Aberrations in oncogenes and tumor suppressors frequently affect the activity of critical signal transduction pathways. To analyze systematically the relationship between the activation status of protein networks and other characteristics of cancer cells, we performed reverse phase protein array (RPPA) profiling of the NCI60 cell lines for total protein expression and activation-specific markers of critical signaling pathways. To extend the scope of the study, we merged those data with previously published RPPA results for the NCI60. Integrative analysis of the expanded RPPA data set revealed 5 major clusters of cell lines and 5 principal proteomic signatures. Comparison of mutations in the NCI60 cell lines with patterns of protein expression demonstrated significant associations for PTEN, PIK3CA, BRAF and APC mutations with proteomic clusters. PIK3CA and PTEN mutation enrichment were not cell lineage-specific but were associated with dominant yet distinct groups of proteins. The five RPPA-defined clusters were strongly associated with sensitivity to standard anti-cancer agents. RPPA analysis identified 27 protein features significantly associated with sensitivity to paclitaxel. The functional status of those proteins was interrogated in a paclitaxel whole genome siRNA library synthetic lethality screen, and confirmed the predicted associations with drug sensitivity. These studies expand our understanding of the activation status of protein networks in the NCI60 cancer cell lines, demonstrate the importance of the direct study of protein expression and activation, and provide a basis for further studies integrating the information with other molecular and pharmacological characteristics of cancer.
NCI60; reverse phase protein arrays; signal transduction
Homologous recombination (HR) repair deficiency predisposes to cancer development, but also sensitizes cancer cells to DNA-damage-inducing therapeutics. Here we identify an HR-defect (HRD) gene signature, which can be used to functionally assess HR repair status without interrogating individual genetic alterations in cells. By using this HRD gene signature as a functional network analysis tool, we discover that simultaneous loss of two major tumor suppressors BRCA1 and PTEN extensively rewire the HR repair-deficient phenotype, which is found in cells with defects in either BRCA1 or PTEN alone. Moreover, the HRD gene signature serves as an effective drug discovery platform to identify agents targeting HR repair as potential chemo/radio-sensitizers. More importantly, this HRD gene signature is able to predict clinical outcomes across multiple cancer lineages. Our findings, therefore, provide a molecular profile of HR repair to assess its status at a functional network level, which can provide both biological insights and have clinical implications in cancer.
The prognosis of epithelial ovarian cancer is poor in part due to the high frequency of chemoresistance. Recent evidence points to the Toll-like receptor-4 (TLR4), and particularly its adaptor protein MyD88, as one potential mediator of this resistance. This study aims to provide further evidence that MyD88 positive cancer cells are clinically significant, stem-like and reproducibly detectable for the purposes of prognostic stratification. Expression of TLR4 and MyD88 was assessed immunohistochemically in 198 paraffin-embedded ovarian tissues and in an embryonal carcinoma model of cancer stemness. In parallel, expression of TLR4 and MyD88 mRNA and regulatory microRNAs (miR-21 and miR-146a) was assessed, as well as in a series of chemosensitive and resistant cancer cells lines. Functional analysis of the pathway was assessed in chemoresistant SKOV-3 ovarian cancer cells. TLR4 and MyD88 expression can be reproducibly assessed via immunohistochemistry using a semi-quantitative scoring system. TLR4 expression was present in all ovarian epithelium (normal and neoplastic), whereas MyD88 was restricted to neoplastic cells, independent of tumour grade and associated with reduced progression-free and overall survival, in an immunohistological specific subset of serous carcinomas, p<0.05. MiR-21 and miR-146a expression was significantly increased in MyD88 negative cancers (p<0.05), indicating their participation in regulation. Significant alterations in MyD88 mRNA expression were observed between chemosensitive and chemoresistant cells and tissue. Knockdown of TLR4 in SKOV-3 ovarian cells recovered chemosensitivity. Knockdown of MyD88 alone did not. MyD88 expression was down-regulated in differentiated embryonal carcinoma (NTera2) cells, supporting the MyD88+ cancer stem cell hypothesis. Our findings demonstrate that expression of MyD88 is associated with significantly reduced patient survival and altered microRNA levels and suggest an intact/functioning TLR4/MyD88 pathway is required for acquisition of the chemoresistant phenotype. Ex vivo manipulation of ovarian cancer stem cell (CSC) differentiation can decrease MyD88 expression, providing a potentially valuable CSC model for ovarian cancer.
Ovarian cancer is now recognized as a number of distinct diseases primarily defined by histological subtype. Both clear cell ovarian carcinomas (CCC) and ovarian endometrioid carcinomas (EC) may arise from endometriosis and frequently harbor mutations in the ARID1A tumor suppressor gene. We studied the influence of histological subtype on protein expression with reverse phase protein array (RPPA) and assessed proteomic changes associated with ARID1A mutation/BAF250a expression in EC and CCC.
