Increased activity of SRC family kinases promotes tumor invasion and metastasis, and overexpression of the mitotic regulator Aurora kinase A (AURKA) drives tumor aneuploidy and chromosomal instability. These actions nominate SRC and AURKA as valuable therapeutic targets for cancer, and inhibitors for SRC and Aurora kinases are now being employed in the clinic. In this study, we demonstrate potent synergy between multiple inhibitors of Aurora and SRC kinases in ovarian and colorectal cancer cell lines, but not in normal ovarian epithelial cell lines. Combination of Aurora and SRC inhibitors selectively killed cells that have undergone a preceding aberrant mitosis, and was associated with a post-mitotic reattachment defect, and selective removal of aneuploid cell populations. Combined inhibition of Aurora kinase and SRC potentiated dasatinib-dependent loss of activated (Y416-phosphorylated) SRC. SRC and AURKA share a common interaction partner, NEDD9, which serves as a scaffolding protein with activities in cell attachment and mitotic control, suggesting SRC and AURKA might interact directly. In vitro, we observed physical interaction and mutual cross-phosphorylation between SRC and AURKA that enhanced SRC kinase activity. Together, these findings suggest that combination of SRC and Aurora-targeting inhibitors in the clinic may be a productive strategy.
Although imatinib mesylate (IM) has transformed the treatment of gastrointestinal stromal tumors (GIST), many patients experience primary/secondary drug resistance. In a previous study, we identified a gene signature, consisting mainly of Kruppel-associated box (KRAB) domain containing zinc finger (ZNF) transcriptional repressors that predict short-term response to IM. To determine if these genes have functional significance, a siRNA library targeting these genes was constructed and applied to GIST cells in vitro. These screens identified seventeen “IM sensitizing genes” in GIST cells (sensitization index (SI) <0.85 ratio of drug/vehicle) with a false discovery rate (FDR) <15%, including twelve ZNF genes, the majority of which are located within the HSA19p12–13.1 locus. These genes were shown to be highly specific to IM and another tyrosine kinase inhibitor (TKI), sunitinib, in GIST cells. In order to determine mechanistically how these ZNFs might be modulating response to IM, RNAi approaches were used to individually silence genes within the predictive signature in GIST cells and expression profiling was performed. Knockdown of the 14 IM-sensitizing genes (10 ZNFs) universally led to downregulation of six genes, including TGFb3, periostin, and NEDD9. These studies implicate a role of KRAB-ZNFs in modulating response to TKIs in GIST.
Targeted therapies have been used to combat many tumor types; however, few have effectively improved the overall survival in women with epithelial ovarian cancer, begging for a better understanding of this deadly disease and identification of essential drivers of tumorigenesis that can be targeted effectively. Therefore, we used a loss-of-function screening approach to help identify molecular vulnerabilities that may represent key points of therapeutic intervention. We employed an unbiased high-throughput lethality screen using a 24,088 siRNA library targeting over 6,000 druggable genes and studied their effects on growth and/or survival of epithelial ovarian cancer (EOC) cell lines. The top 300 “hits” affecting the viability of A1847 cells were rescreened across additional EOC cell lines and non-tumorigenic, human immortalized ovarian epithelial cell lines. Fifty-three gene candidates were found to exhibit effects in all tumorigenic cell lines tested. Extensive validation of these hits refined the list to four high quality candidates (HSPA5, NDC80, NUF2, and PTN). Mechanistic studies show that silencing of three genes leads to increased apoptosis, while HSPA5 silencing appears to alter cell growth through G1 cell cycle arrest. Furthermore, two independent gene expression studies show that NDC80, NUF2 and PTN were significantly aberrantly overexpressed in serous adenocarcinomas. Overall, our functional genomics results integrated with the genomics data provide an important unbiased avenue towards the identification of prospective therapeutic targets for drug discovery, which is an urgent and unmet clinical need for ovarian cancer.
