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Supplementary Figure Legends 1-4 from Knock-in of Mutant K-ras in Nontumorigenic Human Epithelial Cells as a New Model for Studying K-ras–Mediated Transformation

PDF file - 1480K, Figure S1. Double knock in clones have single mutant PIK3CA alleles and equivalent expression of mutant and wild type PIK3CA and form aberrant structures in 3D culture. Figure S2. DKI xenografts retain the same number of KRAS and PIK3CA alleles. Figure S3. Knock out of mutant PIK3CA affects tumorigenicity in nude mouse xenograft assays. Figure S4. DKI cell lines show phosphorylation of Akt and Erk in physiologic but not supra-physiologic concentrations of EGF. Figure S5. DKI cells have increased phosphorylation of S6 ribosomal protein but not 4E-BP1. Figure S6. DKI cells are sensitive to the MEK inhibitor U0126 but not to rapamycin and show differential effects on phosphorylation of Akt, Erk, p70S6K and p90RSK. Figure S7. Transgene expression of mutant PIK3CA cDNA with and without RBD mutations in MCF- 10A and KRAS G12V knock in cell lines. Figure S8. Pdk1 activity is increased in DKI cells.

Supplementary Table 1, Figures 1-4 from 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. [Cancer Res 2007;67(18):8460–7]

Background: The Ras signaling pathway is a key oncogenic growth signaling pathway. The neurofibromatosis gene (NF1) is a tumor suppressor gene and negative regulator of Ras. Approximately 20% of breast cancers experience loss of active NF1 protein, leading to unabated Ras activity. Germline inactivation of NF1 leads to neurofibromatosis, which can be treated with MEK inhibitors. The functional consequences of NF1 loss have not been thoroughly explored in breast cancer. Therefore, we created a cellular model for the loss of NF1 in breast cells in order to identify novel therapies that would target NF1 null breast cells. Methods: We used CRISPR CAS9 to knock out (KO) NF1 through targeted disruption of both NF1 alleles in the human non-tumorigenic breast cell line MCF-10A. Immunoblotting confirmed loss of the protein and this cell line was used to identify changes in cellular signaling that resulted from the loss of NF1. We then used a rational approach to select drugs that may target the cellular changes in NF1 null cells. Finally, the candidate drugs were tested in a tumor xenograft model in mice. Results: Loss of NF1 endowed cells with a more transformed phenotype, including EGF growth independence and anchorage-independent growth. We observed increased activation of MAPK and activated Rho GTPase. The MEK inhibitors trametinib and PD0325901 (IC50=0.25 nM and 0.16 nM, respectively) inhibited growth of NF1-null, but not parent cells. We explored inhibitors of other proteins in the MAPK signaling pathway, including the Raf inhibitors sorafenib and vemurafenib and the dual MAP3K1/MAP2K4 inhibitor LY2228820. However, these compounds did not selectively inhibit growth of NF1-null cells, suggesting that Raf and MAP3K1/MAP2K4 are not vulnerable targets in NF1-null cells. Vincristine and zoledronic acid can indirectly affect Rho GTPase function. NF1-null cells were more sensitive to both as single agents (IC50=1.1 nM and 6.9 μM, respectively) than parental cells. We also tested multiple combinations of these drugs and observed benefits from several combinations that exceeded single agent use. Similar sensitivity was not observed to docetaxel or ixabepilone. We then tested the drugs as single agents and in combination against mouse tumor xenograft models with the human breast cancer cell lines Hs578T (NF1 null) or MCF-7 (wild type NF1). Treatment with single agent vincristine or zoledronic acid for 19 days resulted in statistically-significant decreases of 37.3% and 38.9%, respectively (p Conclusions: Our results suggest that loss of NF1 results in a transformed phenotype. Our results provide the first evidence that vincristine in combination with zoledronic acid may be efficacious in treating breast cancers. Additionally, we provide evidence that other drugs and drug combinations may be more effective at treating breast cancers with NF1 loss than MEK inhibitors, warranting further exploration. Citation Format: Melody A. Cobleigh, Matthew Najor, Satnam Brar, Sanja Turturro, Liam Portt, Timothy Yung, Abde M Abukhdeir. Loss of NF1 leads to rho GTPase activation and sensitivity to multiple agents in breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS18-30.

