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PDF file - 603K, Supplemental Figure S1: Gene maps of the BRCA1 (A) and BRCA2 (B) CpG islands examined with bisulfite pyrosequencing. Supplemental Figure S2: Anchorage independent growth inhibition at 1μM rucaparib in soft agar. Of the 39 tested ovarian cancer cell lines 22 formed colonies in soft agar. Supplemental Figure S3: Microarray hybridizations of the cell line panel were performed using the Agilent Whole Human Genome 4X44K array chip.Supplemental Figure S4: Chemical structures of compounds used for the drug interaction studies.Supplemental Figure S5: A multivariate ANOVA model (accounting for type of chemotherapy, cell line, and drug concentration as single factors) indicates that the drug combination effects differed significantly between individual chemotherapeutic agents (P=0.010). Supplemental Figure S6: A multivariate ANOVA model (accounting for type of chemotherapy, cell line, and drug concentration as single factors) indicates that the best combination effect was achieved at lower topotecan (0.031 μM) and rucaparib (0.625 μM) drug concentrations (P=0.018).

PDF file - 221K, E-cadherin and beta-catenin expression in SKBR3 and SKBR3/100-8 cells

PDF file - 53K, Confirmation of Wnt pathway gene expression by RT-Q-PCR

PDF file, 77K, Comparison of the effect of dacomitinib at 48 hrs and 5 days on cell cycle and apoptosis in BT-474 and SK-BR-3 lines.

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PDF file, 60K, Effect of dacomitinib on apoptosis in BT-474 and SK-BR-3 parental lines and lines resistant to trastuzumab and lapatinib.

PDF file, 68K, Quantification of proteins from Western blots in Figure 3 by densitometry using ImageJ software.

PDF file - 1MB, Pathway signaling differentially expressed in SKBR3/100-8 compared to SKBR3 revealed by IPA

Here, we investigate the potential role of the PARP inhibitor rucaparib (CO-338, formerly known as AG014699 and PF-01367338) for the treatment of sporadic ovarian cancer. We studied the growth inhibitory effects of rucaparib in a panel of 39 ovarian cancer cell lines that were each characterized for mutation and methylation status of BRCA1/2, baseline gene expression signatures, copy number variations of selected genes, PTEN status, and sensitivity to platinum-based chemotherapy. To study interactions with chemotherapy, we used multiple drug effect analyses and assessed apoptosis, DNA fragmentation, and γH2AX formation. Concentration-dependent antiproliferative effects of rucaparib were seen in 26 of 39 (67%) cell lines and were not restricted to cell lines with BRCA1/2 mutations. Low expression of other genes involved in homologous repair (e.g., BCCIP, BRCC3, ATM, RAD51L1), amplification of AURKA or EMSY, and response to platinum-based chemotherapy was associated with sensitivity to rucaparib. Drug interactions with rucaparib were synergistic for topotecan, synergistic, or additive for carboplatin, doxorubicin or paclitaxel, and additive for gemcitabine. Synergy was most pronounced when rucaparib was combined with topotecan, which resulted in enhanced apoptosis, DNA fragmentation, and γH2AX formation. Importantly, rucaparib potentiated chemotherapy independent of its activity as a single agent. PARP inhibition may be a useful therapeutic strategy for a wider range of ovarian cancers bearing deficiencies in the homologous recombination pathway other than just BRCA1/2 mutations. These results support further clinical evaluation of rucaparib either as a single agent or as an adjunct to chemotherapy for the treatment of sporadic ovarian cancer. Mol Cancer Ther; 12(6); 1002–15. ©2013 AACR.

The human EGF (HER) family of receptors has been pursued as therapeutic targets in breast cancer and other malignancies. Trastuzumab and lapatinib are standard treatments for HER2-amplified breast cancer, but a significant number of patients do not respond or develop resistance to these drugs. Here we evaluate the in vitro activity of dacomitinib (PF-00299804), an irreversible small molecule pan-HER inhibitor, in a large panel of human breast cancer cell lines with variable expression of the HER family receptors and ligands, and with variable sensitivity to trastuzumab and lapatinib. Forty-seven human breast cancer and immortalized breast epithelial lines representing the known molecular subgroups of breast cancer were treated with dacomitinib to determine IC50 values. HER2-amplified lines were far more likely to respond to dacomitinib than nonamplified lines (RR, 3.39; P < 0.0001). Furthermore, HER2 mRNA and protein expression were quantitatively associated with response. Dacomitinib reduced the phosphorylation of HER2, EGFR, HER4, AKT, and ERK in the majority of sensitive lines. Dacomitinib exerted its antiproliferative effect through a combined G0–G1 arrest and an induction of apoptosis. Dacomitinib inhibited growth in several HER2-amplified lines with de novo and acquired resistance to trastuzumab. Dacomitinib maintained a high activity in lines with acquired resistance to lapatinib. This study identifies HER2-amplified breast cancer lines as most sensitive to the antiproliferative effect of dacomitinib and provides a strong rationale for its clinical testing in HER2-amplified breast cancers resistant to trastuzumab and lapatinib. Mol Cancer Ther; 11(9); 1978–87. ©2012 AACR.