Immunohistochemistry (IHC) for BAF250a expression was performed on 127 chemotherapy-naive ovarian carcinomas (33 CCC, 29 EC, and 65 high-grade serous ovarian carcinomas (HGSC)). Whole tumor lysates were prepared from frozen banked tumor samples and profiled by RPPA using 116 antibodies. ARID1A mutations were identified by exome sequencing, and PIK3CA mutations were characterized by MALDI-TOF mass spectrometry. SAM (Significance Analysis of Microarrays) was performed to determine differential protein expression by histological subtype and ARID1A mutation status. Multivariate logistic regression was used to assess the impact of ARID1A mutation status/BAF250a expression on AKT phosphorylation (pAKT). PIK3CA mutation type and PTEN expression were included in the model. BAF250a knockdown was performed in 3 clear cell lines using siRNA to ARID1A.
Marked differences in protein expression were observed that are driven by histotype. Compared to HGSC, SAM identified over 50 proteins that are differentially expressed in CCC and EC. These included PI3K/AKT pathway proteins, those regulating the cell cycle, apoptosis, transcription, and other signaling pathways including steroid hormone signaling. Multivariate models showed that tumors with loss of BAF250a expression showed significantly higher levels of AKT-Thr308 and AKT-Ser473 phosphorylation (p < 0.05). In 31 CCC cases, pAKT was similarly significantly increased in tumors with BAF250a loss on IHC. Knockdown of BAF250a by siRNA in three CCC cell lines wild type for ARID1A showed no increase in either pAKT-Thr308 or pAKT-S473 suggesting that pAKT in tumor tissues is indirectly regulated by BAF250a expression.
Proteomic assessment of CCC and EC demonstrates remarkable differences in protein expression that are dependent on histotype, thereby further characterizing these cancers. AKT phosphorylation is associated with ARID1A/BAF250a deficient tumors, however in ovarian cancers the mechanism remains to be elucidated.
Ovarian cancer; Proteomics; ARID1A/BAF250a; PIK3CA mutation; AKT; Phosphorylation
Platinum (Pt)-based antitumor agents are widely used in cancer chemotherapy. Drug resistance is a major obstacle to the successful use of these agents because once drug resistance develops, other effective treatment options are limited. Recently, we have conducted a clinical trial using a copper (Cu)-lowering agent to overcome Pt drug resistance in ovarian cancer patients and the preliminary results are encouraging. In supporting this clinical study, using three pairs of cisplatin (cDDP)-resistant cell lines and two ovarian cancer cell lines derived from patients who had failed in Pt-based chemotherapy, we demonstrated that cDDP resistance associated with reduced expression of the high affinity copper transporter (hCtr1) which is also a cDDP transporter, can be preferentially re-sensitized by copper-lowering agents due to enhanced hCtr1 expression, as compared with their drug-sensitive counterparts. Such a preferential induction of hCtr1 expression in cDDP-resistant variants by Cu chelation can be explained by the mammalian Cu homeostasis regulatory mechanism. Enhanced cell-killing efficacy by a Cu-lowering agent was also observed in animal xenografts bearing cDDP-resistant cells. Finally, by analyzing a public gene expression dataset, we found that ovarian cancer patients with elevated levels of hCtr1 in their tumors, but not ATP7A and ATP7B, had more favorable outcomes after Pt-drug treatment than those expressing low hCtr1 levels. This study reveals the mechanistic basis for using Cu chelation to overcome cDDP resistance in clinical investigations.
Cisplatin; high-affinity copper transporter; Cu-lowering agents; drug-resistance
Motivation: Network inference approaches are widely used to shed light on regulatory interplay between molecular players such as genes and proteins. Biochemical processes underlying networks of interest (e.g. gene regulatory or protein signalling networks) are generally nonlinear. In many settings, knowledge is available concerning relevant chemical kinetics. However, existing network inference methods for continuous, steady-state data are typically rooted in statistical formulations, which do not exploit chemical kinetics to guide inference.
Results: Herein, we present an approach to network inference for steady-state data that is rooted in non-linear descriptions of biochemical mechanism. We use equilibrium analysis of chemical kinetics to obtain functional forms that are in turn used to infer networks using steady-state data. The approach we propose is directly applicable to conventional steady-state gene expression or proteomic data and does not require knowledge of either network topology or any kinetic parameters. We illustrate the approach in the context of protein phosphorylation networks, using data simulated from a recent mechanistic model and proteomic data from cancer cell lines. In the former, the true network is known and used for assessment, whereas in the latter, results are compared against known biochemistry. We find that the proposed methodology is more effective at estimating network topology than methods based on linear models.