Background: The PIK3R1 gene is genetically altered in up to ~5% of triple negative breast cancers (TNBCs). We previously reported that breast cancer cells lacking protein expression of PIK3R1 had elevated levels of activated MEK, sensitizing them to the MEK inhibitor trametinib. To better understand whether MEK inhibition is a therapeutic option for breast cancer patients with mutated PIK3R1, we conducted a pilot study using the newer generation MEK inhibitor, binimetinib, in a PDX mouse model and found that mutations in PIK3R1 sensitized these PDXs to binimetinib. Alpelisib is FDA-approved for use in breast cancers harboring certain mutations in PIK3CA. Since PIK3R1 and PIK3CA form the PI3K complex, we reasoned that alpelisib may also have activity in mutant-PIK3R1 breast cancers. In this study, we aimed to test binimetinib, alpelisib and the combination in PDX models harboring PIK3R1 mutations. Methods: Three PDX models derived from TNBCs were tested in athymic nude-foxn1nu (Immune-compromised) mice. All PDX models had wild-type PIK3CA and PTEN. Model 1 was wild type for PIK3R1, while Models 2 and 3 harbored PIK3R1mutations c.1357_1389del and c.1705_1740del, respectively. PDX models were treated with vehicle control, binimetinib alone (10 mg/kg BID), alpelisib alone (35 mg/kg QD), or binimetinib and alpelisib in combination. Animals were treated orally daily for at least 28 days. Tumor volumes and animal weights were measured twice weekly until study endpoint. Results: At the time of this publication, Models 1 and 2 had not reached the study endpoint. Final results will be presented at the meeting. Interim ANOVA endpoint analysis revealed no significant differences across treatment arms in Model 1 (wild type PIK3R1). However, a significant effect of treatment was observed in Models 2 and 3 (mutant PIK3R1; p=0.040 and p=0.00001, respectively). Tukey’s test revealed that the treatment effect in Model 2 resulted from the vehicle control compared to the combination arm [(binimetinib + alpelisib), p=0.016], while in Model 3, a highly significant effect of treatment was observed between vehicle control and each treatment arm (p Conclusions: Mutations in PIK3R1 sensitize TNBCs to MEK inhibitors like binimetinib. In the absence of functional PIK3R1, aberrant PI3K signaling occurs, which explains the sensitivity of breast cancers that are PIK3CA wild-type and PIK3R1 mutant to alpelisib. The combination of both MEK and PIK3CA inhibition appears to be an effective combination therapy in TNBCs harboring mutations in PIK3R1. AMA is employed by the U.S. FDA, but contributed to this publication in his own capacity. The views expressed are his own and do not necessarily represent the views of the Food and Drug Administration or the United States Government. Citation Format: Abde M. Abukhdeir, Melody Cobleigh. Activity of targeted agents in PIK3R1 mutated patient-derived xenografts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2248.

On June 29, 2020, the FDA approved pertuzumab, trastuzumab, and hyaluronidase-zzxf subcutaneous injection (Phesgo) for the treatment of patients with HER2-positive early-stage and metastatic breast cancer. Patients should be selected for therapy based on an FDA-approved companion diagnostic test. Approval was primarily based on the FeDeriCa trial, a randomized, open-label, multicenter comparability study of pertuzumab, trastuzumab, and hyaluronidase-zzxf subcutaneous injection compared with intravenous pertuzumab and intravenous trastuzumab administered in the neoadjuvant and adjuvant settings with chemotherapy for the treatment of patients with early breast cancer. The pharmacokinetic endpoints were, first, to demonstrate that the exposure of subcutaneous pertuzumab was not inferior to that of intravenous pertuzumab, and then to demonstrate that the exposure of subcutaneous trastuzumab was not inferior to that of intravenous trastuzumab. The primary endpoints were met with the observed lower limit of the two-sided 90% confidence intervals above the prespecified noninferiority margins. The most common adverse reactions were alopecia, nausea, diarrhea, anemia, and asthenia. The totality of the evidence demonstrated comparability of the subcutaneous product to intravenous, allowing for extrapolation and approval of all breast cancer indications for which intravenous trastuzumab and pertuzumab are approved.

