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Table S4

Supplementary Table from Targeting Metabolic Adaptations in the Breast Cancer–Liver Metastatic Niche Using Dietary Approaches to Improve Endocrine Therapy Efficacy

AACR GENIE Data Guide

Supplemental Methods. Supplemental Table 1: ââ,¬â€¹Genomic Data Characterization by Center. Supplemental Table 2: ââ,¬â€¹Gene Panels Submitted by Each Center. Figure S1: Number of putative germline SNPs per sample, before and after uniform germline filtering. Figure S2ââ,¬â€¹. Distribution of total somatic mutation burden per sample stratified by sequencing panel. Figure S3: ââ,¬â€¹Log-scale comparison of mutation frequencies at hotspot sites between GENIE (data aggregated from all sequencing panels) and cancerhotspots.org (CHS) using a binomial test. Figure S4:ââ,¬â€¹ Comparison of mutation frequencies at hotspot sites in each GENIE sequencing panel with cancerhotspots.org (CHS) using a binomial test.

Estrogen receptor–positive (ER+) metastatic tumors contribute to nearly 70% of breast cancer–related deaths. Most patients with ER+ metastatic breast cancer (MBC) undergo treatment with the estrogen receptor antagonist fulvestrant as standard of care. Yet, among such patients, metastasis in liver is associated with reduced overall survival compared with other metastasis sites. The factors underlying the reduced responsiveness of liver metastases to ER-targeting agents remain unknown, impeding the development of more effective treatment approaches to improve outcomes for patients with ER+ liver metastases. We therefore evaluated site-specific changes in MBC cells and determined the mechanisms through which the liver metastatic niche specifically influences ER+ tumor metabolism and drug resistance. We characterized ER activity of MBC cells both in vitro, using a novel system of tissue-specific extracellular matrix hydrogels representing the stroma of ER+ tumor metastatic sites (liver, lung, and bone), and in vivo, in liver and lung metastasis mouse models. ER+ metastatic liver tumors and MBC cells grown in liver hydrogels displayed upregulated expression of glucose metabolism enzymes in response to fulvestrant. Furthermore, differential ERα activity, but not expression, was detected in liver hydrogels. In vivo, increased glucose metabolism led to increased glycogen deposition in liver metastatic tumors, while a fasting-mimicking diet increased efficacy of fulvestrant treatment to reduce the metastatic burden. Our findings identify a novel mechanism of endocrine resistance driven by the liver tumor microenvironment.Implications:These results may guide the development of dietary strategies to circumvent drug resistance in liver metastasis, with potential applicability in other metastatic diseases.

PDF file, 55K, Supplementary table of antibodies used for western blotting.

Urine volumes, DNA concentrations, DNA input amounts, the number of output mutant sequencing reads, mutant copies detected and the 95% Confidence Intervals

Related Article from BEAMing Sheds Light on Drug Resistance

Many estrogen receptor α (ERα)–positive breast cancers develop resistance to endocrine therapy via mutation of ERs whose constitutive activation is associated with shorter patient survival. Because there is now a clinical need for new antiestrogens (AE) against these mutant ERs, we describe here our development and characterization of three chemically novel AEs that effectively suppress proliferation of breast cancer cells and tumors. Our AEs are effective against wild-type and Y537S and D538G ERs, the two most commonly occurring constitutively active ERs. The three new AEs suppressed proliferation and estrogen target gene expression in WT and mutant ER-containing cells and were more effective in D538G than in Y537S cells and tumors. Compared with WT ER, mutants exhibited approximately 10- to 20-fold lower binding affinity for AE and a reduced ability to be blocked in coactivator interaction, likely contributing to their relative resistance to inhibition by AE. Comparisons between mutant ER–containing MCF7 and T47D cells revealed that AE responses were compound, cell-type, and ERα-mutant dependent. These new ligands have favorable pharmacokinetic properties and effectively suppressed growth of WT and mutant ER–expressing tumor xenografts in NOD/SCID-γ mice after oral or subcutaneous administration; D538G tumors were more potently inhibited by AE than Y537S tumors. These studies highlight the differential responsiveness of the mutant ERs to different AEs and make clear the value of having a toolkit of AEs for treatment of endocrine therapy–resistant tumors driven by different constitutively active ERs. Cancer Res; 77(20); 5602–13. ©2017 AACR.