PDF file, 247K, The effects of dacomitinib on protein expression in lines resistant to trastuzumab and lapatinib.

PDF file - 43K, siRNA knocked down EGFR in SKBR3/100-8 cells

PDF file, 75K, Dacomitinib response is marginally inversely correlated with relative HER3 mRNA levels.

PDF file, 50K, Effect of dacomitinib on cell cycle in BT-474 and SK-BR-3 parental lines and lines resistant to trastuzumab and lapatinib.

PDF file, 45K, Dacomitinib response is associated with the total HER2 protein levels in HER2-amplified cell lines.

PDF file, 66K, Quantification of proteins from Western blots in Supplementary Figure S7 by densitometry using ImageJ software.

PDF file - 66K, Cell lines and culture media.

PDF file - 59K, siRNA knocked down HER3 sensitizes SKBR3/100-8 cell response to trastuzumab-induced cell growth inhibition

PDF file, 60K, Dacomitinib response is associated with relative HER2 mRNA levels.

Supplementary Table 2 from Activated Phosphoinositide 3-Kinase/AKT Signaling Confers Resistance to Trastuzumab but not Lapatinib

Supplementary Table 1 from Activated Phosphoinositide 3-Kinase/AKT Signaling Confers Resistance to Trastuzumab but not Lapatinib

Selumetinib (AZD6244; ARRY-142886) is a tight-binding, uncompetitive inhibitor of mitogen-activated protein kinase kinases (MEK) 1 and 2 currently in clinical development. We evaluated the effects of selumetinib in 31 human breast cancer cell lines and 43 human non–small cell lung cancer (NSCLC) cell lines to identify characteristics correlating with in vitro sensitivity to MEK inhibition. IC50 raf mutations in breast cancer cell lines (P = 0.022) and ras mutations in NSCLC cell lines (P = 0.045). Evaluation of 27 of the NSCLC cell lines with Western blots showed no clear association between MEK and phosphoinositide 3-kinase pathway activation and sensitivity to MEK inhibition. Baseline gene expression profiles were generated for each cell line using Agilent gene expression arrays to identify additional predictive markers. Genes associated with differential sensitivity to selumetinib were seen in both histologies, including a small number of genes in which differential expression was common to both histologies. In total, these results suggest that clinical trials of selumetinib in breast cancer and NSCLC might select patients whose tumors harbor raf and ras mutations, respectively. Mol Cancer Ther; 9(7); 1985–94. ©2010 AACR.

Supplementary Data from Identification of Common Predictive Markers of In vitro Response to the Mek Inhibitor Selumetinib (AZD6244; ARRY-142886) in Human Breast Cancer and Non–Small Cell Lung Cancer Cell Lines

Supplementary Figure S1; Supplementary Table S1; Supplementary Tables S3-S4.

Purpose: PD-0332991 is a selective inhibitor of the CDK4/6 kinases with the ability to block retinoblastoma (Rb) phosphorylation in the low nanomolar range. Here we investigate the role of CDK4/6 inhibition in human ovarian cancer.Experimental Design: We examined the effects of PD-0332991 on proliferation, cell-cycle, apoptosis, and Rb phosphorylation using a panel of 40 established human ovarian cancer cell lines. Molecular markers for response prediction, including p16 and Rb, were studied using gene expression profiling, Western blot, and array CGH. Multiple drug effect analysis was used to study interactions with chemotherapeutic drugs. Expression of p16 and Rb was studied using immunohistochemistry in a large clinical cohort of ovarian cancer patients.Results: Concentration-dependent antiproliferative effects of PD-0332991 were seen in all ovarian cancer cell lines, but varied significantly between individual lines. Rb-proficient cell lines with low p16 expression were most responsive to CDK4/6 inhibition. Copy number variations of CDKN2A, RB, CCNE1, and CCND1 were associated with response to PD-0332991. CDK4/6 inhibition induced G0/G1 cell cycle arrest, blocked Rb phosphorylation in a concentration-and time-dependent manner, and enhanced the effects of chemotherapy. Rb-proficiency with low p16 expression was seen in 97/262 (37%) of ovarian cancer patients and was independently associated with poor progression-free survival (adjusted relative risk 1.49, 95% CI 1.00–2.24, P = 0.052).Conclusions: PD-0332991 shows promising biologic activity in ovarian cancer cell lines. Assessment of Rb and p16 expression may help select patients most likely to benefit from CDK4/6 inhibition in ovarian cancer. Clin Cancer Res; 17(6); 1591–602. ©2011 AACR.