HER2 amplification results in increased tumor growth and aggressiveness in breast cancer. Despite advancements in HER2-targeted therapy, treatment resistance, cancer recurrence, and metastasis remain significant obstacles in combating this deadly disease. PARP inhibitors, have emerged as a promising class of drugs targeting cancers deficient in homologous recombination repair. Recent preclinical evidence suggests that PARP inhibitors exhibit sensitivity in cancers with high replication stress and genomic instability, as well as some HER2 positive breast cancer cell lines. To investigate the relationship between HER2 amplification and PARP inhibitor sensitivity, we utilized isogenic models of HER2-positive breast cancer, whereby cells were either sensitive or resistant to lapatinib and treated with the PARP inhibitor olaparib used as a single agent, or in combination with HER2-targeted therapies. Our results show that HER2 overexpressing MCF-10A cells are highly sensitivity to single-agent olaparib. The addition of trastuzumab and lapatinib resulted in a synergistic increase in toxicity. To determine if olaparib could be used to overcome resistance to lapatinib, we obtained SKBR3 cells conditioned to be resistant to lapatinib. Despite resistance to lapatinib, these cells continue to exhibit sensitivity to olaparib. Reverse phase protein analysis revealed that lapatinib resistant clones exhibited a statistically-significant increase in protein expression of the apoptosis inhibitor MCL-1, which is decreased upon olaparib treatment resulting in apoptosis. Taken together, our findings suggest that HER2 overexpression may predict sensitivity to olaparib alone, or in combination with trastuzumab and lapatinib. Lapatinib resistant HER2-positive breast cancer cells are sensitive to olaparib, possibly through downregulation of MCL-1.

Expression of the voltage gated proton channel (HV1) as identified by immunocytochemistry has been previously reported in breast cancer tissue. Increased expression of HV1 was correlated with poor prognosis and decreases overall and disease-free survival but the mechanism of its involvement in the disease is unknown. Here we present electrophysiological recordings of HV1 channel activity thus, demonstrating their presence and functional properties in the plasma membrane of a breast cancer cell line, MDA-MB-231. With western blotting we also identify significant levels of HV1 expression in 3 out of 8 “triple negative” breast cancer cell lines (estrogen, progesterone, and HER2 receptor expression negative). We examine the function of HV1 in breast cancer using MDA-MB-231 cells as a model by suppressing the expression of HV1 using shRNA (“knock-down; “KD”) and by eliminating HV1 using CRISPR/Cas9 gene editing (“knock-out”; “KO”). However, these two approaches produced different effects. Knock-down of HV1 using shRNA resulted in slower cell migration in a scratch assay and a significant reduction in H2O2 release. In contrast, HV1 KO cells do not show reduction in migration or H2O2 release. HV1 KO but not knock-down cells showed an increased glycolytic rate with an accompanied increase in p-AKT (Phospho-AKT, Ser473) activity. The expression of CD171/LCAM-1, an adhesion molecule and prognostic indicator for breast cancer, was reduced in the absence of HV1 expression. When we compared MDA-MB-231 xenograft growth rates in immunocompromised mice, tumors from HV1 KO cells grew less in mass with lower staining for the Ki-67 maker for cell proliferation rate. Therefore, deletion of HV1 expression in MDA-MB-231 cells limits tumor growth rate. The limited growth thus, appears to be independent of oxidant production by NADPH oxidase molecules and be determined through cell adhesion activity. While HV1 KO results in cell mechanisms different from KD, both implicate HV1-mediated pathways for control of tumor growth in the MDA-MB-231 cell line.

Background: Approximately 15-20% of breast cancers overexpress HER2. These patients are eligible for trastuzumab in combination with chemotherapy. However, some patients are extreme responders to single agent trastuzumab and we wanted to identify differences in cancer gene expression that could predict response to single agent trastuzumab. Methods: We performed paired-end RNAseq on an isogenic cellular model of trastuzumab sensitivity and resistance. We reasoned that the isogenic nature of the cellular clones used in this study would enrich for differentially-expressed genes (DEGs) that were associated with response to single-agent trastuzumab. DEGs where chosen based upon either i) large fold changes in resistant vs. sensitive clones, ii) high frequency in human HER2+ breast cancers, or iii) were found to be enriched with other DEGs in signaling pathways selected by Ingenuity Pathway Analysis (IPA). DEGs were further scrutinized based upon associations with overall survival (OS) in HER2+ human breast cancers. The resulting genes were validated using qPCR and in several independent sample sets containing gene expression profiles of human breast cancers. Results: Discovery: RNAseq yielded 3,241 statistically-significant DEGs. We used two independent filtering pipelines to obtain 175 DEGs. Ingenuity Pathway Analysis found signaling pathways associated with eukaryotic initiation factor, lysine specific demethylase 5B, and estrogen receptor alpha to be enriched in DEGs associated with trastuzumab resistance. Of these DEGs, six genes correlated with a statistically significant change in OS in the training dataset, and were validated by qPCR in the cell lines used for the analysis. We further determined that the six-gene signature was a negative predictor of overall survival in HER2+ breast cancer patients whose cancers carried at least one DEG. Validation: Using independent cohorts from TCGA and the website KMplot.com, we validated the predictive power of the six-gene signature. Of the 47 HER2+ patients from TCGA, eight patients carried two more DEGs, while 39 carried ≤ 1 DEG. Although the numbers are small, of the 8 patients followed for four or more years, only one patient was alive as compared with 7 out of 39 patients without the signature. Similarly, Kaplan Meier analysis of gene expression data from KMplot.com revealed that only 1 out of 23 patients (4.3%) who carried high mean expression of the six-gene signature were free of distant metastases after 87 months, compared to 4 out of 43 patients (9.3%) from the cohort carrying low mean expression of the six-gene signature. In both validation cohorts, the six DEG signature was not predictive in HER2-negative breast cancers. Discussion: Patients whose tumors lack this gene expression signature are more likely to experience a favorable response to trastuzumab therapy. This signature requires validation in a clinical cohort treated with trastuzumab monotherapy. Citation Format: Abukhdeir AM, Najor MS, Turturro SB, Armstrong AR, McDonald A, Fogg L, Cobleigh MA. A gene expression signature that predicts for trastuzumab response in HER2+ breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P2-09-30.