Purpose:Prognostic value of Oncotype DX Breast Recurrence Score (RS) in male patients with breast cancer is understudied. We evaluated associations of RS with overall mortality in male patients with breast cancer and compared it with female counterparts.Experimental Design:With a cohort of 848 male and 110,898 female patients with breast cancer identified from the National Cancer Database (2010–2014), we estimated HRs and 95% confidence intervals (CI) for overall mortality associated with RS using Cox regression models. RS was evaluated continuously, as well as by categorization following respective traditional (≤17, 18–30, and ≥31) and TAILORx (≤10, 11–25, and ≥26) cutoffs.Results:RS was positively associated with mortality in male patients (HR = 1.13; 95% CI, 1.02–1.26 per unit RS increment) up to RS > 21, after which the risk plateaued. Among female patients, mortality began to increase with RS only when RS > 23 (HR = 1.02; 95% CI, 1.01–1.02 per unit of RS increment). The intermediate- (HR = 5.37; 95% CI, 1.79–16.11) and high-risk diseases (HR = 4.28; 95% CI, 1.22–14.97) defined by TAILORx, but not traditional cutoffs established for female patients, were associated with elevated mortality risk in men even after adjustment for demographic, clinical characteristics, and treatments, except chemotherapy.Conclusions:RS is associated with mortality in male patients with breast cancer at a much lower threshold than that for female patients. Studies are needed to establish specific guidelines for RS thresholds for male patients with breast cancer.

Purpose: Noninvasive drug biomarkers for the early assessment of tumor response can enable adaptive therapeutic decision-making and proof-of-concept studies for investigational drugs. Circulating tumor DNA (ctDNA) is released into the circulation by tumor cell turnover and has been shown to be detectable in urine.Experimental Design: We tested the hypothesis that dynamic changes in EGFR activating (exon 19del and L858R) and resistance (T790M) mutation levels detected in urine could inform tumor response within days of therapy for advanced non–small cell lung cancer (NSCLC) patients receiving osimertinib, a second-line third-generation anti-EGFR tyrosine kinase inhibitor.Results: Eight of nine evaluable NSCLC patients had detectable T790M-mutant DNA fragments in pretreatment baseline samples. Daily monitoring of mutations in urine indicated a pattern of intermittent spikes throughout week 1, suggesting apoptosis with an overall decrease in fragment numbers from baselines to day 7 preceding radiographic response assessed at 6 to 12 weeks.Conclusions: These findings suggest drug-induced tumor apoptosis within days of initial dosing. Daily sampling of ctDNA may enable early assessment of patient response and proof-of-concept studies for drug development. The modeling of tumor lysis through the day-to-day kinetics of ctDNA released into the blood and then into the urine is demonstrated in this proof-of-concept study in lung cancer patients receiving anti-EGFR tyrosine kinase inhibitors. This strategy may determine the specific clonal populations of cells which undergo apoptosis within the first week of therapy. This has important implications for developing combinational strategies to address inter- and intralesional heterogeneity and characterizing residual disease after initial drug exposure. Clin Cancer Res; 23(16); 4716–23. ©2017 AACR.

Supplementary Table S1. Characteristics of male breast cancer patients by receipt of Oncotype DX Recurrence Score (RS) testing Supplementary Table S2. HRs (95% CI) for total mortality associated with the Recurrence Score categories among patients {less than or equal to} 50 years Supplementary Table S3. HRs (95% CI) for total mortality associated with the Recurrence Score categories among patients > 50 years Supplementary Table S4. HRs (95% CI) for total mortality associated with chemotherapy status among intermediate-risk patients by age Supplementary Table S5. HRs (95% CI) for total mortality associated with Recurrence Score categories (sensitivity analysis)

A: Analysis of dilution series of indicated mutant EGFR variants spiked into 60 ng (â‰^18,180 genome equivalents) of WT DNA. Each data point represents one preparative within 6 independent dilutions series prepared and analyzed by two operators on two different instruments on three non-consecutive days for a total of 18 samples per dilution point. An analysis algorithm was applied to transform the mutant EGFR sequencing reads into the absolute mutant copies detected. The box-and-whisker plots show the median (center line), 25th and 75th percentiles (box) with the connecting "whiskers" extending from the first quartile minus 1.5 of the interquartile range (IQR, the third quartile less the first quartile) and the third quartile plus 1.5 of the IQR. A positive Spearman's correlation close to 1 indicates a strong, positive relationship between the absolute mutant EGFR copies detected and the absolute mutant EGFR copies per input. B: Inter-run reproducibility of the EGFR exon 19 deletions, L858R and T790M enrichment PCR-NGS assays for the dilution series shown in panel A. The Coefficient of Variation Percent (CV%) was calculated as the ratio of the standard deviation to the mean of the absolute EGFR copies detected within each absolute copy per input level and is reported as a percentage.