Epithelial ovarian cancer (EOC) remains the most lethal gynecological malignancy in the US. Thus, there is an urgent need to develop novel therapeutics for this disease. Cellular senescence is an important tumor suppression mechanism that has recently been suggested as a novel mechanism to target for developing cancer therapeutics. Wnt5a is a non-canonical Wnt ligand that plays a context-dependent role in human cancers. Here, we investigate the role of Wnt5a in regulating senescence of EOC cells. We demonstrate that Wnt5a is expressed at significantly lower levels in human EOC cell lines and in primary human EOCs (n = 130) compared with either normal ovarian surface epithelium (n = 31; p = 0.039) or fallopian tube epithelium (n = 28; p < 0.001). Notably, a lower level of Wnt5a expression correlates with tumor stage (p = 0.003) and predicts shorter overall survival in EOC patients (p = 0.003). Significantly, restoration of Wnt5a expression inhibits the proliferation of human EOC cells both in vitro and in vivo in an orthotopic EOC mouse model. Mechanistically, Wnt5a antagonizes canonical Wnt/β-catenin signaling and induces cellular senescence by activating the histone repressor A (HIRA)/promyelocytic leukemia (PML) senescence pathway. In summary, we show that loss of Wnt5a predicts poor outcome in EOC patients and Wnt5a suppresses the growth of EOC cells by triggering cellular senescence. We suggest that strategies to drive senescence in EOC cells by reconstituting Wnt5a signaling may offer an effective new strategy for EOC therapy.
Ovarian Cancer; Cellular senescence; Wnt5a; HIRA; PML
The 19p13.1 breast cancer susceptibility locus is a modifier of breast cancer risk in BRCA1 mutation carriers and is also associated with risk of ovarian cancer. Here we investigated 19p13.1 variation and risk of breast cancer subtypes, defined by estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor-2 (HER2) status, using 48,869 breast cancer cases and 49,787 controls from the Breast Cancer Association Consortium (BCAC). Variants from 19p13.1 were not associated with breast cancer overall or with ER-positive breast cancer but were significantly associated with ER-negative breast cancer risk [rs8170 Odds Ratio (OR)=1.10, 95% Confidence Interval (CI) 1.05 – 1.15, p=3.49 × 10-5] and triple negative (TN) (ER, PR and HER2 negative) breast cancer [rs8170 OR=1.22, 95% CI 1.13 – 1.31, p=2.22 × 10-7]. However, rs8170 was no longer associated with ER-negative breast cancer risk when TN cases were excluded [OR=0.98, 95% CI 0.89 – 1.07, p=0.62]. In addition, a combined analysis of TN cases from BCAC and the Triple Negative Breast Cancer Consortium (TNBCC) (n=3,566) identified a genome-wide significant association between rs8170 and TN breast cancer risk [OR=1.25, 95% CI 1.18 – 1.33, p=3.31 × 10-13]. Thus, 19p13.1 is the first triple negative-specific breast cancer risk locus and the first locus specific to a histological subtype defined by ER, PR, and HER2 to be identified. These findings provide convincing evidence that genetic susceptibility to breast cancer varies by tumor subtype and that triple negative tumors and other subtypes likely arise through distinct etiologic pathways.
genetic susceptibility; association study; subtype; neoplasms; common variant
Patients with persistent/recurrent epithelial ovarian cancer/primary peritoneal cancer (EOC/PPC) have limited treatment options. AKT and PI3K pathway activation is common in EOC/PPC, resulting in constitutive activation of downstream mTOR. The GOG conducted a phase II evaluation of efficacy and safety for the mTOR inhibitor, temsirolimus in EOC/PPC and explored circulating tumor cells (CTC) and AKT/mTOR/downstream tumor markers.
Eligible women with measurable, persistent/recurrent EOC/PPC who had received 1–3 prior regimens were treated with 25 mg weekly IV temsirolimus until progression or intolerable toxicity. Primary endpoints were progression-free survival (PFS) ≥6-months, tumor response, and toxicity. CellSearch® system was used to examine CTC, and AKT/mTOR/downstream markers were evaluated by archival tumor immunohistochemistry. Kendall’s tau-b correlation coefficient (r) and Cox regression modeling were used to explore marker associations with baseline characteristics and outcome.
Sixty patients were enrolled in a two-stage sequential design. Of 54 eligible and evaluable patients, 24.1% (90%CI 14.9%–38.6%) had PFS ≥6 months (median 3.1 months), 9.3% (90%CI 3.7%–23.4%) experienced a partial response. Grade 3/4 adverse events included metabolic(8), gastrointestinal(8), pain(6), constitutional(5) and pulmonary(4). Suggested associations were between cyclin D1 and PFS ≥6 months, PFS or survival; positive CTC pre-treatment and lack of response; and high CTC expression of M30 and PFS ≥6 months/longer PFS.
Temsirolimus appears to have modest activity in persistent/recurrent EOC/PPC; however, PFS is just below that required to warrant inclusion in phase III studies in unselected patients. Cyclin D1 as a selection marker and CTC measures merit further study.