Risk biomarkers for estrogen receptor (ER)-negative breast cancer have clear value for breast cancer prevention. We previously reported a set of lipid metabolism (LiMe) genes with high expression in the contralateral unaffected breasts (CUBs) of ER-negative cancer cases. We now further examine LiMe gene expression in both tumor and CUB, and investigate the role of Pre-B-cell leukemia homeobox-1 (PBX1) as a candidate common transcription factor for LiMe gene expression. mRNA was extracted from laser-capture microdissected epithelium from tumor and CUB of 84 subjects (28 ER-positive cases, 28 ER-negative cases, 28 healthy controls). Gene expression was quantitated by qRT-PCR. Logistic regression models were generated to predict ER status of the contralateral cancer. Protein expression of HMGCS2 and PBX1 was measured using immunohistochemistry. The effect of PBX1 on LiMe gene expression was examined by overexpressing PBX1 in MCF10A cells with or without ER, and by suppressing PBX1 in MDA-MB-453 cells. The expression of DHRS2, HMGCS2, UGT2B7, UGT2B11, ALOX15B, HPGD, UGT2B28 and GLYATL1 was significantly higher in ER-negative versus ER-positive CUBs, and predicted ER status of the tumor in test and validation sets. In contrast, LiMe gene expression was significantly lower in ER-negative than ER-positive tumors. PBX1 overexpression in MCF10A cells up-regulated most LiMe genes, but not in MCF10A cells overexpressing ER. Suppressing PBX1 in MDA-MB-453 cells resulted in decrease of LiMe gene expression. Four binding sites of PBX1 and cofactor were identified in three lipid metabolism genes using ChIP-qPCR. These data suggest a novel role for PBX1 in the regulation of lipid metabolism genes in benign breast, which may contribute to ER-negative tumorigenesis.

A review of the literature finds that women diagnosed with breast cancer, who were on an aspirin regimen, experienced a decreased risk of distant metastases and death. Several recent studies have reported an improvement in overall survival in colorectal cancer patients who harbored mutations in the oncogene PIK3CA and received a daily aspirin regimen. Breast cancer patients on a daily aspirin regimen experienced decreased risk of distant metastases and death. PIK3CA is the most frequently mutated oncogene in breast cancer, occurring in up to 45 % of all breast cancers. In order to determine if mutations in PIK3CA sensitized breast cancers to aspirin treatment, we employed the use of isogenic cellular clones of the non-tumorigenic, breast epithelial cell line MCF-10A that harbored mutations in either PIK3CA or KRAS or both. We report that mutations in both PIK3CA and KRAS are required for the greatest aspirin sensitivity in breast cancer, and that the GSK3β protein was hyperphosphorylated in aspirin-treated double knockin cells, but not in other clones/treatments. A more modest effect was observed with single mutant PIK3CA, but not KRAS alone. These observations were further confirmed in a panel of breast cancer cell lines. Our findings provide the first evidence that mutations in PIK3CA sensitize breast cancer cells to aspirin. Electronic supplementary material The online version of this article (doi:10.1007/s10549-016-3729-8) contains supplementary material, which is available to authorized users.