Supplementary data are available at Bioinformatics online.
Breast cancers of unusual histological subtype with an approximate incidence ≤1% are discussed. Each tumor subtype described represents a small but real cohort of patients with breast cancer, and although inferences may be made from this review, the management of each patient must be considered in the context of their unique clinical presentation and correlated with the evidence-based principles that apply to more common breast cancer histologies.
There is increased understanding of the heterogeneity of breast tumors, with greater emphasis now being placed on histological and molecular profiles and, in particular, their implications for prognosis and therapy. This review addresses breast cancers of unusual histological subtype with an approximate incidence ≤1%. Given the rarity of these tumors, the literature contains primarily case reports, small series, and population-based studies. Data are heterogeneous and almost entirely retrospective, frequently gathered over long time periods, in the context of changing pathological techniques and reporting. In addition, our understanding of the disease biology and therapeutic context has also evolved significantly over this time. There is often limited information about the specific therapies used and the rationale for choosing such an approach. Meaningful comparisons of treatment modalities are not feasible and it is not possible to define management guidelines. Instead, this review correlates the available information to give an impression of how each subgroup behaves—of the favored surgical technique, responses to therapy, and prognosis—as well as the emerging molecular data, highlighting new research areas for potential target in clinical trials. Each tumor subtype described represents a small but real cohort of patients with breast cancer, and although inferences may be made from this review, we are mindful of the paucity of data. The management of each patient must be considered in the context of their unique clinical presentation and correlated with the evidence-based principles that apply to more common breast cancer histologies.
Management; Histological types; Breast cancer
Most patients with non–small cell lung cancer (NSCLC) have responded poorly to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs). We investigated the involvement of insulin-like growth factor 1 receptor (IGF-1R) signaling in primary resistance to EGFR TKIs and the molecular determinants of resistance to IGF-1R TKIs.
Phosphorylated IGF-1R/insulin receptor (pIGF-1R/IR) was immunohistochemically evaluated in a NSCLC tissue microarray. We analyzed the antitumor effects of an IGF-1R TKI (PQIP or OSI-906), either alone or in combination with a small-molecular inhibitor (PD98059 or U0126) or with siRNA targeting K-Ras or MAPK/extracellular signal-regulated kinase kinase (MEK), in vitro and in vivo in NSCLC cells with variable histologic features and EGFR or K-Ras mutations.
pIGF-1R/IR expression in NSCLC specimens was associated with a history of tobacco smoking, squamous cell carcinoma histology, mutant (mut) K-Ras, and wild-type (wt) EGFR, all of which have been strongly associated with poor response to EGFR TKIs. IGF-1R TKIs exhibited significant antitumor activity in NSCLC cells with wt EGFR and wt K-Ras but not in those with mutations in these genes. Introduction of mut K-Ras attenuated the effects of IGF-1R TKIs on NSCLC cells expressing wt K-Ras. Conversely, inactivation of MEK restored sensitivity to IGF-TKIs in cells carrying mut K-Ras.
The mutation status of both EGFR and K-Ras could be predictive markers of response to IGF-1R TKIs. Also, MEK antagonism can abrogate primary resistance of NSCLC cells to IGF-1R TKIs.
EGFR; K-Ras; IGF-1R; lung cancer; TKI
On the basis of reversal of taxane resistance with AKT inhibition, we initiated a phase I trial of the AKT inhibitor perifosine with docetaxel in taxane and platinum-resistant or refractory epithelial ovarian cancer.
Patients with pathologically confirmed high-grade epithelial ovarian cancer (taxane resistant, n = 10; taxane refractory, n = 11) were enrolled. Peripheral blood samples and tumor biopsies were obtained and 18F-FDG-PET and DCE-MRI scans were performed for pharmacodynamic and imaging studies.
Patients received a total of 42 treatment cycles. No dose-limiting toxicity was observed. The median progression-free survival and overall survival were 1.9 months and 4.5 months, respectively. One patient with a PTEN mutation achieved a partial remission (PR) for 7.5 months, and another patient with PIK3CA mutation had stable disease (SD) for 4 months. Two other patients without apparent PI3K pathway aberrations achieved SD. Two patients with RAS mutations demonstrated rapid progression. Decreased phosphorylated S6 correlated with 18F-FDG-PET responses.
Patients tolerated perifosine 150 mg PO daily plus docetaxel at 75 mg/m2 every 4 weeks. Further clinical evaluation of effects of perifosine with docetaxel on biological markers and efficacy in patients with ovarian cancer with defined PI3K pathway mutational status is warranted.
