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1.  Single copies of mutant KRAS and mutant PIK3CA cooperate in immortalized human epithelial cells to induce tumor formation 
Cancer research  2013;73(11):3248-3261.
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/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.
doi:10.1158/0008-5472.CAN-12-1578
PMCID: PMC3674106  PMID: 23580570
Tumorigenicity; cancer; KRAS; PIK3CA
2.  Simple Monitoring of Gene Targeting Efficiency in Human Somatic Cell Lines Using the PIGA Gene 
PLoS ONE  2012;7(10):e47389.
Gene targeting in most of human somatic cell lines has been labor-intensive because of low homologous recombination efficiency. The development of an experimental system that permits a facile evaluation of gene targeting efficiency in human somatic cell lines is the first step towards the improvement of this technology and its application to a broad range of cell lines. In this study, we utilized phosphatidylinositol glycan anchor biosynthesis class A (PIGA), a gene essential for the synthesis of glycosylphosphatidyl inositol (GPI) anchors, as a reporter of gene targeting events in human somatic cell lines. Targeted disruption of PIGA was quantitatively detected with FLAER, a reagent that specifically binds to GPI anchors. Using this PIGA-based reporter system, we successfully detected adeno-associated virus (AAV)-mediated gene targeting events both with and without promoter-trap enrichment of gene-targeted cell population. The PIGA-based reporter system was also capable of reproducing previous findings that an AAV-mediated gene targeting achieves a remarkably higher ratio of homologous versus random integration (H/R ratio) of targeting vectors than a plasmid-mediated gene targeting. The PIGA-based system also detected an approximately 2-fold increase in the H/R ratio achieved by a small negative selection cassette introduced at the end of the AAV-based targeting vector with a promoter-trap system. Thus, our PIGA-based system is useful for monitoring AAV-mediated gene targeting and will assist in improving gene targeting technology in human somatic cell lines.
doi:10.1371/journal.pone.0047389
PMCID: PMC3466256  PMID: 23056640
3.  Urinary levels of Hepatocarcinoma-intestine-pancreas/Pancreatitis-associated protein as a diagnostic biomarker in patients with bladder cancer 
BMC Urology  2012;12:24.
Background
To assess the possibility of hepatocarcinoma-intestine-pancreas/pancreatitis-associated protein (HIP/PAP) as a biological marker for detecting Bladder cancer (BCa), we examined the expression of HIP/PAP in both BCa specimens and BCa cell lines and measured HIP/PAP levels in urine from patients with BCa.
Methods
HIP/PAP expression in BCa samples was evaluated by western blot analysis, and urinary levels of HIP/PAP in patients with BCa were measured by enzyme-linked immunosorbent assay. Urine samples were collected from 10 healthy volunteers and 109 with benign urological disorders as controls, and from 101 patients who were diagnosed with BCa.
Results
HIP/PAP was highly expressed in BCa samples as compared with control bladder. Urinary HIP/PAP concentrations were significantly higher in BCa patients than in controls (median value; 3.184 pg/mL vs. 55.200 pg/mL, P <0.0001, by Mann–Whitney U test). Urinary HIP/PAP levels in BCa patients correlated positively with pathological T stages and progression-risk groups among non-muscle invasive BCa (P = 0.0008, by Kruskal-Wallis test). Regarding the recurrence-risk classifications of non-muscle invasive BCa, the urinary levels of HIP/PAP were significantly higher in the intermediate than in the low risk group (P = 0.0002, by Mann–Whitney U test). Based on a cut-off of 8.5 pg/mL, the ability of urinary HIP/PAP levels to detect BCa had a sensitivity of 80.2%, specificity of 78.2%, positive predictive value (PPV) of 75.7%, and negative predictive value (NPV) of 82.3%.
Conclusions
HIP/PAP was abundantly expressed in BCa, and the urinary levels of HIP/PAP could be a novel and potent biomarker for detection of BCa, and also for predicting the risks of recurrence- and progression-risk of non-muscle invasive BCa. A large scale study will be needed to establish the usefulness of this biomarker.
doi:10.1186/1471-2490-12-24
PMCID: PMC3487857  PMID: 22943287
Bladder cancer; Urinary marker; HIP/PAP; ELISA; ROC
4.  The growth response to androgen receptor signaling in ERα-negative human breast cells is dependent on p21 and mediated by MAPK activation 
Introduction
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.
Methods
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.
Results
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.
Conclusions
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.
doi:10.1186/bcr3112
PMCID: PMC3496145  PMID: 22321971
5.  PIK3CA mutations and EGFR overexpression predict for lithium sensitivity in human breast epithelial cells 
Cancer Biology & Therapy  2011;11(3):358-367.
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.
doi:10.4161/cbt.11.3.14227
PMCID: PMC3047087  PMID: 21124076
breast cancer; AKT; PI3K; Pten; EGFR
6.  Knock in of the AKT1 E17K mutation in human breast epithelial cells does not recapitulate oncogenic PIK3CA mutations 
Oncogene  2010;29(16):2337-2345.
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 employing 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 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.
doi:10.1038/onc.2009.516
PMCID: PMC3042798  PMID: 20101210
Akt1; oncogene; knock in; gene targeting; breast epithelial cells
8.  p21 Gene Knock Down Does Not Identify Genetic Effectors Seen with Gene Knock Out 
Cancer biology & therapy  2007;6(7):1025-1030.
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 (TGFβ) 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.
PMCID: PMC2667557  PMID: 17611398
RNAi; gene knock down; gene knock out; p21; TGFβ

Results 1-8 (8)