Approximately 20% of breast cancers are HER2-positive. Trastuzumab has improved patient outcomes significantly for these cancers. However, acquired resistance remains a major hurdle in the clinical management of these patients. Therefore, identifying molecular changes that cause trastuzumab resistance is worthwhile. STAT6 is a transcription factor that regulates a variety of genes involved in cell cycle regulation, growth inhibition, and apoptosis. STAT6 expression is lost in approximately 3% of breast cancers, but little work has been done in the context of trastuzumab resistance in breast cancer. In isogenic cell line pairs, we observed that trastuzumab-resistant cells expressed significantly lower levels of STAT6 compared to trastuzumab-sensitive cells. Therefore, in order to study the consequences of STAT6 loss in HER2+ breast cancer, we knocked out both alleles of the STAT6 gene using somatic cell gene targeting. Interestingly, loss of STAT6 resulted in anchorage-independent growth and changes in several genes involved in epithelial to mesenchymal transition. This study suggests that STAT6 may play a role in the pathophysiology of HER2+ human breast cancer.

Pancreatic ductal adenocarcinoma (PDA) is characterized by epithelial mutations in KRAS and prominent tumor-associated inflammation, including macrophage infiltration. But knowledge of early interactions between neoplastic epithelium and macrophages in PDA carcinogenesis is limited. Using a pancreatic organoid model, we found that the expression of mutant KRAS in organoids increased i) ductal to acinar gene expression ratios, ii) epithelial cells proliferation, and iii) colony formation capacity in vitro, and endowed pancreatic cells with the ability to generate neoplastic tumors in vivo. KRAS mutations induced a pro-tumorigenic phenotype in macrophages. Altered macrophages decreased epithelial Pigment Epithelial Derived Factor (PEDF) expression and induced a cancerous phenotype. We validated our findings using annotated patient samples from The Cancer Genome Atlas (TCGA) as well as in our human PDA specimens. Epithelium-macrophage cross talk occurs early in pancreatic carcinogenesis where KRAS directly induces cancer-related phenotypes in epithelium, and also promotes a pro-tumorigenic phenotype in macrophages, in turn augmenting neoplastic growth.

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.

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 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.

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 Res 2007;67(18):8460–7]

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 (TGFbeta) 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-1alpha (IL-1alpha), 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.

Tamoxifen is effective for treating estrogen receptor-alpha (ER) positive breast cancers. However, few molecular mediators of tamoxifen resistance have been elucidated. Here we describe a previously unidentified gene, MACROD2 that confers tamoxifen resistance and estrogen independent growth. We found MACROD2 is amplified and overexpressed in metastatic tamoxifen-resistant tumors. Transgene overexpression of MACROD2 in breast cancer cell lines results in tamoxifen resistance, whereas RNAi-mediated gene knock down reverses this phenotype. MACROD2 overexpression also leads to estrogen independent growth in xenograft assays. Mechanistically, MACROD2 increases p300 binding to estrogen response elements in a subset of ER regulated genes. Primary breast cancers and matched metastases demonstrate MACROD2 expression can change with disease evolution, and increased expression and amplification of MACROD2 in primary tumors is associated with worse overall survival. These studies establish MACROD2 as a key mediator of estrogen independent growth and tamoxifen resistance, as well as a potential novel target for diagnostics and therapy.

Li+ is the most effective drug used to treat bipolar disorder; however, its exact mechanism of action has yet to be elucidated. One hypothesis is that Li+ competes with Mg2+ for the Mg2+ binding sites on guanine-nucleotide binding proteins (G-proteins). Using 7Li T1 relaxation measurements and fluorescence spectroscopy with the Mg2+ fluorophore furaptra, we detected Li+/Mg2+ competition in three preparations: the purified G-protein transducin (Gt), stripped rod outer segment membranes (SROS), and SROS with purified Gt reattached (ROS-T). When purified ROS-T, SROS or transducin were titrated with Li+ in the presence of fixed amounts of Mg2+, the apparent Li+ binding constant decreased due to Li+/Mg2+ competition. Whereas for SROS the competition mechanism was monophasic, for Gt, the competition was biphasic, suggesting that in Gt, Li+/Mg2+ competition occurred with different affinities for Mg2+ in two types of Mg2+ binding sites. Moreover, as [Li+] increased, the fluorescence excitation spectra of both ROS-T and Gt were blue shifted, indicating an increase in free [Mg2+] compatible with Li+ displacement of Mg2+ from two low affinity Mg2+ binding sites of Gt. Gt release from ROS-T membrane was also inhibited by Li+ addition. In summary, we found evidence of Li+/Mg2+ competition in Gt-containing preparations. http://www.sciencedirect.com/science/article/B6TGG-4BJ2538-2/1/c5cc8bd5992b6629aba06705e0861119

Immunohistochemical evidence for voltage gated proton channels (hHV1) was recently reported in breast cancer cells, but not healthy breast tissue. Higher levels of hHV1 expression were correlated with advanced stages of the disease, worse prognosis, and decreased survival [Wang et al, 2012, J.Biol.Chem. 287:13877]. However, its function and mechanism of action have yet to be elucidated. Here, we examine a number of breast cancer cell lines for expression of hHV1 and explored the consequences of hHV1 genetic knockout.