Platelet hyperreactivity is associated with an increased risk of thrombosis. Cancer patients are at an increased risk of thrombosis, a risk that increases with disease progression. While cancer patients show evidence of platelet activation in vivo, few studies have extensively assessed whether these patients display platelet hyperreactivity. We hypothesized that patients with metastatic cancer would display platelet hyperreactivity, reflecting their associated high risk of thrombosis. In a cohort of patients with metastatic cancer (n = 13), we assessed platelet function using well-established assays of platelet reactivity (agonist-induced platelet aggregation, spontaneous platelet aggregation, and agonist-induced P-selectin expression). In comparison with healthy controls (n = 10), patients with metastatic cancer displayed global platelet hyperreactivity. Agonist-induced platelet aggregation responses to ADP (adenosine diphosphate), epinephrine, collagen, arachidonic acid, and PAR-1 (protease-activated receptor-1) activating peptide, as well as spontaneous platelet aggregation, were significantly increased in patients with metastatic cancer. Furthermore, agonist-induced platelet P-selectin expression was also significantly increased within the patient cohort. We demonstrate that patients with metastatic cancer are characterized by global platelet hyperreactivity, a factor that may contribute to their increased risk of thrombosis.
We assessed platelet function in a cohort of patients with metastatic cancer (n = 13) using well-established assays of platelet reactivity. Agonist-induced platelet aggregation and activation in response to platelet agonists, as well as spontaneous platelet aggregation, was significantly increased in cancer patients compared with healthy controls. We demonstrate that patients with metastatic cancer are characterized by global platelet hyperreactivity, a factor that may contribute to their increased risk of thrombosis.
Activation; aggregation; hyperreactivity; metastasis; platelets
To demonstrate the functional, clinical and biological significance of JNK-1 in ovarian carcinoma.
Analysis of the impact of JNK on 116 epithelial ovarian cancers was conducted. The role of JNK in vitro and in experimental models of ovarian cancer was assessed. We studied the role of WBZ_4, a novel JNK inhibitor redesigned from imatinib based on targeting wrapping defects, in cell lines and in experimental models of ovarian cancer.
We found a significant association of pJNK with progression free survival in the 116 epithelial ovarian cancers obtained at primary debulking therapy. WBZ_4 led to cell growth inhibition and increased apoptosis in a dose dependent fashion in four ovarian cancer cell lines. In vivo, while imatinib had no effect on tumor growth, WBZ_4 inhibited tumor growth in orthotopic murine models of ovarian cancer. The anti-tumor effect was further increased in combination with docetaxel. Silencing of JNK-1 with systemically administered siRNA led to significantly reduced tumor weights as compared to non-silencing siRNA controls, indicating that indeed the antitumor effects observed were due to JNK-1 inhibition.
These studies identify JNK-1 as an attractive therapeutic target in ovarian carcinoma and that the re-designed WBZ_4 compound should be considered for further clinical development.
JNK-1; Ovarian Cancer; Wrapping defects; Tyrosine Kinase inhibitor; Dehydrons
The phosphatidylinositol 3-kinase (PI3K)/PTEN/AKT/mTOR and Ras/Raf/MEK/ERK pathways have been implicated in endometrial tumorigenesis. In this candidate pathway analysis, we investigated associations between genetic variations in these two pathways and both risk and clinical outcomes of endometrial cancer.
We genotyped a total of 48 potentially functional SNPs in 11 key genes (AKT1, AKT2, AKT3, BRAF, FRAP1, KRAS, PDPK1, PIK3CA, PIK3CB, PIK3R1, and PTEN) with the Sequenom genotyping platform in 115 endometrial cancer patients and 230 cancer-free women to evaluate their associations with risk, survival, and recurrence of endometrial cancer.
We found the following: (1) PIK3CA rs6443624 and rs9838411 variants either borderline or significantly decreased risk of endometrial cancer in a dominant model (adjusted odds ratio [OR], 0.62; 95% CI, 0.39–1.00 and 0.59; 95% CI, 0.36–0.95, respectively). Furthermore, there was a statistically significant multiplicative interaction (Pint = 0.036) between these two loci in risk of endometrial cancer. In contrast, the AKT1 rs2498801 genotype significantly increased risk of endometrial cancer (adjusted OR, 1.94; 95% CI, 1.02–3.67 in a recessive model). (2) In Cox regression analyses, three SNPs (PIK3R1 rs1862162, AKT2 rs892119, and PIK3CA rs2699887) showed significant associations with survival of endometrial cancer patients. (3) KRAS rs7312175 and PIK3CA rs6443624 had significant effects on recurrence of endometrial cancer individually and combined in a locus–dosage manner (adjusted Ptrend = 0.003).