Trastuzumab and lapatinib provide clinical benefit to women with human epidermal growth factor receptor 2 (HER)–positive breast cancer. However, not all patients whose tumors contain the HER2 alteration respond. Consequently, there is an urgent need to identify new predictive factors for these agents. The aim of this study was to investigate the role of receptor tyrosine kinase signaling and phosphoinositide 3-kinase (PI3K)/AKT pathway activation in conferring resistance to trastuzumab and lapatinib. To address this question, we evaluated response to trastuzumab and lapatinib in a panel of 18 HER2-amplified cell lines, using both two- and three-dimensional culture. The SUM-225, HCC-1419, HCC-1954, UACC-893, HCC-1569, UACC-732, JIMT-1, and MDA-453 cell lines were found to be innately resistant to trastuzumab, whereas the MDA-361, MDA-453, HCC-1569, UACC-732, JIMT-1, HCC-202, and UACC-893 cells are innately lapatinib resistant. Lapatinib was active in de novo (SUM-225, HCC-1419, and HCC-1954) and in a BT-474 cell line with acquired resistance to trastuzumab. In these cells, trastuzumab had little effect on AKT phosphorylation, whereas lapatinib retained activity through the dephosphorylation of AKT. Increased phosphorylation of HER2, epidermal growth factor receptor, HER3, and insulin-like growth factor IR correlated with response to lapatinib but not trastuzumab. Loss of PTEN or the presence of activating mutations in PI3K marked resistance to trastuzumab, but lapatinib response was independent of these factors. Thus, increased activation of the PI3K/AKT pathway correlates with resistance to trastuzumab, which can be overcome by lapatinib. In conclusion, pharmacologic targeting of the PI3K/AKT pathway may provide benefit to HER2-positive breast cancer patients who are resistant to trastuzumab therapy. Mol Cancer Ther; 9(6); 1489–502. ©2010 AACR.

Supplementary Table S2.

Background Lapatinib has clinical efficacy in the treatment of trastuzumab-refractory HER2-positive breast cancer. However, a significant proportion of patients develop progressive disease due to acquired resistance to the drug. Induction of apoptotic cell death is a key mechanism of action of lapatinib in HER2-positive breast cancer cells. Methods We examined alterations in regulation of the intrinsic and extrinsic apoptosis pathways in cell line models of acquired lapatinib resistance both in vitro and in patient samples from the NCT01485926 clinical trial, and investigated potential strategies to exploit alterations in apoptosis signalling to overcome lapatinib resistance in HER2-positive breast cancer. Results In this study, we examined two cell lines models of acquired lapatinib resistance (SKBR3-L and HCC1954-L) and showed that lapatinib does not induce apoptosis in these cells. We identified alterations in members of the BCL-2 family of proteins, in particular MCL-1 and BAX, which may play a role in resistance to lapatinib. We tested the therapeutic inhibitor obatoclax, which targets MCL-1. Both SKBR3-L and HCC1954-L cells showed greater sensitivity to obatoclax-induced apoptosis than parental cells. Interestingly, we also found that the development of acquired resistance to lapatinib resulted in acquired sensitivity to TRAIL in SKBR3-L cells. Sensitivity to TRAIL in the SKBR3-L cells was associated with reduced phosphorylation of AKT, increased expression of FOXO3a and decreased expression of c-FLIP. In SKBR3-L cells, TRAIL treatment caused activation of caspase 8, caspase 9 and caspase 3/7. In a second resistant model, HCC1954-L cells, p-AKT levels were not decreased and these cells did not show enhanced sensitivity to TRAIL. Furthermore, combining obatoclax with TRAIL improved response in SKBR3-L cells but not in HCC1954-L cells. Conclusions Our findings highlight the possibility of targeting altered apoptotic signalling to overcome acquired lapatinib resistance, and identify potential novel treatment strategies, with potential biomarkers, for HER2-positive breast cancer that is resistant to HER2 targeted therapies. Electronic supplementary material The online version of this article (10.1186/s12885-018-4852-1) contains supplementary material, which is available to authorized users.