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. Cancer Res; 73(11); 3248–61. ©2013 AACR.

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.

BACKGROUND. Small cell carcinoma of the prostate is an uncommon neoplasm, the origin of which has been controversial. To address this, we performed transcriptome profiling and TP53 sequencing of concurrent small cell and prostatic adenocarcinoma to determine the relationship between these entities. METHODS. We identified an unusual case of primary prostate cancer that contained adjacent acinar adenocarcinoma (Gleason score 4 þ 3 ¼ 7) and small cell carcinoma. We performed laser capture microdissection to isolate tumor components and performed gene expression and TP53 gene sequence analysis on each component, with results validated by immunohistochemistry for PSA, PSAP, PSMA, androgen receptor, NKX 3.1 and neuroendocrine markers. RESULTS. Transcriptome profiling of the carcinoma components identified 99 genes with a greater than 10-fold differential expression between prostatic adenocarcinoma and small cell carcinoma, many of which have not been previously reported in prostate cancer. The small cell carcinoma component demonstrated upregulation of proliferative and neuroendocrine markers and tyrosine kinase receptors, and downregulation of cell adhesion molecules, supporting the aggressive nature of this form of carcinoma. Sequencing of the TP53 gene suggested a common clonal origin for both components. CONCLUSIONS. This is the first report of a primary small cell carcinoma of the prostate subjected to extensive molecular analysis and the first to show a clonal relation between two morphologically distinct prostate cancer types. The evidence of progression to small cell carcinoma may yield important insights into the pathogenesis of this entity and provide a novel spectrum of molecular markers to further dissect cellular pathways important in tumor progression. Prostate 69: 603–609, 2009. # 2009 Wiley-Liss, Inc.

Human cancers arise from an imbalance of cell growth and cell death. Key proteins that govern this balance are those that mediate the cell cycle. Several different molecular effectors have been identified that tightly regulate specific phases of the cell cycle, including cyclins, cyclin-dependent kinases (CDKs) and CDK inhibitors. Notably, loss of expression or function of two G1-checkpoint CDK inhibitors – p21 (CDKN1A) and p27 (CDKN1B) – has been implicated in the genesis or progression of many human malignancies. Additionally, there is a growing body of evidence suggesting that functional loss of p21 or p27 can mediate a drug-resistance phenotype. However, reports in the literature have also suggested p21 and p27 can promote tumours, indicating a paradoxical effect. Here, we review historic and recent studies of these two CDK inhibitors, including their identification, function, importance to carcinogenesis and finally their roles in drug resistance.

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.

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-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.

A minority of all breast cancers will express increased levels of the ERBB2 protein. They are eligible for trastuzumab-based therapy. Some will respond, but all will progress. Thus, the problem of resistance to trastuzumab has generated an urgent need to determine the underlying mechanisms of that resistance. Cancer is a genetic disease and the mechanism of trastuzumab resistance is likely also genetic in nature. However, the significant genomic heterogeneity between and within patient tumors greatly complicates the identification of a genetic mechanism of resistance for trastuzumab. In order to overcome some of these challenges, we looked to an isogenic model of trastuzumab resistance. We acquired the trastuzumab-sensitive breast cancer cell line, BT474 and two clones of this cell line that were conditioned to exhibit trastuzumab resistance. To investigate a possible genetic mechanism of trastuzumab-resistance, we performed whole exome sequencing using Ampliseq chemistry on the Ion Torrent platform from Life Technologies and paired-end RNA-sequencing on the Illumina HiSeq platform. Next-generation sequencing data was bioinformatically analyzed using tools that allowed us to filter relevant variants based on statistical and functional significance. Variants of interest were those that that arose during drug treatment, which were identified as those in each of the resistant clones, which were novel compared to the parent clone. Proteins from whole cell lysates were resolved in two dimensions using 3-10 nonlinear strips for isoelectric focusing followed by resolution via 4-20% SDS-PAGE. Proteins were visualized via Gelcode blue and cored with a biopsy punch, trypsinized, and submitted for protein ID on an LTQ XL mass spectrometer. We performed functional validation of genetic alterations through the use of somatic cell gene targeting of an ERBB2-expressing clone of the MCF-10A cell lines, a non-tumorigenic model of breast cancer, which is sensitive to trastuzumab. Exome sequencing initially yielded more than 10,000 unique DNA variants across the three clones, which after bioinformatic analysis resulted in ∼1000 variants of interest. Two-dimensional gel electrophoresis revealed 25-30 differentially expressed proteins per sample. Correlation between the sequencing and proteomics data provided us with a candidate gene list of less than 100 genes and several cellular pathways related to growth signaling and immunity. Genetic alterations were tested for their ability to cause trastuzumab resistance in MCF-10A clones. We describe herein a detailed molecular analysis for a model of trastuzumab resistance. Validated genetic alterations will be investigated in a unique collection of archival specimens, which we hope will open the path towards the development of novel agents to augment the effects of trastuzumab. Citation Format: Abde M Abukhdeir, Matthew Najor, Sanja Turturro, Melissa R Pergande, Jeffrey A Borgia, Hanif G Khalak, Melody Cobleigh. Elucidating molecular resistance to trastuzumab using next generation sequencing in isogenic cell models [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P5-05-07.