These results suggest that common genetic variations in these pathways may modulate risk and clinical outcomes of endometrial cancer. Further replication and functional studies are needed to confirm these findings.
PI3K/PTEN/AKT/mTOR and RAS/RAF/MEK/ERK pathways; Polymorphisms; Endometrial cancer risk; Survival; Recurrence
PTEN is a tumor suppressor that negatively regulates the PI3K-AKT signaling pathway, which is implicated in the pathogenesis of endometrial carcinoma. Sanger sequencing has been considered to be the gold standard for detection of PTEN sequence abnormalities. However, this approach fails to address the epigenetic mechanisms that contribute to functional PTEN loss. Using a study cohort of 154 endometrioid and non-endometrioid endometrial carcinomas, we performed full-length PTEN sequencing and PTEN immunohistochemistry on each tumor. PTEN sequence abnormalities were detected in a significantly lower proportion of cases (43%) than PTEN protein loss (64%, p= 0.0004). Endometrioid tumors had a significantly higher proportion of PTEN sequence abnormalities and PTEN protein loss than non-endometrioid tumors. Within the latter group, PTEN sequence abnormalities and PTEN protein loss were most frequent in undifferentiated carcinomas, followed by mixed carcinomas; they were least frequent in carcinosarcomas. Overall, at least one PTEN sequence abnormality was detected in each exon, and the greatest number of sequence abnormalities was detected in exon 8. Pure endometrioid tumors had a significantly higher frequency of sequence abnormalities in exon 7 than did the non-endometrioid tumors (p=0.0199). Importantly, no mutational hotspots were identified. While PTEN protein loss by immunohistochemistry was identified in 89% of cases with a PTEN sequence abnormality, PTEN protein loss was detected by immunohistochemistry in 44% of cases classified as PTEN wildtype by sequencing. For the first time, we demonstrate that PTEN immunohistochemistry is able to identify the majority of cases with functional PTEN loss. However, PTEN immunohistochemistry also detects additional cases with PTEN protein loss that would otherwise be undetected by gene sequencing. Therefore, for clinical purposes, immunohistochemistry appears to be a preferable technique for identifying endometrial tumors with loss of PTEN function.
Translation initiation is activated in cancer through increase in eukaryotic initiation factor 4E (eIF4E), eIF4G, phosphorylated eIF4E-binding protein (p4E-BP1) and phosphorylated ribosomal protein S6 (pS6), and decreased programmed cell death protein 4 (pdcd4), a translational inhibitor. Further, translation elongation is deregulated though alterations in eukaryotic elongation factor 2 (eEF2) and eEF2 kinase (eEF2K). We sought to determine the association of these translational aberrations with clinical-pathologic factors and survival outcomes in hormone receptor-positive breast cancer.
Primary tumors were collected from 190 patients with Stage I to III hormone receptor-positive breast cancer. Expression of eIF4E, eIF4G, 4E-BP1, p4E-BP1 T37/46, p4E-BP1 S65, p4E-BP1 T70, S6, pS6 S235/236, pS6 S240/244, pdcd4, eEF2 and eEF2K was assessed by reverse phase protein arrays. Univariable and multivariable analyses for recurrence-free survival (RFS) and overall survival (OS) were performed.
High eEF2, S6, pS6 S240/244, p4E-BP1 T70, and low pdcd4 were significantly associated with node positivity. Median follow-up for living patients was 96 months.
High p4E-BP1 T36/47, p4E-BP1 S65, p4E-BP1 T70 and 4E-BP1 were associated with worse RFS. High p4E-BP1 T70 and pS6 S235/236, and low pdcd4, were associated with worse OS. In multivariable analysis, in addition to positive nodes, p4E-BP1 S65 remained a significant predictor of RFS (HR = 1.62, 95% CI = 1.13-2.31; P = 0.008). In addition to age, pS6 S235/236 (HR = 1.73, 95% CI = 1.03-2.90, P = 0.039), eEF2K (HR = 2.19, 95% CI = 1.35-3.56, P = 0.002) and pdcd4 (HR = 0.42, 95% CI = 0.25-0.70, P = 0.001) were associated with OS.
Increased pS6, p4E-BP1, eEF2K and decreased pdcd4 are associated with poor prognosis in hormone receptor-positive breast cancer, suggesting their role as prognostic markers and therapeutic targets.
Protein extraction from formalin-fixed paraffin-embedded (FFPE) tissues is challenging due to extensive molecular crosslinking that occurs upon formalin fixation. Reverse-phase protein array (RPPA) is a high-throughput technology, which can detect changes in protein levels and protein functionality in numerous tissue and cell sources. It has been used to evaluate protein expression mainly in frozen preparations or FFPE-based studies of limited scope. Reproducibility and reliability of the technique in FFPE samples has not yet been demonstrated extensively. We developed and optimized an efficient and reproducible procedure for extraction of proteins from FFPE cells and xenografts, and then applied the method to FFPE patient tissues and evaluated its performance on RPPA.