Circulating tumor cells; ovarian clinical trial; AKT/PI3K pathway; mTOR inhibitor
TREX (transcription/export) is a multiprotein complex that plays a key role in the transcriptional elongation and transport of mRNA from the nucleus to the cytoplasm. We previously reported the purification of the human TREX protein and found that expression of a member of this complex, p84N5 (referred to as hTREX84 or hHPR1), a RB binding protein, correlated with breast tumor size and metastasis. Here we examine the mechanisms of aberrant expression of hTREX84 in breast and ovarian cancer cells and evaluate its role in tumorigenesis. We show that ovarian tumor cells over-express hTREX84 4-fold and 10-fold compared to immortal, non-tumorigenic and primary ovarian surface epithelial cells, respectively. Reduction of hTREX84 levels by small interfering RNA result in inhibition of cellular proliferation and G2/M arrest. Even though we observed that hTREX84 expression was induced by treatment with a demethylation agent, 5-aza-2′-deoxycytidine (5-aza-dC), sodium bisulfite DNA sequencing and methylation specific PCR found no evidence of changes in DNA methylation in the CpG islands in the regulator region of hTREX84. We subsequently identify several transcriptional factors, including NF-κB binding sites in the hTREX84 gene promoter and demonstrate by chromatin immunoprecipation (ChIP) and site directed mutagenesis that RelA/p65 binds the NF-kB binding sites and induces hTREX84 expression. Finally, we show by immunohistochemistry (IHC) that RelA/p65 is abundantly expressed in malignant cells that aberrantly express hTREX84 indicating that RelA/p65 might play a pivotal role in regulating hTREX84 expression in cancer. Our results indicate that overexpression of hTREX84 is associated with cancer cell transformation, proliferation and may be regulated by RelA/p65.
Identification and characterization of underlying genetic aberrations could facilitate diagnosis and treatment of ovarian cancer. Copy number analysis using array Comparative Genomic Hybridization (aCGH) on 93 primary ovarian tumors identified PI3K/AKT pathway as the most frequently altered cancer related pathway. Furthermore, survival analyses to correlate gene copy number and mutation data with patient outcome showed that copy number gains of PIK3CA, PIK3CB and PIK3R4 in these tumors were associated with decreased survival. To confirm these findings at the protein level, immunohistochemistry (IHC) for PIK3CA product p110α and p-Akt was performed on tissue microarrays from 522 independent serous ovarian cancers. Overexpression of either of these two proteins was found to be associated with decreased survival. Multivariant analysis from these samples further showed that overexpression of p-AKT and /or p110α is an independent prognostic factor for these tumors. siRNAs targeting altered PI3K/AKT pathway genes inhibited proliferation and induced apoptosis in ovarian cancer cell lines. In addition, the effect of the siRNAs in different cell lines seemed to correlate with the particular genetic alterations that the cell line carries. These results strongly support the utilization of PI3K pathway inhibitors in ovarian cancer. They also suggest identifying the specific component in the PI3K pathway that is genetically altered has the potential to help select the most effective therapy. Both mutation as well as copy number changes can be used as predictive markers for this purpose.
Siglecs (Sialic acid-binding Immunoglobulin Superfamily Lectins) are cell surface signaling receptors of the I-type lectin group that recognize sialic acid-bearing glycans. CD33-related-Siglecs are a subset with expression primarily in cells of hematopoietic origin and functional relevance to immune reactions. Earlier we reported a human-specific gene conversion event that markedly changed the coding region for the extracellular domain of Siglec-11, associated with human-specific expression in microglia (Hayakawa T, Angata T, Lewis AL, Mikkelsen TS, Varki NM, Varki A. 2005. A human-specific gene in microglia. Science. 309:1693). Analyzing human gene microarrays to define new patterns of expression, we observed high levels of SIGLEC11 transcript in the ovary and adrenal cortex. Thus, we examined human and chimpanzee tissues using a well-characterized anti-Siglec-11 mouse monoclonal antibody. Although adrenal expression was variable and confined to infiltrating macrophages in capillaries, ovarian expression of Siglec-11 in both humans and chimpanzees was on fibroblasts, the first example of Siglec expression on mesenchyme-derived stromal cells. Cytokines from such ovarian stromal fibroblasts play important roles in follicle development and ovulation. Stable transfection of SIGLEC11 into a primary human ovarian stromal fibroblast cell line altered the secretion of growth-regulated oncogene α, interleukin (IL)-10, IL-7, transforming growth factor β1 and tumor necrosis factor-α, cytokines involved in ovarian physiology. Probing for Siglec-11 ligands revealed distinct and strong mast cell expression in human ovaries, contrasting to diffuse stromal ligands in chimpanzee ovaries. Interestingly, there was a trend of increased Siglec-11 expression in post-menopausal ovaries compared with pre-menopausal ones. Siglec-11 expression was also found on human ovarian stromal tumors and in polycystic ovarian syndrome, a human-specific disease. These results indicate potential roles for Siglec-11 in ovarian physiology and human evolution.