Transforming growth factor-beta type 1 (TGF-beta) has been implicated as both a tumor suppressor and a tumor promoter in many solid epithelial cancers. We have previously demonstrated that the cyclin dependent kinase (CDK) inhibitor p21 acts as a molecular switch in determining a growth inhibitory versus growth proliferative response to TGF-beta in the spontaneously immortalized human mammary epithelial cell line MCF-10A. We now demonstrate that this proliferative effect of TGF-beta is mediated through the proinflammatory cytokine, interleukin-1alpha (IL-1alpha). Using gene expression array analysis, we identified IL-1alpha as a cytokine specifically upregulated only in cells lacking p21 and only upon TGF-beta stimulation. Cell proliferation assays verified that recombinant IL-1alpha was capable of inducing a growth proliferative response in p21 null MCF-10A cells, while neutralizing antibodies against IL-1alpha prevented the growth proliferative effects of TGF-beta. Mechanistically, both the CDK and proliferating cell nuclear antigen (PCNA) inhibitory functions of p21 were responsible for preventing TGF-beta induced cell proliferation, but only PCNA inhibition by p21 regulated IL-1alpha gene expression. These studies demonstrate a novel role for IL-1alpha in mediating a proliferative response to TGF-beta signaling, and suggest that therapies directed against IL-1alpha could abate the growth proliferative effects of TGF-beta without compromising its tumor suppressive function.

Li+ binding in subcellular fractions of human neuroblastoma SH-SY5Y cells was investigated using 7Li NMR spin-lattice (T1) and spin-spin (T2) relaxation measurements, as the T1/T2 ratio is a sensitive parameter of Li+ binding. The majority of Li+ binding occurred in the plasma membrane, microsomes, and nuclear membrane fractions as demonstrated by the Li+ binding constants and the values of the T1/T2 ratios, which were drastically larger than those observed in the cytosol, nuclei, and mitochondria. We also investigated by 31P NMR spectroscopy the effects of chronic Li+ treatment for 4-6 weeks on the phospholipid composition of the plasma membrane and the cell homogenate and found that the levels of phosphatidylinositol and phosphatidylserine were significantly increased and decreased, respectively, in both fractions. From these observations, we propose that Li+ binding occurs predominantly to membrane domains, and that chronic Li+ treatment alters the phospholipid composition at these membrane sites. These findings support those from clinical studies that have indicated that Li+ treatment of bipolar patients results in irregularities in Li+ binding and phospholipid metabolism. Implications of our observations on putative mechanisms of Li+ action, including the cell membrane abnormality, the inositol depletion and the G-protein hypotheses, are discussed. http://www.sciencedirect.com/science/article/B6T1Y-4GTVVV4-1/1/78da35833eca5a0d76829b754d64687c