PDF file, 526K, Supplementary Figure 1. Genomic DNA and cDNA sequencing verification of additional gene-targeted MCF-7 derivatives. Supplementary Figure 2. PI3K pathway signaling in low serum conditions. Supplementary Figure 3. Estrogenic stimulation of growth. Supplementary Figure 4. GDC-0941 and MK-2206 treatment of MCF-7 cells and three gene-targeted derivatives with a single mutant copy of PIK3CA (Het 1-3). Supplementary Figure 5. MK-2206 treatment of isogenic MCF-10A cells. Supplementary Figure 6. Perifosine treatment of MCF-7 and cells with wild type PIK3CA and AKT1, mutant PIK3CA, or mutant AKT1 genes.

Targeted therapies against somatically altered genes are currently used for the treatment of many human cancers. The nascent technology known as BEAMing has the potential to increase the clinical utility of these agents because it allows for the detection of cancer mutations in peripheral blood, providing a rapid assessment of tumor mutation status. Clin Cancer Res; 17(24); 7508–10. ©2011 AACR.

PDF file - 395KB, Figure S1. Pre-surgery plasma ddPCR analysis for PIK3CA mutations. Figure S2. Positive and negative controls for droplet thresholds and counts. Figure S3. Lower Limit of Detection for PIK3CA Exon 9. Supplementary Table S1: Primers used to amplify and sequence FFPE specimens. Supplementary Table S2: Primers and probes used for ddPCR on FFPE samples. Supplementary Table S3: Primers and probes for ddPCR of plasma DNA. Supplementary Table S4: Fractional abundance and 95% confidence intervals of positive ptDNA samples.

Purpose: Detecting circulating plasma tumor DNA (ptDNA) in patients with early-stage cancer has the potential to change how oncologists recommend systemic therapies for solid tumors after surgery. Droplet digital polymerase chain reaction (ddPCR) is a novel sensitive and specific platform for mutation detection.Experimental Design: In this prospective study, primary breast tumors and matched pre- and postsurgery blood samples were collected from patients with early-stage breast cancer (n = 29). Tumors (n = 30) were analyzed by Sanger sequencing for common PIK3CA mutations, and DNA from these tumors and matched plasma were then analyzed for PIK3CA mutations using ddPCR.Results: Sequencing of tumors identified seven PIK3CA exon 20 mutations (H1047R) and three exon 9 mutations (E545K). Analysis of tumors by ddPCR confirmed these mutations and identified five additional mutations. Presurgery plasma samples (n = 29) were then analyzed for PIK3CA mutations using ddPCR. Of the 15 PIK3CA mutations detected in tumors by ddPCR, 14 of the corresponding mutations were detected in presurgical ptDNA, whereas no mutations were found in plasma from patients with PIK3CA wild-type tumors (sensitivity 93.3%, specificity 100%). Ten patients with mutation-positive ptDNA presurgery had ddPCR analysis of postsurgery plasma, with five patients having detectable ptDNA postsurgery.Conclusions: This prospective study demonstrates accurate mutation detection in tumor tissues using ddPCR, and that ptDNA can be detected in blood before and after surgery in patients with early-stage breast cancer. Future studies can now address whether ptDNA detected after surgery identifies patients at risk for recurrence, which could guide chemotherapy decisions for individual patients. Clin Cancer Res; 20(10); 2643–50. ©2014 AACR.

Purpose: Mutations in the estrogen receptor (ER)α gene, ESR1, have been identified in breast cancer metastases after progression on endocrine therapies. Because of limitations of metastatic biopsies, the reported frequency of ESR1 mutations may be underestimated. Here, we show a high frequency of ESR1 mutations using circulating plasma tumor DNA (ptDNA) from patients with metastatic breast cancer.Experimental Design: We retrospectively obtained plasma samples from eight patients with known ESR1 mutations and three patients with wild-type ESR1 identified by next-generation sequencing (NGS) of biopsied metastatic tissues. Three common ESR1 mutations were queried for using droplet digital PCR (ddPCR). In a prospective cohort, metastatic tissue and plasma were collected contemporaneously from eight ER-positive and four ER-negative patients. Tissue biopsies were sequenced by NGS, and ptDNA ESR1 mutations were analyzed by ddPCR.Results: In the retrospective cohort, all corresponding mutations were detected in ptDNA, with two patients harboring additional ESR1 mutations not present in their metastatic tissues. In the prospective cohort, three ER-positive patients did not have adequate tissue for NGS, and no ESR1 mutations were identified in tissue biopsies from the other nine patients. In contrast, ddPCR detected seven ptDNA ESR1 mutations in 6 of 12 patients (50%).Conclusions: We show that ESR1 mutations can occur at a high frequency and suggest that blood can be used to identify additional mutations not found by sequencing of a single metastatic lesion. Clin Cancer Res; 22(4); 993–9. ©2015 AACR.