Fresh frozen and FFPE preparations from cell lines, xenografts and breast cancer and renal tissues were included in the study. Serial FFPE cell or xenograft sections were deparaffinized and extracted by six different protein extraction protocols. The yield and level of protein degradation were evaluated by SDS-PAGE and Western Blots. The most efficient protocol was used to prepare protein lysates from breast cancer and renal tissues, which were subsequently subjected to RPPA. Reproducibility was evaluated and Spearman correlation was calculated between matching fresh frozen and FFPE samples.
The most effective approach from six protein extraction protocols tested enabled efficient extraction of immunoreactive protein from cell line, breast cancer and renal tissue sample sets. 85% of the total of 169 markers tested on RPPA demonstrated significant correlation between FFPE and frozen preparations (p < 0.05) in at least one cell or tissue type, with only 23 markers common in all three sample sets. In addition, FFPE preparations yielded biologically meaningful observations related to pathway signaling status in cell lines, and classification of renal tissues.
With optimized protein extraction methods, FFPE tissues can be a valuable source in generating reproducible and biologically relevant proteomic profiles using RPPA, with specific marker performance varying according to tissue type.
Formalin-fixed; Paraffin-embedded tissue; Protein extraction; Reverse phase protein array; Breast cancer; Renal cancer
A large fraction of ductal carcinoma in situ (DCIS), a non-invasive precursor lesion of invasive breast cancer, overexpresses the HER2/neu oncogene. The ducts of DCIS are abnormally filled with cells that evade apoptosis, but the underlying mechanisms remain incompletely understood. We overexpressed HER2 in mammary epithelial cells and observed growth factor-independent proliferation. When grown in extracellular matrix as 3- dimensional spheroids, control cells developed a hollow lumen, but HER2-overexpressing cells populated the lumen by evading apoptosis. We demonstrate that HER2 overexpression in this cellular model of DCIS drives transcriptional up-regulation of multiple components of the Notch survival pathway. Importantly, luminal filling required up-regulation of a signaling pathway comprising Notch3, its cleaved intracellular domain (NICD) and the transcriptional regulator HES1, resulting in elevated levels of c-MYC and Cyclin D1. In line with HER2- Notch3 collaboration, drugs intercepting either arm reverted the DCIS-like phenotype. In addition, we report up-regulation of Notch3 in hyperplastic lesions of HER2 transgenic animals, as well as an association between HER2 levels and expression levels of components of the Notch pathway in tumor specimens of breast cancer patients. Therefore, it is conceivable that the integration of the Notch and HER2 signaling pathways contributes to the pathophysiology of DCIS.
breast cancer; DCIS; growth factor; spheroids; receptor tyrosine kinase; signal transduction
We demonstrate that phosphatidylinositol 3-kinase (PI3K) pathway aberrations occur in >80% of endometrioid endometrial cancers, with coordinate mutations of multiple PI3K pathway members being more common than predicted by chance. PIK3R1 (p85α) mutations occur at a higher rate in endometrial cancer than in any other tumor lineage, and PIK3R2 (p85β), not previously demonstrated to be a cancer gene, is also frequently mutated. The dominant activation event in the PI3K pathway appears to be PTEN protein loss. However, in tumors with retained PTEN protein, PI3K pathway mutations phenocopy PTEN loss, resulting in pathway activation. KRAS mutations are common in endometrioid tumors activating independent events from PI3K pathway aberrations. Multiple PIK3R1 and PIK3R2 mutations demonstrate gain of function including disruption of a novel mechanism of pathway regulation wherein p85α dimers bind and stabilize PTEN. Taken together, the PI3K pathway represents a critical driver of endometrial cancer pathogenesis and a novel therapeutic target.
Endometrial Cancer; PTEN; PIK3CA; PIK3R1; PIK3R2
Sequential treatment with azacitidine can induce re-expression of epigenetically silenced genes through genomic DNA hypomethylation and reverse carboplatin resistance of epithelial ovarian cancer cells. We initiated a phase Ib-IIa clinical trial of this sequential combination of azacitidine and carboplatin in platinum-resistant or refractory epithelial ovarian cancer.
Patients with pathologically confirmed intermediate- or high-grade epithelial ovarian cancer who had disease progression within 6 months (resistant, n = 18) or during a platinum-based therapy (refractory, n = 12) were eligible. All patients had measurable disease.