We studied the efficacy of the tris-glycinatocobaltate(II) complex ([Co(gly)(3)](-)) as a shift reagent (SR) for chloride by (35)Cl NMR spectroscopy and compared to that of Co(2+)((aq)). Due to the relatively low thermodynamic stability of [Co(gly)(3)](-), a 1:3 Co(II)/gly stoichiometric solution at physiological pH is approximately a 2:1 mixture of [Co(gly)(2)(H(2)O)(2)] and [Co(gly)(H(2)O)(4)](+). This SR was found to be stable up to higher pH values than Co(2+)((aq)), better preventing Co(OH)(2) formation at alkaline pH. No significant differences in the (35)Cl(-) NMR chemical shift induced by Co(II)/gly or Co(2+)((aq)) were observed in the presence of physiological concentrations of either Ca(2+) or Mg(2+), or of either Na(+) or K(+). Although Co(2+)((aq)) was almost twice as effective as Co(II)/gly in shifting the (35)Cl(-) NMR resonance at the same high rho ([SR]/[Cl(-)]) value and low ionic strength, Co(2+)((aq)) showed a significant decrease (p < 0.05) in the (35)Cl(-) chemical shift at higher ionic strength. Line widths at half-height were significantly (p < 0.05) less for Co(II)/gly than for Co(2+)((aq)) at rho values in the range 0.066-0.40. Intracellular chloride was clearly detectable by (35)Cl NMR spectroscopy in human skin fibroblast cells suspended in medium containing 40 mM Co(II)/gly SR. We determined that, although Co(2+)((aq)) provides a larger shift than Co(II)/gly at the same rho value, there are significant advantages for using Co(II)/gly, such as pH stability, ionic strength independent chemical shifts, narrow (35)Cl(-) NMR resonances, and reduced cellular toxicity, as a SR in biological systems.

Li(+)/Mg(2+) competition has been implicated in the therapeutic action of Li(+) treatment in bipolar illness. We hypothesized that this competition depended on cell-specific properties. To test this hypothesis, we determined the degree of Li(+) transport, immobilization, and Li(+)/Mg(2+) competition in lymphoblastomas, neuroblastomas, and erythrocytes. During a 50 mM/L Li(+)-loading incubation, Li(+) accumulation at 30 min (mmoles Li(+)/L cells) was the greatest in lymphoblastomas (11.1+/-0.3), followed by neuroblastomas (9.3+/-0.5), and then erythrocytes (4.0+/-0.5). Li(+) binding affinities to the plasma membrane in all three cell types were of the same order of magnitude; however, Li(+) immobilization in intact cells was greatest in neuroblastomas and least in erythrocytes. When cells were loaded for 30 min in a 50 mM/L Li(+)-containing medium, the percentage increase in free intracellular [Mg(2+)] in neuroblastoma and lymphoblastoma cells ( approximately 55 and approximately 52%, respectively) was similar, but erythrocytes did not exhibit any substantial increase ( approximately 6%). With the intracellular [Li(+)] at 15 mM/L, the free intracellular [Mg(2+)] increased by the greatest amount in neuroblastomas ( approximately 158%), followed by lymphoblastomas ( approximately 75%), and then erythrocytes ( approximately 50%). We conclude that Li(+) immobilization and transport are related to free intracellular [Mg(2+)] and to the extent of Li(+)/Mg(2+) competition in a cell-specific manner.

Multiple myeloma (MM) is an incurable hematological malignancy characterized by recurrent chromosomal translocations. The t(4;14)(p16;q32) is associated with the worst prognosis of any patient subgroup in MM, although the basis for this poor prognosis is unknown. The t(4;14) is unusual in that it involves two potential target genes on chromosome 4: fibroblast growth factor receptor 3 (FGFR3) and multiple myeloma SET domain (MMSET). MMSET is universally over-expressed in t(4;14) MM, whereas FGFR3 expression is lost in one third of cases, suggesting a role for MMSET in myeloma pathogenesis. 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. Using homologous recombination-mediated gene targeting, we disrupted the N-terminal and full-length isoforms of MMSET in t(4;14)+ KMS-11 MM cells. Disruption of the translocated MMSET allele revealed that this allele accounts for most of the MMSET transcription in t(4;14) MM cells. Accordingly, selective targeting of the translocated allele, but not the non-translocated allele, led to reduced colony formation in methylcellulose and reduced tumor formation in nude mouse xenografts. Down-regulation of MMSET expression in t(4;14) MM cell lines by stable RNA interference (RNAi) led to a slower growth in liquid culture, a significant reduction in colony formation in methylcellulose, and decreased tumorigenicity in vivo. Additionally, MMSET knockdown led to partial cell cycle arrest of adherent MM cells and reduced the ability of MM cells to adhere to extracellular matrix. Cells with targeted disruption or knockdown of MMSET exhibited changes in expression levels of potential target genes, including several adhesion molecules. These results provide the first direct evidence that translocation-mediated overexpression of MMSET plays a critical role in t(4;14) MM and suggest that therapies targeting this gene could impact this particular subset of poor-prognosis patients.

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.