor-positive breast cancer. Aromatase inhibitors have succeeded tamoxifen as the endocrine manipulation of choice in the treatment of postmenopausal patients. However, their use as monotherapy has traditionally been contraindicated in premenopausal women because the suppression of peripheral aromatase results in reduced feedback to the hypothalamus, and an increase in ovarian stimulation. The Austrian Breast and Colorectal Cancer Study Group trial 12 randomized premenopausal women with endocrine-responsive early breast cancer receiving the luteinizing hormone-releasing hormone agonist goserelin to tamoxifen or anastrozole without finding a significant efficacy advantage for either arm. A further randomization within the study assigned patients to receive zoledronic acid or not. Intriguingly, the addition of the bisphosphonate to adjuvant endocrine therapy significantly improved disease-free survival in the trial population. These data support the hypothesis that bisphosphonates can reduce rates of distant metastases by manipulation of the bone microenvironment, or potentially, by a direct anti-tumor effect. We discuss the potential impact of this study on clinical practice and the important issue of bone health in young women with breast cancer.