Supplementary Methods Figure S1 ESR1 Y537SY537N and D538G probes are mutation specific. Figure S2 Patient 1 and Patient 14's plasma DNA. Table S1 Additional patient characteristics. Table S2 Primers used in this study.

Figure S1 shows the dose-dependent inhibition of proliferation of T47D cells with wild type or mutant ERα by antiestrogen compounds. Figure S2 shows that the three antiestrogen compounds (K-07, K-09, K-62) and trans-hydroxytamoxifen suppress binding of the coregulator SRC3 to wild type ER and mutant ERs (Y537S, D538G). Figure S3 shows that antiestrogen compounds suppress the proliferation of MCF7 cells or T47D cells containing half mutant and half wild type ERα. Figure S4 shows that antiestrogen compounds induce ERα down-regulation in MCF7 and T47D cells containing half mutant and half wild type ERα. Figure S5 shows that antiestrogen compounds suppress ERα-regulated gene expressions in MCF7 and T47D cells containing half mutant and half wild type ERα. Figure S6 shows that treatment with antiestrogen compounds does not affect the body weights of mice bearing MCF7 xenograft tumors.

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

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 - 1821 KB, Inducible expression of v-SRC induces invasive behavior in MCF10A cells. (A) Constitutive SRC activity induces epithelial invasion in MCF10A acinar cultures.

PDF - 1578 KB, BEZ235 functions as mTOR inhibitor, without evidence of PI3K inhibition, and induces invasive protrusions in MCF10A cultures. (A) BEZ235 inhibits mTORC1/mTORC2 without inhibition of PI3K in MCF10A acini.

Supplementary Figure 1 from Deletion of PTEN Promotes Tumorigenic Signaling, Resistance to Anoikis, and Altered Response to Chemotherapeutic Agents in Human Mammary Epithelial Cells

Supplementary Figure Legends 1-2 from Deletion of PTEN Promotes Tumorigenic Signaling, Resistance to Anoikis, and Altered Response to Chemotherapeutic Agents in Human Mammary Epithelial Cells

Supplementary Figure 2 from Deletion of PTEN Promotes Tumorigenic Signaling, Resistance to Anoikis, and Altered Response to Chemotherapeutic Agents in Human Mammary Epithelial Cells

Oncogenic signaling pathways are tightly regulated by negative feedback circuits and relief of these circuits represents a common mechanism of tumor drug resistance. Although the significance of these feedback pathways for signal transduction is evident, their relevance for cellular differentiation and morphogenesis in a genetically defined context is unclear. In this study, we used isogenic benign mammary organotypic cultures to interrogate the role of mTOR-mediated negative feedback in the specific setting of PTEN inactivation. We found that mTOR signaling promoted basal-like differentiation and repressed nuclear hormone receptor expression after short-term PTEN loss in murine cell cultures analyzed ex vivo. Unexpectedly, we found that PTEN inactivation inhibited growth factor-induced epithelial invasion and that downstream mTOR-mediated signaling feedback was both necessary and sufficient for this effect. Mechanistically, using isogenic MCF10A cells with and without somatic PTEN deletion, we showed that mTOR inhibition promoted EGF-mediated epithelial invasion by derepressing upstream EGF receptor, SRC tyrosine kinase, and phosphoinositide 3-kinase signaling. In addition to offering new signal transduction insights, these results bring to light a number of important and potentially clinically relevant cellular consequences of mTOR inhibition in the specific context of PTEN loss, including modulation of hormone and growth factor responsiveness and promotion of epithelial invasion. Our findings prompt future investigations of the possibility that mTOR inhibitor therapy may not only be ineffective but even deleterious in tumors with PTEN loss. Cancer Res; 73(16); 5218–31. ©2013 AACR.

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.

MOV - 6269 KB, Growth factor-induced epithelial budding and invasion of ECM in mammary organoid system. 100-hour time-lapse DIC imaging (days 3-7 after addition of growth factor) of FGF2-induced epithelial budding in a wild-type mammary organoid. Luminal epithelial buds invade beyond the myoepithelial layer and into surrounding Matrigel, recapitulating normal ductal morphogenesis and the early stages of invasive breast cancer.