cytokine; mast cells; microarray; ovarian stroma; PCOS
Susceptibility to testicular germ cell tumors (TGCT) has a significant heritable component, and genome-wide association studies (GWASs) have identified association with variants in several genes, including KITLG, SPRY4, BAK1, TERT, DMRT1 and ATF7IP. In our GWAS, we genotyped 349 TGCT cases and 919 controls and replicated top hits in an independent set of 439 cases and 960 controls in an attempt to find novel TGCT susceptibility loci. We identified a second marker (rs7040024) in the doublesex and mab-3-related transcription factor 1 (DMRT1) gene that is independent of the previously described risk allele (rs755383) at this locus. In combined analysis that mutually conditions on both DMRT1 single nucleotide polymorphism markers, TGCT cases had elevated odds of carriage of the rs7040024 major A allele [per-allele odds ratio (OR) = 1.48, 95% confidence interval (CI) 1.23, 1.78; P = 2.52 × 10−5] compared with controls, while the association with rs755383 persisted (per allele OR = 1.26, 95% CI 1.08, 1.47, P = 0.0036). In similar analyses, the association of rs7040024 among men with seminomatous tumors did not differ from that among men with non-seminomatous tumors. In combination with KITLG, the strongest TGCT susceptibility locus found to date, men with TGCT had greatly elevated odds (OR = 14.1, 95% CI 5.12, 38.6; P = 2.98 × 10−7) of being double homozygotes for the risk (major) alleles at DMRT (rs7040024) and KITLG (rs4474514) when compared with men without TGCT. Our findings continue to corroborate that genes influencing male germ cell development and differentiation have emerged as the major players in inherited TGCT susceptibility.
The molecular phenotype of tumor vasculature is different from normal vasculature, offering new opportunities for diagnosis and therapy of cancer, but the identification of tumor-restricted targets remains a challenge. We investigated 13 tumor vascular markers (TVMs) from 50 candidates identified through expression profiling of ovarian cancer vascular cells and selected to be either transmembrane or secreted, and to be either absent or expressed at low levels in normal tissues while overexpressed in tumors, based on analysis of 1,110 normal and tumor tissues from publicly available Affymetrix microarray data. Tumor-specific expression of each TVM was confirmed at the protein level in tumor tissue and/or in serum. Among the 13 TVMs, 11 were expressed on tumor vascular endothelium; the remaining 2 TVMs were expressed by tumor leukocytes. Our results demonstrate that certain transmembrane TVMs such as ADAM12 and CDCP1 are selectively expressed in tumor vasculature and represent promising targets for vascular imaging or anti-vascular therapy of epithelial ovarian cancer, while secreted or shed molecules such as TNFRSF21/DR6 can function as serum biomarkers. We have identified novel tumor-specific vasculature markers which appear promising for cancer serum diagnostics, molecular imaging and/or therapeutic targeting applications and warrant further clinical development.
ovarian; cancer; vascular; biomarkers; diagnostics; serum; expression; profiling
BRCA1 and BRCA2 mutation carriers have a lifetime breast cancer risk of 40% to 80%, suggesting the presence of risk modifiers. We previously identified significant associations in genetic variants in the insulin-like growth factor (IGF) signaling pathway. Here, we investigate additional IGF signaling genes as risk modifiers for breast cancer development in BRCA carriers.
A cohort of 1,019 BRCA1 and 500 BRCA2 mutation carriers were genotyped for 99 single-nucleotide polymorphisms (SNP) in 13 genes. Proportional hazards regression was used to model time from birth to diagnosis of breast cancer for BRCA1 and BRCA2 carriers separately. For linkage disequilibrium (LD) blocks with multiple SNPs, an additive genetic model was used. For an SNP analysis, no additivity assumptions were made.