Aurora kinases play important roles in cell division and are frequently overexpressed in human cancer. AMG 900 is a novel pan-Aurora kinase inhibitor currently being tested in Phase I clinical trials. We aimed to evaluate the in vitro activity of AMG 900 in a panel of 44 human breast cancer and immortalized cell lines and identify predictors of response. AMG 900 inhibited proliferation at low nanomolar concentrations in all cell lines tested. Response was further classified based on the induction of lethality. 25 cell lines were classified as highly sensitive (lethality at 10 nM of AMG 900 10 %), 19 cell lines as less sensitive to AMG 900 (lethality at 10 nM of AMG 900 10 %). Traditional molecular subtypes of breast cancer did not predict for this differential response. There was a weak association between AURKA amplification and response to AMG 900 (response ratio = 2.53, p = 0.09). mRNA expression levels of AURKA, AURKB, and AURKC and baseline protein levels of Aurora kinases A and B did not significantly associate with response. Cell lines with TP53 loss of function mutations (RR = 1.86, p = 0.004) and low baseline p21 protein levels (RR = 2.28, p = 0.0004) were far more likely to be classified as highly sensitive to AMG 900. AMG 900 induced p53 and p21 protein expression in cell lines with wt TP53. AMG 900 caused the accumulation of cells with4 N DNA content in a majority of cell lines independently of sensitivity and p53 status. AMG 900 induced more pronounced apoptosis in highly sensitive p53-dysfunctional cell lines. We have found that AMG 900 is highly active in breast cancer cell lines and that TP53 loss of function mutations as well as low baseline expression of p21 protein predict strongly for increased sensitivity to this compound in vitro.

Here, we investigate the potential role of the PARP inhibitor rucaparib (CO-338, formerly known as AG014699 and PF-01367338) for the treatment of sporadic ovarian cancer. We studied the growth inhibitory effects of rucaparib in a panel of 39 ovarian cancer cell lines that were each characterized for mutation and methylation status of BRCA1/2, baseline gene expression signatures, copy number variations of selected genes, PTEN status, and sensitivity to platinum-based chemotherapy. To study interactions with chemotherapy, we used multiple drug effect analyses and assessed apoptosis, DNA fragmentation, and γH2AX formation. Concentration-dependent antiproliferative effects of rucaparib were seen in 26 of 39 (67%) cell lines and were not restricted to cell lines with BRCA1/2 mutations. Low expression of other genes involved in homologous repair (e.g., BCCIP, BRCC3, ATM, RAD51L1), amplification of AURKA or EMSY, and response to platinum-based chemotherapy was associated with sensitivity to rucaparib. Drug interactions with rucaparib were synergistic for topotecan, synergistic, or additive for carboplatin, doxorubicin or paclitaxel, and additive for gemcitabine. Synergy was most pronounced when rucaparib was combined with topotecan, which resulted in enhanced apoptosis, DNA fragmentation, and γH2AX formation. Importantly, rucaparib potentiated chemotherapy independent of its activity as a single agent. PARP inhibition may be a useful therapeutic strategy for a wider range of ovarian cancers bearing deficiencies in the homologous recombination pathway other than just BRCA1/2 mutations. These results support further clinical evaluation of rucaparib either as a single agent or as an adjunct to chemotherapy for the treatment of sporadic ovarian cancer. Mol Cancer Ther; 12(6); 1002–15. ©2013 AACR.

Hepatocellular carcinoma (HCC) is the fifth most common malignancy and is the third leading cause of cancer death worldwide. Recently, the multitargeted kinase inhibitor sorafenib was shown to be the first systemic agent to improve survival in advanced HCC. Unlike other malignancies such as breast cancer, in which molecular subtypes have been clearly defined (i.e., luminal, HER2 amplified, basal, etc.) and tied to effective molecular therapeutics (hormone blockade and trastuzumab, respectively), in HCC this translational link does not exist. Molecular profiling studies of human HCC have identified unique molecular subtypes of the disease. We hypothesized that a panel of human HCC cell lines would maintain molecular characteristics of the clinical disease and could then be used as a model for novel therapeutics. Twenty human HCC cell lines were collected and RNA was analyzed using the Agilent microarray platform. Profiles from the cell lines in vitro recapitulate previously described subgroups from clinical material. Next, we evaluated whether molecular subgroup would have predictive value for response to the Src/Abl inhibitor dasatinib. The results demonstrate that sensitivity to dasatinib was associated with a progenitor subtype. Dasatinib was effective at inducing cell cycle arrest and apoptosis in “progenitor-like” cell lines but not in resistant lines. Conclusion: These findings suggest that cell line models maintain the molecular background of HCC and that subtype may be important for selecting patients for response to novel therapies. In addition, it highlights a potential role for Src family signaling in this progenitor subtype of HCC. (HEPATOLOGY 2013)

