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Grace M Wang,
Hong-Yuen Wong,
Hiroyuki Konishi,
Brian G Blair,
Abde Abukhdeir,
John P Gustin,
D Marc Rosen,
Samuel Denmeade,
Zeshaan Rasheed,
William Matsui, [......],
Danijela Jelovac,
Sarah Croessmann,
Rory Cochran,
Sivasundaram Karnan,
Yuko Konishi,
Akinobu Ota,
Yoshitaka Hosokawa,
Pedram Argani,
Josh Lauring,
Ben Ho Park
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ABSTRACT: The selective pressures leading to cancers with mutations in both KRAS and PIK3CA are unclear. Here we demonstrate that somatic cell knock in of both KRAS G12V and oncogenic PIK3CA mutations in human breast epithelial cells results in cooperative activation of the PI3 Kinase and MAP Kinase pathways in vitro, and leads to tumor formation in immunocompromised mice. Xenografts from double knock in 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/p110 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.
Cancer Research 04/2013; · 7.86 Impact Factor
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Joseph P Garay,
Bedri Karakas,
Abde M Abukhdeir,
David P Cosgrove,
John P Gustin,
Michaela J Higgins,
Hiroyuki Konishi,
Yuko Konishi,
Josh Lauring,
Morassa Mohseni, [......],
Ashani Weeraratna,
Cheryl A Sherman Baust,
Patrice J Morin,
Antoun Toubaji,
Alan Meeker,
Angelo M De Marzo,
Gloria Lewis,
Andrea Subhawong,
Pedram Argani,
Ben H Park
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ABSTRACT: Although 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.
To 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.
We 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.
These 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.
Breast cancer research: BCR 02/2012; 14(1):R27. · 5.24 Impact Factor
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ABSTRACT: Breast cancer occurs at a high frequency in women and, given this fact, a primary focus of breast cancer research has been the study of estrogen receptor α (ER) signaling. However, androgens are known to play a role in normal breast physiology and therefore androgen receptor (AR) signaling is becoming increasingly recognized as an important contributor towards breast carcinogenesis. Moreover, the high frequency of AR expression in breast cancer makes it an attractive therapeutic target, but the ability to exploit AR for therapy has been difficult. Here we review the historical use of androgen/anti-androgen therapies in breast cancer, the challenges of accurately modeling nuclear hormone receptor signaling in vitro, and the presence and prognostic significance of AR in breast cancer.
American journal of cancer research. 01/2012; 2(4):434-45.
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Hiroyuki Konishi,
Morassa Mohseni,
Akina Tamaki, Joseph P Garay,
Sarah Croessmann,
Sivasundaram Karnan,
Akinobu Ota,
Hong Yuen Wong,
Yuko Konishi,
Bedri Karakas, [......],
Josh Lauring,
Amy L Gross,
Christopher M Heaphy,
Yositaka Hosokawa,
Edward Gabrielson,
Alan K Meeker,
Kala Visvanathan,
Pedram Argani,
Kurtis E Bachman,
Ben Ho Park
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ABSTRACT: 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.
Proceedings of the National Academy of Sciences 10/2011; 108(43):17773-8. · 9.68 Impact Factor
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Michaela J Higgins,
Julia A Beaver,
Hong Yuen Wong,
John P Gustin,
Josh D Lauring, Joseph P Garay,
Hiroyuki Konishi,
Morassa Mohseni,
Grace M Wang,
Justin Cidado,
Danijela Jelovac,
David P Cosgrove,
Akina Tamaki,
Abde M Abukhdeir,
Ben Ho Park
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ABSTRACT: A high frequency of somatic mutations has been found in breast cancers within the gene encoding the catalytic p110α subunit of PI3K, PIK3CA. Using isogenic human breast epithelial cells, we have previously demonstrated that oncogenic PIK3CA "hotspot" mutations predict for response to the toxic effects of lithium. However, other somatic genetic alterations occur within this pathway in breast cancers, and it is possible that these changes may also predict for lithium sensitivity. We overexpressed the epidermal growth factor receptor (EGFR) into the non-tumorigenic human breast epithelial cell line MCF-10A, and compared these cells to isogenic cell lines previously created via somatic cell gene targeting to model Pten loss, PIK3CA mutations, and the invariant AKT1 mutation, E17K. EGFR overexpressing clones were capable of cellular proliferation in the absence of EGF and were sensitive to lithium similar to the results previously seen with cells harboring PIK3CA mutations. In contrast, AKT1 E17K cells and PTEN -/- cells displayed resistance or partial sensitivity to lithium, respectively. Western blot analysis demonstrated that lithium sensitivity correlated with significant decreases in both PI3K and MAPK signaling that were observed only in EGFR overexpressing and mutant PIK3CA cell lines. These studies demonstrate that EGFR overexpression and PIK3CA mutations are predictors of response to lithium, whereas Pten loss and AKT1 E17K mutations do not predict for lithium sensitivity. Our findings may have important implications for the use of these genetic lesions in breast cancer patients as predictive markers of response to emerging PI3K pathway inhibitors.