Thirty patients received a total of 163 cycles of treatment. This regimen produced 1 CR, 3 PR (ORR: 13.8%), and 10 SD among 29 evaluable patients. For those who achieved clinical benefits, the median duration of the treatment was 7.5 months. The median PFS and OS for all patients were 3.7 months and 14 months, respectively. Patients with platinum resistant disease achieved an ORR of 22%, with a median PFS of 5.6 months and a median OS of 23 months. The predominant toxicities were fatigue and myelosuppression. Correlative studies showed that DR4 methylation in peripheral blood leukocytes was decreased during treatment in 3 of 4 objective responders (75%), but in only 5 of 13 non-responders (38%).
To our knowledge, this study provides the first clinical evidence that a hypomethylating agent may partially reverse platinum resistance in ovarian cancer. Further clinical evaluation of hypomethylating agents in combination with carboplatin is warranted.
To investigate the incidence of germline and somatic BRCA1/2 mutations in unselected patients with triple negative breast cancer (TNBC), and determine the prognostic significance of carrying a mutation.
DNA was obtained from 77 TNBC and normal tissues. BRCA1/2 exons/flanking regions were sequenced from tumor and patients classified as mutant or wild type (WT). Sequencing was repeated from normal tissue to identify germline and somatic mutations. Patient characteristics were compared with chi-square. Survival was estimated by Kaplan-Meier method and compared with log-rank. Cox proportional hazards models were fit to determine the independent association of mutation status with outcome.
Median age was 51 years (27-83 years). Fifteen patients (19.5%) had BRCA mutations: 12 (15.6%) in BRCA1 (one somatic), and 3 (3.9%) in BRCA2. Patients with BRCA mutations tended to be younger than WT, (p=0.005). Grade, histology and stage were not associated with mutation status. At a median follow-up of 43 months (7-214 months), there were 33 (42.9%) recurrences and 35 (45.5%) deaths. Five-year recurrence-free survival estimates were 51.7% for WT vs. 86.2% for patients with mutations, (p=0.031); and 5-year overall survival estimates were 52.8% for WT vs. 73.3% for patients with mutations, (p=0.225). After adjustment, patients with BRCA mutations had a significantly better RFS (HR:0.19, 95% CI:0.045-0.79, p=0.016) compared to WT.
In this unselected cohort of TNBC, we found a 19.5% incidence of BRCA mutations. Genetic testing should be discussed with patients with TNBC. Patients with TNBC with BRCA mutations had a significantly lower risk of relapse.
Ovarian cancer frequently acquires resistance to platinum chemotherapy, representing a major challenge for improving patient survival. Recent work suggests resistant clones exist within a larger drug sensitive cell-population prior to chemotherapy, implying that resistance is selected for rather than generated by treatment. We sought to compare clinically-derived, intra-patient paired models of initial platinum response and subsequent resistant relapse to define molecular determinants of evolved resistance. Transcriptional analysis of a matched cell-line series from three patients with high-grade serous ovarian cancer before and after development of clinical platinum resistance (PEO1/PEO4/PEO6, PEA1/PEA2, PEO14/PEO23) identified 91 up- and 126 down-regulated genes common to acquired resistance. Significantly enhanced apoptotic response to platinum treatment in resistant cells was observed following knockdown of HDAC4, FOLR2, PIK3R1 or STAT1 (p<0.05). Interestingly, HDAC4 and STAT1 were found to physically interact. Acetyl-STAT1 was detected in platinum sensitive but not HDAC4 over-expressing platinum resistant cells from the same patient. In resistant cells, STAT1 phosphorylation/nuclear translocation was seen following platinum exposure, whereas silencing of HDAC4 increased acetyl-STAT1 levels, prevented platinum induced STAT1 activation and restored cisplatin sensitivity. Conversely, matched sensitive cells were refractory to STAT1 phosphorylation on platinum treatment. Analysis of 16 paired tumor biopsies taken before and after development of clinical platinum resistance showed significantly increased HDAC4 expression in resistant tumors (n=7/16[44%]; p=0.04). Therefore, clinical selection of HDAC4 overexpressing tumor cells upon exposure to chemotherapy promotes STAT1 deacetylation and cancer cell survival. Together, our findings identify HDAC4 as a novel, therapeutically tractable target to counter platinum resistance in ovarian cancer.