Significant associations were found between risk of breast cancer and LD blocks in IGF2 for BRCA1 and BRCA2 mutation carriers (global P values of 0.009 for BRCA1 and 0.007 for BRCA2), HTRA1 for BRCA1 carriers (global P value of 0.005), and MMP3 for BRCA2 carriers (global P = 0.0000007 for BRCA2).
We identified significant associations of genetic variants involved in IGF signaling. With the known interaction of BRCA1 and IGF signaling and the loss of PTEN in a majority of BRCA1 tumors, this suggests that signaling through AKT may modify breast cancer risk in BRCA1 carriers.
These results suggest potential avenues for future research targeting the IGF signaling pathway in modifying risk in BRCA1and BRCA2 mutation carriers.
Antimüllerian hormone (AMH) is extensively studied in ovarian aging and pathology; however, little is known about correlates in healthy premenopausal women. We found that AMH levels are strongly inversely associated with age and differed significantly between oral contraceptive pill users and nonusers, whereas no significant associations were seen between AMH and other clinical, behavioral, and anthropometric characteristics and laboratory variables, making it an attractive hormone for clinical applications.
Müllerian-inhibiting substance (MIS); antimüllerian hormone (AMH); ovary; premenopausal; healthy
PI3K is an important therapeutic target. Mutations in PIK3CA, which encodes p110α, the catalytic subunit of PI3K, occur in endometrioid and non-endometrioid endometrial cancers (EECs and NEECs). The goal of this study was to determine whether PIK3R1, which encodes p85α, the inhibitory subunit of PI3K, is mutated in endometrial carcinoma. We performed exonic sequencing of PIK3R1 from 42 EECs and 66 NEECs. The pattern of PIK3R1 mutations was compared to the patterns of PIK3CA, PTEN and KRAS mutations. The biochemical effect of seven PIK3R1 mutations was examined by stable expression in U2OS cells, followed by coimmunoprecipitation analysis of p110α, and Western blotting of phospho-AKTSer473. We found that PIK3R1 was somatically mutated in 43% of EECs and 12% of NEECs. The majority of mutations (93.3%) localized to the p85α-nSH2 and -iSH2 domains. Several mutations were recurrent. PIK3R1 mutations were significantly (P=0.0015) more frequent in PIK3CA-wild type EECs (70%) than in PIK3CA-mutant EECs (18%). Introduction of wild type p85α into U2OS cells reduced the level of phospho-AKTSer473 compared to the vector control. Five p85α mutants, p85αdelH450-E451, p85αdelK459, p85αdelY463-L466, p85αdelR574-T576, and the p85αN564D positive control, were shown to bind p110α and led to increased levels of p-AKTSer473. The p85αR348X and p85αK511VfsX2 mutants did not bind p110α and showed no appreciable change in p-AKTSer473 levels. In conclusion, our study has revealed a new mode of PI3K alteration in primary endometrial tumors and warrants future studies to determine whether PIK3R1 mutations correlate with clinical outcome to targeted therapies directed against the PI3K pathway in EEC and NEEC.
Endometrial; cancer; p85α; PIK3R1; mutation
Gamma-synuclein is a neuronal protein found in peripheral and motor nerve systems. It becomes highly expressed in metastatic but not in primary tumor or normal tissues. The close association between gamma-synuclein expression and cancer spreading has been demonstrated in a broad range of malignancies. Our previous study showed that exogenous expression of gamma-synuclein in ovarian and breast cancer cells significantly enhanced cell migration and resistance to paclitaxel-induced apoptotic death. In our current research, we found that gamma-synuclein can affect microtubule properties and act as a functional microtubule associated protein. In vitro assays revealed that gamma-synuclein can bind and promote tubulin polymerization, induce the microtubule bundling and alter microtubules morphology developed in the presence of microtubule associated protein 2 (MAP2). Using cancer cell lysate, gamma-synuclein protein was found to be localized in both cytosolic compartment and extracted cytoskeleton portion. Immunofluorescence staining demonstrated that gamma-synuclein can colocalize with microtubule in HeLa cells and decrease rigidity of microtubule bundles caused by paclitaxel. In human ovarian cancer epithelial A2780 cells, gamma-synuclein overexpression improved cell adhesion and microtubule structure upon paclitaxel treatment. Importantly, it led to microtubule-dependent mitochondria clustering at perinuclear area. These observations suggest that overexpression of gamma-synuclein may reduce cell chemo-sensitivity of tumor cells through decreasing microtubule rigidity. In summery, our studies suggested that gamma-synuclein can directly participate in microtubule regulation.