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]

PDF - 915 KB, Short-term PTEN loss induces independence from endogenous HER signaling in mammary organoids.

PDF file - 2823K, Supplementary Figure 1. Estrogen metabolites cause DNA double strand breaks in S- phase cells. Supplementary Figure 2. BRCA1 supresses estrogen metabolite mediated DSBs and is required for their repair. Supplementary Figure 3. Estrogen and its metabolites induce DNA DSBs in a dose and time dependent manner. Supplementary Figure 4. BRCA1 suppresses estrogen metabolites induced DNA damage in BRCA1 mutant breast cancer cells. Supplementary Figure 5. BRCA1 and BRCA2 are required for repair of estrogen metabolite induced DSBs in MCF10A and MCF7 breast cells. Supplementary Figure 6. BRCA1, but not BRCA2, suppresses estrogen metabolite mediated DNA damage by repressing CYP1A1 expression in breast cells.

PDF - 4796K, In vitro Cre induction is efficient and does not inhibit morphogenesis using the R26CreER system in mammary organoid cultures.

PDF - 3219 KB, (A) DIC imaging of MCF10A clones cultured for 24 hours on in pure Matrigel. Scale bars = 100 and 20 microm. (B) Quantification of invasive protrusion formation after 24 hours of culture on Matrigel. (represents mean and SEM for 3 independent replicates).

PDF file - 124K

Germline mutations in BRCA1 predispose carriers to a high incidence of breast and ovarian cancers. BRCA1 functions to maintain genomic stability through critical roles in DNA repair, cell-cycle arrest, and transcriptional control. A major question has been why BRCA1 loss or mutation leads to tumors mainly in estrogen-regulated tissues, given that BRCA1 has essential functions in all cell types. Here, we report that estrogen and estrogen metabolites can cause DNA double-strand breaks (DSB) in estrogen receptor-α–negative breast cells and that BRCA1 is required to repair these DSBs to prevent metabolite-induced genomic instability. We found that BRCA1 also regulates estrogen metabolism and metabolite-mediated DNA damage by repressing the transcription of estrogen-metabolizing enzymes, such as CYP1A1, in breast cells. Finally, we used a knock-in human cell model with a heterozygous BRCA1 pathogenic mutation to show how BRCA1 haploinsufficiency affects these processes. Our findings provide pivotal new insights into why BRCA1 mutation drives the formation of tumors in estrogen-regulated tissues, despite the general role of BRCA1 in DNA repair in all cell types. Cancer Res; 74(10); 2773–84. ©2014 AACR.

MOV - 1654 KB, Acinar morphogenesis in PTEN-/- MCF10A cells treated with rapamycin reveals abundant, stabilized invasive protrusions into ECM. 54-hour time-lapse DIC imaging (roughly days 1-3 after plating) of PTEN-/- MCF10A clone #1 treated with rapamycin (200 nM). Invasive cytoplasmic protrusions into surrounding collagen-Matrigel ECM are evident from the beginning and fail to retract, forming a web of interconnected ducts with cell migration and proliferation.

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Many cancers, including breast cancer, harbor loss-of-function mutations in the catalytic domain of phosphatase and tensin homologue deleted on chromosome 10 (PTEN) or have reduced PTEN expression through loss of heterozygosity and/or epigenetic silencing mechanisms. However, specific phenotypic effects of PTEN inactivation in human cancer cells remain poorly defined without a direct causal connection between the loss of PTEN function and the development or progression of cancer. To evaluate the biological and clinical relevance of reduced or deleted PTEN expression, a novel in vitro model system was generated using human somatic cell knockout technologies. Targeted homologous recombination allowed for a single and double allelic deletion, which resulted in reduced and deleted PTEN expression, respectively. We determined that heterozygous loss of PTEN in the nontumorigenic human mammary epithelial cell line MCF-10A was sufficient for activation of the phosphoinositide 3-kinase/AKT and mitogen-activated protein kinase pathways, whereas the homozygous absence of PTEN expression led to a further increased activation of both pathways. The deletion of PTEN was able to confer growth factor–independent proliferation, which was confirmed by the resistance of the PTEN−/− MCF-10A cells to small-molecule inhibitors of the epidermal growth factor receptor. However, neither heterozygous nor homozygous loss of PTEN expression was sufficient to promote anchorage-independent growth, but the loss of PTEN did confer apoptotic resistance to cell rounding and matrix detachment. Finally, MCF-10A cells with the reduction or loss of PTEN showed increased susceptibility to the chemotherapeutic drug doxorubicin but not paclitaxel. [Cancer Res 2009;69(21):8275–83]