To understand the mechanisms leading to trastuzumab resistance in HER2-overexpressing breast tumors, we created trastuzumab-insensitive cell lines (SKBR3/100-8 and BT474/100-2). The cell lines maintain HER2 receptor overexpression and show increase in EGF receptor (EGFR). Upon trastuzumab treatment, SKBR3/100-8 and BT474/100-2 cell lines displayed increased growth rate and invasiveness. The trastuzumab resistance in SKBR3/100-8 and BT474/100-2 was accompanied with activation of the Wnt/β-catenin signaling pathway. Further investigation found that Wnt3 overexpression played a key role toward the development of trastuzumab resistance. The expression of Wnt3 in trastuzumab-resistant cells increased nuclear expression of β-catenin and transactivated expression of EGFR. The increased Wnt3 in the trastuzumab-resistant cells also promoted a partial EMT-like transition (epithelial-to-mesenchymal transition); increased N-cadherin, Twist, Slug; and decreased E-cadherin. Knockdown of Wnt3 by siRNA restored cytoplasmic expression of β-catenin and decreased EGFR expression in trastuzumab-resistant cells. Furthermore, the EMT markers were decreased, E-cadherin was increased, and the cell invasiveness was inhibited in response to the Wnt3 downregulation. Conversely, SKBR3 cells which had been stably transfected with full-length Wnt3 exhibited EMT-like transition. The Wnt3 transfectants, SKBR3/Wnt3-7 and SKBR3/Wnt3-9, showed a significant decrease in E-cadherin and increase in N-cadherin, Twist, and Slug. The cells were less sensitive to trastuzumab than parental SKBR3 and vector-transfected cells. In summary, our data suggest that Wnt3 overexpression activates Wnt/β-catenin signaling pathway that leads to transactivation of EGFR and promotes EMT-like transition. This could be an important mechanism leading to trastuzumab resistance in HER2-overexpressing breast cancer cells. Mol Cancer Res; 10(12); 1597–606. ©2012 AACR.

The human EGF (HER) family of receptors has been pursued as therapeutic targets in breast cancer and other malignancies. Trastuzumab and lapatinib are standard treatments for HER2-amplified breast cancer, but a significant number of patients do not respond or develop resistance to these drugs. Here we evaluate the in vitro activity of dacomitinib (PF-00299804), an irreversible small molecule pan-HER inhibitor, in a large panel of human breast cancer cell lines with variable expression of the HER family receptors and ligands, and with variable sensitivity to trastuzumab and lapatinib. Forty-seven human breast cancer and immortalized breast epithelial lines representing the known molecular subgroups of breast cancer were treated with dacomitinib to determine IC50 values. HER2-amplified lines were far more likely to respond to dacomitinib than nonamplified lines (RR, 3.39; P < 0.0001). Furthermore, HER2 mRNA and protein expression were quantitatively associated with response. Dacomitinib reduced the phosphorylation of HER2, EGFR, HER4, AKT, and ERK in the majority of sensitive lines. Dacomitinib exerted its antiproliferative effect through a combined G0–G1 arrest and an induction of apoptosis. Dacomitinib inhibited growth in several HER2-amplified lines with de novo and acquired resistance to trastuzumab. Dacomitinib maintained a high activity in lines with acquired resistance to lapatinib. This study identifies HER2-amplified breast cancer lines as most sensitive to the antiproliferative effect of dacomitinib and provides a strong rationale for its clinical testing in HER2-amplified breast cancers resistant to trastuzumab and lapatinib. Mol Cancer Ther; 11(9); 1978–87. ©2012 AACR.

Breast cancer is a heterogeneous disease that comprises multiple subtypes. Luminal subtype tumors confer a more favorable patient prognosis, which is, in part, attributed to estrogen receptor (ER)-α positivity and antihormone responsiveness. Expression of the forkhead box transcription factor, FOXA1, similarly correlates with the luminal subtype and patient survival, but is also present in a subset of ER-negative tumors. FOXA1 is also consistently expressed in luminal breast cancer cell lines even in the absence of ER. In contrast, breast cancer cell lines representing the basal subtype do not express FOXA1. To delineate an ER-independent role for FOXA1 in maintaining the luminal phenotype, and hence a more favorable prognosis, we performed expression microarray analyses on FOXA1-positive and ER-positive (MCF7, T47D), or FOXA1-positive and ER-negative (MDA-MB-453, SKBR3) luminal cell lines in the presence or absence of transient FOXA1 silencing. This resulted in three FOXA1 transcriptomes: (1) a luminal signature (consistent across cell lines), (2) an ER-positive signature (restricted to MCF7 and T47D) and (3) an ER-negative signature (restricted to MDA-MB-453 and SKBR3). Gene set enrichment analyses revealed FOXA1 silencing causes a partial transcriptome shift from luminal to basal gene expression signatures. FOXA1 binds to a subset of both luminal and basal genes within luminal breast cancer cells, and loss of FOXA1 increases enhancer RNA transcription for a representative basal gene (CD58). These data suggest FOXA1 directly represses a subset of basal signature genes. Functionally, FOXA1 silencing increases migration and invasion of luminal cancer cells, both of which are characteristics of basal subtype cells. We conclude FOXA1 controls plasticity between basal and luminal breast cancer cells, not only by inducing luminal genes but also by repressing the basal phenotype, and thus aggressiveness. Although it has been proposed that FOXA1-targeting agents may be useful for treating luminal tumors, these data suggest that this approach may promote transitions toward more aggressive cancers.