Protein kinase C (PKC) activation contributes to proliferation and angiogenesis in epithelial ovarian or primary peritoneal carcinoma (EOC/PPC). A multi-institutional phase II trial was conducted to evaluate the efficacy and safety of PKCβ inhibitor enzastaurin in persistent or recurrent EOC/PPC and to explore potential prognostic and predictive biomarkers.
Eligible women with measurable platinum-sensitive and resistant EOC/PPC were treated with continuous administration of oral enzastaurin until disease progression or unacceptable toxicity. A two-stage sequential design was used to evaluate progression-free survival (PFS) ≥ 6-months, tumor response, and toxicity. Translational studies included sequencing of the TP53, PTEN, PIK3CA and PKCβII genes for somatic mutations, quantitative PCR assays for AKT2 and PTEN copy number alterations, and measurement of circulating VEGF-A plasma levels.
Among 27 eligible and evaluable patients, 3 women with PFS ≥ 6-months (11%) and 2 women with partial responses (7%) were observed. One of them achieved a durable response and remains on the study. No grade 4 adverse events were observed. Most common grade 3 adverse events were constitutional (4) and gastrointestinal (3). Mutations in the TP53 gene and abnormal copy number in the PTEN gene were common (56% and 48% of cases, respectively).
Enzastaurin was tolerable but had insufficient activity to proceed with the second stage of accrual. However, 1 patient has been progression-free for 44 months. No association between a biomarker and response to enzastaurin has been found.
Exploratory analysis suggested an association between survival and PTEN copy number losses.
Enzastaurin; ovarian cancer; VEGF PKCβ; TP53; PTEN; PIK3CA; AKT2
Cis-acting regulatory single nucleotide polymorphisms (SNPs) at specific loci may modulate penetrance of germline mutations at the same loci by introducing different levels of expression of the wild-type allele. We have previously reported that BRCA2 shows differential allelic expression and we hypothesize that the known variable penetrance of BRCA2 mutations might be associated with this mechanism.
We combined haplotype analysis and differential allelic expression of BRCA2 in breast tissue to identify expression haplotypes and candidate cis-regulatory variants. These candidate variants underwent selection based on in silico predictions for regulatory potential and disruption of transcription factor binding, and were functionally analyzed in vitro and in vivo in normal and breast cancer cell lines. SNPs tagging the expression haplotypes were correlated with the total expression of several genes in breast tissue measured by Taqman and microarray technologies. The effect of the expression haplotypes on breast cancer risk in BRCA2 mutation carriers was investigated in 2,754 carriers.
We identified common haplotypes associated with differences in the levels of BRCA2 expression in human breast cells. We characterized three cis-regulatory SNPs located at the promoter and two intronic regulatory elements which affect the binding of the transcription factors C/EBPα, HMGA1, D-binding protein (DBP) and ZF5. We showed that the expression haplotypes also correlated with changes in the expression of other genes in normal breast. Furthermore, there was suggestive evidence that the minor allele of SNP rs4942440, which is associated with higher BRCA2 expression, is also associated with a reduced risk of breast cancer (per-allele hazard ratio (HR) = 0.85, 95% confidence interval (CI) = 0.72 to 1.00, P-trend = 0.048).
Our work provides further insights into the role of cis-regulatory variation in the penetrance of disease-causing mutations. We identified small-effect genetic variants associated with allelic expression differences in BRCA2 which could possibly affect the risk in mutation carriers through altering expression levels of the wild-type allele.
Triple negative breast cancers are an aggressive subtype of breast cancer with poor survival, but there remains little known about the etiological factors which promote its initiation and development. Commonly inherited breast cancer risk factors identified through genome wide association studies (GWAS) display heterogeneity of effect among breast cancer subtypes as defined by estrogen receptor (ER) and progesterone receptor (PR) status. In the Triple Negative Breast Cancer Consortium (TNBCC), 22 common breast cancer susceptibility variants were investigated in 2,980 Caucasian women with triple negative breast cancer and 4,978 healthy controls. We identified six single nucleotide polymorphisms (SNPs) significantly associated with risk of triple negative breast cancer, including rs2046210 (ESR1), rs12662670 (ESR1), rs3803662 (TOX3), rs999737 (RAD51L1), rs8170 (19p13.11) and rs8100241 (19p13.11). Together, our results provide convincing evidence of genetic susceptibility for triple negative breast cancer.
genetic susceptibility; neoplasms; association study; subtypes; common variant
The goal of this study was to comprehensively define the incidence of mutations in all exons of PIK3CA in both endometrioid endometrial cancer (EEC) and non-endometrioid endometrial cancer (NEEC).