Cancer biology & therapy 02/2011; 11(3):358-67. · 2.64 Impact Factor
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John P Gustin,
Bedri Karakas,
Michele B Weiss,
Abde M Abukhdeir,
Josh Lauring, Joseph P Garay,
David Cosgrove,
Akina Tamaki,
Hiroyuki Konishi,
Yuko Konishi,
Morassa Mohseni,
Grace Wang,
D Marc Rosen,
Samuel R Denmeade,
Michaela J Higgins,
Michele I Vitolo,
Kurtis E Bachman,
Ben Ho Park
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ABSTRACT: 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 GSK3beta phosphorylation. Paradoxically, the GSK3beta 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 GSK3beta 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 GSK3beta 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.
Proceedings of the National Academy of Sciences 03/2009; 106(8):2835-40. · 9.68 Impact Factor
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ABSTRACT: Multiple myeloma (MM) is an incurable hematologic malignancy characterized by recurrent chromosomal translocations. Patients with t(4;14)(p16;q32) are the worst prognostic subgroup in MM, although the basis for this poor prognosis is unknown. The t(4;14) is unusual in that it involves 2 potential target genes: fibroblast growth factor receptor 3 (FGFR3) and multiple myeloma SET domain (MMSET). MMSET is universally overexpressed in t(4;14) MM, whereas FGFR3 expression is lost in one-third of cases. Nonetheless, the role of MMSET in t(4;14) MM has remained unclear. Here we demonstrate a role for MMSET in t(4;14) MM cells. Down-regulation of MMSET expression in MM cell lines by RNA interference and by selective disruption of the translocated MMSET allele using gene targeting dramatically reduced colony formation in methylcellulose but had only modest effects in liquid culture. In addition, MMSET knockdown led to cell-cycle arrest of adherent MM cells and reduced the ability of MM cells to adhere to extracellular matrix. Finally, MMSET knockdown and knockout reduced tumor formation by MM xenografts. These results provide the first direct evidence that MMSET plays a significant role in t(4;14) MM and suggest that therapies targeting this gene could impact this particular subset of poor-prognosis patients.
Blood 02/2008; 111(2):856-64. · 9.90 Impact Factor
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Abde M Abukhdeir,
Michele I Vitolo,
Pedram Argani,
Angelo M De Marzo,
Bedri Karakas,
Hiroyuki Konishi,
John P Gustin,
Josh Lauring, Joseph P Garay,
Courtney Pendleton,
Yuko Konishi,
Brian G Blair,
Keith Brenner,
Elizabeth Garrett-Mayer,
Hetty Carraway,
Kurtis E Bachman,
Ben Ho Park
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ABSTRACT: 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 p21's cyclin-dependent kinase inhibitory property results in hyperphosphorylation of estrogen receptor-alpha, 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.
Proceedings of the National Academy of Sciences 02/2008; 105(1):288-93. · 9.68 Impact Factor
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ABSTRACT: 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.
Cancer Research 10/2007; 67(18):8460-7. · 7.86 Impact Factor
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ABSTRACT: Here, we describe a method of systematic PCR screening with multiround sample pooling for the isolation of rare PCR-positive samples. As an example, we have applied this protocol to the recovery of gene-targeted clones in human somatic cells comprising only 0.02-0.17% of cells transduced with targeting vectors. Initially, cells infected with targeting vectors are seeded and grown in fourteen 96-well tissue culture plates. Samples are then collected from these plates and subjected to two rounds of pooling to yield twelve 'superpools' used for an initial PCR. After identifying PCR-positive samples, de-pooling is carried out with successive rounds of PCR screening, using samples of decreasing complexity. Single-cell cloning is subsequently performed to isolate gene-targeted clones. The entire protocol can be completed in 4-8 weeks depending on the proliferative capacity of the cell line.
Nature Protocol 02/2007; 2(11):2865-74. · 8.36 Impact Factor