Objective Perostin (PN) has been found to be overexpressed in a variety of human malignancies including ovarian cancer. In the present study, we investigated PN expression status in a large cohort of ovarian tumors with the focus on biological influence of PN related on ovarian tumor angiogenesis and metastasis. Methods PN expression was determined by cDNA microarray, PN northern blot and PN IHC tissue array analyses. Exogenous PN expression in ovarian cancer cells OVCAR-3 and OV2008 were achieved through retroviral transfection and confirmed by PN western blot and ELISA. The effects of exogenous PN expression on tumor angiogenesis and metastatic growth were accessed in orthotopic mouse models. The in vitro cell adhesion, migration and invasion assays were performed to investigate the potential mechanisms involved in PN's in vivo effects. Results PN was frequently overexpressed in ovarian tumors. Higher PN levels significantly correlated with clinical late stages (III/IV) and cancer recurrence. PN was produced by engineered PN-overexpressing cells at levels comparable to that of A2780 cells, an ovarian carcinoma cell line with endogenous PN expression. PN overexpression did not change cell growth rates in vitro ; however it significantly promoted intraperitoneal tumor metastatic growth in immunodeficient mice, which was associated with increased tumor angiogenesis and decreased tumor cell apoptosis. In vitro purified PN promoted cell adhesion, migration, and invasion of both human umbilical endothelial cells (HUVECs) and/or ovarian cancer cells. Conclusions Our data indicate PN plays a critical role in both ovarian tumor angiogenesis and metastasis. Thus PN may represent a clinically effective new target for therapy of ovarian cancer.

Trastuzumab and lapatinib provide clinical benefit to women with human epidermal growth factor receptor 2 (HER)–positive breast cancer. However, not all patients whose tumors contain the HER2 alteration respond. Consequently, there is an urgent need to identify new predictive factors for these agents. The aim of this study was to investigate the role of receptor tyrosine kinase signaling and phosphoinositide 3-kinase (PI3K)/AKT pathway activation in conferring resistance to trastuzumab and lapatinib. To address this question, we evaluated response to trastuzumab and lapatinib in a panel of 18 HER2-amplified cell lines, using both two- and three-dimensional culture. The SUM-225, HCC-1419, HCC-1954, UACC-893, HCC-1569, UACC-732, JIMT-1, and MDA-453 cell lines were found to be innately resistant to trastuzumab, whereas the MDA-361, MDA-453, HCC-1569, UACC-732, JIMT-1, HCC-202, and UACC-893 cells are innately lapatinib resistant. Lapatinib was active in de novo (SUM-225, HCC-1419, and HCC-1954) and in a BT-474 cell line with acquired resistance to trastuzumab. In these cells, trastuzumab had little effect on AKT phosphorylation, whereas lapatinib retained activity through the dephosphorylation of AKT. Increased phosphorylation of HER2, epidermal growth factor receptor, HER3, and insulin-like growth factor IR correlated with response to lapatinib but not trastuzumab. Loss of PTEN or the presence of activating mutations in PI3K marked resistance to trastuzumab, but lapatinib response was independent of these factors. Thus, increased activation of the PI3K/AKT pathway correlates with resistance to trastuzumab, which can be overcome by lapatinib. In conclusion, pharmacologic targeting of the PI3K/AKT pathway may provide benefit to HER2-positive breast cancer patients who are resistant to trastuzumab therapy. Mol Cancer Ther; 9(6); 1489–502. ©2010 AACR.