We resequenced all coding exons of PIK3CA and PTEN, and exons 1 and 2 of KRAS, from 108 primary endometrial tumors. Somatic mutations were confirmed by sequencing matched normal DNAs. The biochemical properties of a subset of novel PIK3CA mutations were determined by exogenously expressing wildtype and mutant constructs in U2OS cells and measuring levels of AKTSer473 phosphorylation.
Somatic PIK3CA mutations were detected in 52.4% of 42 EECs and 33.3% of 66 NEECs. Half (29 of 58) of all nonsynonymous PIK3CA mutations were in exons 1–7 and half were in exons 9 and 20. The exons 1–7 mutations localized to the ABD, ABD-RBD linker and C2 domains of p110α. Within these regions, Arg88, Arg93, Gly106, Lys111, Glu365, and Glu453, were recurrently mutated; Arg88, Arg93 and Lys111 formed mutation hotspots. The p110α-R93W, -G106R, -G106V, -K111E, -delP449-L455, and -E453K mutants led to increased levels of phospho-AKTSer473 compared to wild-type p110α. Overall, 62% of exons 1–7 PIK3CA mutants and 64% of exon 9–20 PIK3CA mutants were activating; 72% of exon 1–7 mutations have not previously been reported in endometrial cancer.
Our study identified a new subgroup of endometrial cancer patients with activating mutations in the amino-terminal domains of p110α; these patients might be appropriate for consideration in clinical trials of targeted therapies directed against the PI3K pathway.
Endometrial; cancer; PIK3CA; mutation; spectrum
Hesca-2, a monoclonal antibody (mAb) IgM raised to the human embryonic stem cell (hESC) line BG-01v, binds with high affinity (nM) to the disaccharide epitope (Galβ1-3GlcNAc) on a glycan microarray. This epitope was expressed on pluripotent progenitor hESCs in culture, but not in various differentiated cells derived from hESC based on immunofluorescence microscopy. Hesca-2 stains a limited subset of cells in adult human tissues (eg, esophagus and breast). This mAb also crossreacts in immunofluorescence microscopy studies with several human ovarian cancer cell lines and is cytotoxic to them based on the release of cytosolic enzyme lactate dehydrogenase into the media. Hesca-2 immunohistochemically stained tissue from a number of human tumors, including ovary, breast, colon, and esophageal cancer. These data suggest that Hesca-2 recognizes a surface marker found both in stem cells and certain cancer cells.
Establishing a cancer screening biomarker’s intended performance requires “phase III” specimens obtained in asymptomatic individuals before clinical diagnosis rather than “phase II” specimens obtained from symptomatic individuals at diagnosis. We used specimens from the Prostate, Lung, Colorectal, and Ovarian (PLCO) cancer screening trial to evaluate ovarian cancer biomarkers previously assessed in phase II sets.
Phase II specimens from 180 ovarian cancer cases and 660 benign disease or general population controls were assembled from four Early Detection Research Network (EDRN) or Ovarian Cancer Specialized Program of Research Excellence (SPORE) sites and used to rank 49 biomarkers. Thirty-five markers, including 6 additional markers from a fifth site, were then evaluated in PLCO proximate specimens from 118 women with ovarian cancer and 474 matched controls.
Top markers in phase II specimens included CA125, HE4, transthyretin, CA15.3, and CA72.4 with sensitivity at 95% specificity ranging from 0.73 to 0.40. Except for transthyretin, these markers had similar or better sensitivity when moving to phase III specimens that had been drawn within six months of the clinical diagnosis. Performance of all markers declined in phase III specimens more remote than 6 months from diagnosis.
Despite many promising new markers for ovarian cancer, CA125 remains the single-best biomarker in the phase II and phase III specimens tested in this study.
Ovarian neoplasms; CA125; HE4; Screening tests; CA72.4
GP88 (progranulin) has been implicated in tumorigenesis and resistance to anti-estrogen therapies for estrogen receptor positive (ER+) breast cancer. Previous pathological studies showed that GP88 is expressed in invasive ductal carcinoma (IDC), but not in normal mammary epithelial tissue, benign lesions or lobular carcinoma. Based on these results, the present study examines GP88 prognostic significance in association with recurrence and death risks for ER+ IDC patients.