ESR1 is one of the most important transcription factors and therapeutic targets in breast cancer. By applying systems-level re-analysis of publicly available gene expression data, we uncovered a potential regulator of ESR1. We demonstrated that orphan nuclear receptor NR2E3 regulates ESR1 via direct binding to the ESR1 promoter with concomitant recruitment of PIAS3 to the promoter in breast cancer cells, and is essential for physiological cellular activity of ESR1 in estrogen receptor (ER)-positive breast cancer cells. Moreover, expression of NR2E3 was significantly associated with recurrence-free survival and a favourable response to tamoxifen treatment in women with ER-positive breast cancer. Our results provide mechanistic insights on the regulation of ESR1 by NR2E3 and the clinical relevance of NR2E3 in breast cancer.
breast cancer; genomics; microarray; NR2E3; nuclear receptor
Mammalian target of rapamycin (mTOR) is a PI3K-related kinase that regulates cell growth, proliferation, and survival via mTOR complex 1 (mTORC1) and mTORC2. The mTOR pathway is often aberrantly activated in cancers. While hypoxia, nutrient deprivation, and DNA damage restrain mTORC1 activity, multiple genetic events constitutively activate mTOR in cancers. Here we provide a brief overview of the signaling pathways up- and downstream of mTORC1 and -2, and discuss the insights into therapeutic anticancer targets — both those that have been tried in the clinic with limited success and those currently under clinical development — that knowledge of these pathways gives us.
The lack of large panels of validated antibodies, tissue handling variability, and intratumoral heterogeneity potentially hamper comprehensive study of the functional proteome in non-microdissected solid tumors. The purpose of this study was to address these concerns and to demonstrate clinical utility for the functional analysis of proteins in non-microdissected breast tumors using reverse phase protein arrays (RPPA).
Herein, 82 antibodies that recognize kinase and steroid signaling proteins and effectors were validated for RPPA. Intraslide and interslide coefficients of variability were <15%. Multiple sites in non-microdissected breast tumors were analyzed using RPPA after intervals of up to 24 h on the benchtop at room temperature following surgical resection.
Twenty-one of 82 total and phosphoproteins demonstrated time-dependent instability at room temperature with most variability occurring at later time points between 6 and 24 h. However, the 82-protein functional proteomic “fingerprint” was robust in most tumors even when maintained at room temperature for 24 h before freezing. In repeat samples from each tumor, intratumoral protein levels were markedly less variable than intertumoral levels. Indeed, an independent analysis of prognostic biomarkers in tissue from multiple tumor sites accurately and reproducibly predicted patient outcomes. Significant correlations were observed between RPPA and immunohistochemistry. However, RPPA demonstrated a superior dynamic range. Classification of 128 breast cancers using RPPA identified six subgroups with markedly different patient outcomes that demonstrated a significant correlation with breast cancer subtypes identified by transcriptional profiling.
Thus, the robustness of RPPA and stability of the functional proteomic “fingerprint” facilitate the study of the functional proteome in non-microdissected breast tumors.
Functional proteome; RPPA; Breast cancer; Kinase signaling; Steroid signaling
The prevalence of BRCA½ mutations in germline DNA from unselected ovarian cancer patients is 11% to 15.3%. It is important to determine the frequency of somatic BRCA½ changes, given the sensitivity of BRCA-mutated cancers to poly (ADP ribose) polymerase-1 (PARP1) inhibitors and platinum analogs.
Patients and Methods
In 235 unselected ovarian cancers, BRCA½ was sequenced in 235, assessed by copy number analysis in 95, and tiling arrays in 65. 113 tumors were sequenced for TP53. BRCA½ transcript levels were assessed by quantitative polymerase chain reaction in 220. When available for tumors with BRCA½ mutations, germline DNA was sequenced.
Forty-four mutations (19%) in BRCA1 (n = 31)/BRCA2 (n = 13) were detected, including one homozygous BRCA1 intragenic deletion. BRCA½ mutations were particularly common (23%) in high-grade serous cancers. In 28 patients with available germline DNA, nine (42.9%) of 21 and two (28.6%) of seven BRCA1 and BRCA2 mutations were demonstrated to be somatic, respectively. Five mutations not previously identified in germline DNA were more commonly somatic than germline (four of 11 v one of 17; P = .062). There was a positive association between BRCA1 and TP53 mutations (P = .012). BRCA½ mutations were associated with improved progression-free survival (PFS) after platinum-based chemotherapy in univariate (P = .032; hazard ratio [HR] = 0.65; 95% CI, 0.43 to 0.98) and multivariate (P = .019) analyses. BRCA½ deficiency, defined as BRCA½ mutations or expression loss (in 24 [13.3%] BRCA½–wild-type cancers), was present in 67 ovarian cancers (30%) and was also significantly associated with PFS in univariate (P = .026; HR = 0.67; 95% CI, 0.47 to 0.96) and multivariate (P = .008) analyses.
BRCA½ somatic and germline mutations and expression loss are sufficiently common in ovarian cancer to warrant assessment for prediction of benefit in clinical trials of PARP1 inhibitors.