Purpose To identify a biomarker of ovarian cancer response to chemotherapy. Patients and Methods Study participants had epithelial ovarian cancer treated with surgery followed by platinum-based chemotherapy. DNA and RNA were isolated from frozen tumors and normal DNA was isolated from matched peripheral blood. A whole-genome loss of heterozygosity (LOH) analysis was performed using a high-density oligonucleotide array. Candidate genomic areas that predicted enhanced response to chemotherapy were identified with Cox proportional hazards methods. Gene expression analyses were performed through microarray experiments. Candidate genes were tested for independent effects on survival using Cox proportional hazards models, Kaplan-Meier survival curves, and the log-rank test. Results Using a whole-genome approach to study the molecular determinants of ovarian cancer response to platinum-based chemotherapy, we identified LOH of a 13q region to predict prolonged progression-free survival (PFS; hazard ratio, 0.23; P = .006). ERCC5 was identified as a candidate gene in this region because of its known function in the nucleotide excision repair pathway, the unique DNA repair pathway that removes platinum-DNA adducts. We found LOH of the ERCC5 gene locus and downregulation of ERCC5 gene expression to predict prolonged PFS. Integration of genomic and gene expression data shows a correlation between 13q LOH and ERCC5 gene downregulation. Conclusion ERCC5 is a novel biomarker of ovarian cancer prognosis and a potential therapeutic target of ovarian cancer response to platinum chemotherapy.

Molecular classification of high-grade serous ovarian cancer (HGSOC) using transcriptional profiling has proven to be complex and difficult to validate across studies. We determined gene expression profiles of 174 well-annotated HGSOCs and demonstrate prognostic significance of the prespecified TCGA Network gene signatures. Furthermore, we confirm the presence of four HGSOC transcriptional subtypes using a de novo classification. Survival differed statistically significantly between de novo subtypes (log rank, P = .006) and was the best for the immunoreactive-like subtype, but statistically significantly worse for the proliferative- or mesenchymal-like subtypes (adjusted hazard ratio = 1.89, 95% confidence interval = 1.18 to 3.02, P = .008, and adjusted hazard ratio = 2.45, 95% confid ence interval = 1.43 to 4.18, P = .001, respectively). More prognostic information was provided by the de novo than the TCGA classification (Likelihood Ratio tests, P = .003 and P = .04, respectively). All statistical tests were two-sided. These findings were replicated in an external data set of 185 HGSOCs and confirm the presence of four prognostically relevant molecular subtypes that have the potential to guide therapy decisions. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Objective To identify molecular prognosticators and therapeutic targets for high-grade serous epithelial ovarian cancers (EOCs) using genetic analyses driven by biologic features of EOC pathogenesis. Methods Ovarian tissue samples (n = 172; 122 serous EOCs, 30 other EOCs, 20 normal/benign) collected prospectively from sequential patients undergoing gynecologic surgery were analyzed using RNA expression microarrays. Samples were classified based on expression of genes with potential relevance in ovarian cancer. Gene sets were defined using Rosetta Similarity Search Tool (ROAST) and analysis of variance (ANOVA). Gene copy number variations were identified by array comparative genomic hybridization. Results No distinct subgroups of EOC could be identified by unsupervised clustering, however, analyses based on genes correlated with periostin (POSTN) and estrogen receptor-alpha (ESR1) yielded distinct subgroups. When 95 high-grade serous EOCs were grouped by genes based on ANOVA comparing ESR1/WT1 and POSTN/TGFBI samples, overall survival (OS) was significantly shorter for 43 patients with tumors expressing genes associated with POSTN/TGFBI compared to 52 patients with tumors expressing genes associated with ESR1/WT1 (median 30 versus 49 months, respectively; P = 0.022). Several targets with therapeutic potential were identified within each subgroup. BRCA germline mutations were more frequent in the ESR1/WT1 subgroup. Proliferation-associated genes and TP53 status (mutated or wild-type) did not correlate with survival. Findings were validated using independent ovarian cancer datasets. Conclusions Two distinct molecular subgroups of high-grade serous EOCs based on POSTN/TGFBI and ESR1/WT1 expressions were identified with significantly different OS. Specific differentially expressed genes between these subgroups provide potential prognostic and therapeutic targets. © 2013 Elsevier Inc. All rights reserved.

PD-0332991 is an inhibitor of cyclin-dependent kinases (CDK) 4 and 6, and was evaluated to determine its anti-proliferative effects in 25 renal cell carcinoma (RCC) cell lines.Half-maximal inhibitory concentrations (IC50) of PD-0332991 were determined with cell line proliferation assays, as were its effects on the cell cycle, apoptosis, and retinoblastoma (RB) phosphorylation. Molecular markers for response prediction, including p16, p15, cyclin D1 (CCND1), cyclin E1 (CCNE1), E2F transcription factor 1 (E2F1), RB, CDK4 and CDK6, were studied using array comparative genomic hybridization (CGH) and gene expression.IC50 values for PD-0332991 ranged from 25.0 nM to 700 nM, and the agent demonstrated G0/G1 cell-cycle arrest, induction of late apoptosis, and blockade of RB phosphorylation. Through genotype and expression data p16, p15 and E2F1 were identified as having significant association between loss and sensitivity to PD-0332991: p16 (p=0.021), p15 (p=0.047), and E2F1 (p=0.041).PD-0332991 has antiproliferative activity in RCC cell lines, and molecular markers predict for sensitivity to this agent.

The design, construction and operation of a four-color capillary array electrophoresis scanner are presented. The use of sensitive energy transfer primers facilitates four-color detection of the DNA sequencing fragments following excitation at a single laser wavelength (488 nm). This scanner collects fluorescence data from up to 25 capillaries in parallel. The resulting four-color image files are automatically reduced to four-color line plots, and a base-calling program (Sax) is used to call the sequence. The performance of this system for DNA sequencing is demonstrated by examining twelve different motifs of the hypervariable region I of human mitochondrial (mt) DNA obtained from a Sierra Leone population.

Purpose: PD-0332991 is a selective inhibitor of the CDK4/6 kinases with the ability to block retinoblastoma (Rb) phosphorylation in the low nanomolar range. Here we investigate the role of CDK4/6 inhibition in human ovarian cancer. Experimental Design: We examined the effects of PD-0332991 on proliferation, cell-cycle, apoptosis, and Rb phosphorylation using a panel of 40 established human ovarian cancer cell lines. Molecular markers for response prediction, including p16 and Rb, were studied using gene expression profiling, Western blot, and array CGH. Multiple drug effect analysis was used to study interactions with chemotherapeutic drugs. Expression of p16 and Rb was studied using immunohistochemistry in a large clinical cohort of ovarian cancer patients. Results: Concentration-dependent antiproliferative effects of PD-0332991 were seen in all ovarian cancer cell lines, but varied significantly between individual lines. Rb-proficient cell lines with low p16 expression were most responsive to CDK4/6 inhibition. Copy number variations of CDKN2A, RB, CCNE1, and CCND1 were associated with response to PD-0332991. CDK4/6 inhibition induced G0/G1 cell cycle arrest, blocked Rb phosphorylation in a concentration-and time-dependent manner, and enhanced the effects of chemotherapy. Rb-proficiency with low p16 expression was seen in 97/262 (37%) of ovarian cancer patients and was independently associated with poor progression-free survival (adjusted relative risk 1.49, 95% CI 1.00–2.24, P = 0.052). Conclusions: PD-0332991 shows promising biologic activity in ovarian cancer cell lines. Assessment of Rb and p16 expression may help select patients most likely to benefit from CDK4/6 inhibition in ovarian cancer. Clin Cancer Res; 17(6); 1591–602. ©2011 AACR.

Well-differentiated/de-differentiated liposarcomas (WDLS/DDLS) encompass an intriguing disease model in which a temporal intersection occurs between the malignant transformation of mesenchymal cells and the process of adipogenesis. Deciphering the molecular events that trigger and are characteristic of the intersection of these oncogenic and normal processes is critical to affect the often morbid and lethal consequences of malignant tumors of fat. High-resolution genome-wide oligonucleotide array-based comparative genomic hybridization (aCGH) with matched gene expression analyses was performed on seven lipomas, one hibernoma, and 38 WD and DDLS to define and compare the genomic events associated with these tumors. WD and DDLS had complex karyotypes. On average, WDLS had 11.1 and DDLS had 22.7 chromosomal copy number aberrations. All of the liposarcomas had 12q13-q15 amplifications with varying peaks at CDK4 (12q14.1), HMGA2 (12q14.3), and MDM2 (12q15); 24% of the DDLS and no WDLS had 1p32.2 (JUN) amplifications; 33% WDLS and 35% DDLS had 1q24.3 amplifications involving DNM3 and miR-214/miR-199a2; 24% of the liposarcomas had 6q23-q24 amplifications (including MAP3K5). Amplifications in GLI1 (12q13.3), JUN, and MAP3K5 (6q23.3) were mutually exclusive and occurred predominately in the DDLS. 6q amplifications occurred primarily in retroperitoneal tumors and females represented the majority of those patients who developed fatty tumors prior to the age of 50 years old. This detailed genetic mapping provides insight into the heterogeneity of WD and DDLS and the chromosomal and genetic abnormalities that are present in and distinguish these mesenchymal malignancies.

Selumetinib (AZD6244; ARRY-142886) is a tight-binding, uncompetitive inhibitor of mitogen-activated protein kinase kinases (MEK) 1 and 2 currently in clinical development. We evaluated the effects of selumetinib in 31 human breast cancer cell lines and 43 human non–small cell lung cancer (NSCLC) cell lines to identify characteristics correlating with in vitro sensitivity to MEK inhibition. IC50