Two retrospective multi-site clinical studies examined GP88 expression by immunohistochemistry (IHC) analysis of paraffin-embedded breast tumor tissue sections from ER+ IDC patients (lymph node positive and negative, stage 1 to 3) in correlation with patients' survival outcomes. The training study established a GP88 cut-off value associated with decreased disease-free (DFS) and overall (OS) survivals. The validation study verified the GP88 cut-off value and compared GP88 prognostic information with other prognostic factors, particularly tumor size, grade, disease stage and lymph node status in multivariate analysis.
GP88 expression is associated with a statistically significant increase in recurrence risk for ER+ IDC patients. The training study established that GP88 3+ score was associated with decreased DFS (P = 0.0004) and OS (P = 0.0036). The independent validation study verified that GP88 3+ score was associated with a 5.9-fold higher hazard of disease recurrence and a 2.5-fold higher mortality hazard compared to patients with tumor GP88 < 3+. GP88 remained an independent risk predictor after considering age, ethnicity, nodal status, tumor size, tumor grade, disease stage, progesterone receptor expression and treatments.
The survival factor GP88 is a novel prognostic biomarker, predictive of recurrence risk and increased mortality for non-metastatic ER+ IDC patients. Of importance, our data show that GP88 continues to be a prognostic factor even after five years. These results also provide evidence that GP88 provides prognostic information independent of tumor and clinical characteristics and would support prospective study to examine whether GP88 expression could help stratify patients with ER+ tumors for adjuvant therapy.
EZH2 is the catalytic subunit of the polycomb repressive complex 2 (PRC2) that includes non-catalytic subunits SUZ12 and EED. When present in PRC2, EZH2 catalyzes trimethylation on lysine 27 residue of histone H3 (H3K27Me3), resulting in epigenetic silencing of gene expression. Here, we investigated the expression and function of EZH2 in epithelial ovarian cancer (EOC). When compared to primary human ovarian surface epithelial (pHOSE) cells, EZH2, SUZ12 and EED were expressed at higher levels in all eight human EOC cell lines tested. Consistently, H3K27Me3 was also overexpressed in human EOC cell lines compared to pHOSE cells. EZH2 was significantly overexpressed in primary human EOCs (n=134) when compared to normal ovarian surface epithelium (n=46) (p<0.001). EZH2 expression positively correlated with expression of Ki67 (p<0.001) (a marker of cell proliferation) and tumor grade (p=0.034) but not tumor stage (p=0.908) in EOC. There was no correlation of EZH2 expression with overall (p=0.3) or disease-free survival (p=0.2) in high-grade serous histotype EOC patients (n=98). Knockdown of EZH2 expression reduced the level of H3K27Me3 and suppressed the growth of human EOC cells both in vitro and in vivo in xenograft models. EZH2 knockdown induced apoptosis of human EOC cells. Finally, we showed that EZH2 knockdown suppressed the invasion of human EOC cells. Together, these data demonstrate that EZH2 is frequently overexpressed in human EOC cells and its overexpression promotes the proliferation and invasion of human EOC cells, suggesting that EZH2 is a potential target for developing EOC therapeutics.
Epithelial ovarian cancer; EZH2; cell proliferation; apoptosis; invasion
The distal-less homeobox gene (dlx) 5 encodes a transcription factor that controls jaw formation and appendage differentiation during embryonic development. We previously found that Dlx5 is overexpressed in an Akt2 transgenic model of T-cell lymphoma. To investigate if DLX5 is involved in human cancer, we screened its expression in the NCI 60 cancer cell line panel. DLX5 was frequently up regulated in cell lines derived from several tumor types, including ovarian cancer. We next validated its up regulation in primary ovarian cancer specimens. Stable knockdown of DLX5 by lentivirus-mediated transduction of short hairpin RNAi (shRNA) resulted in reduced proliferation of ovarian cancer cells due to inhibition of cell cycle progression in connection with down regulation of cyclins A, B1, D1, D2 and E and decreased phosphorylation of AKT. Cell proliferation resumed following introduction of a DLX5 cDNA harboring wobbled mutations at the shRNA-targeting sites. Cell proliferation was also rescued by transduction of a constitutively active form of AKT. Intriguingly, down regulation of IRS-2 and MET contributed to the suppression of AKT signaling. Moreover, DLX5 was found to directly bind to the IRS-2 promoter and augmented its transcription. Knockdown of DLX5 in xenografts of human ovarian cancer cells resulted in markedly diminished tumor size. In addition, DLX5 was found to cooperate with HRAS in the transformation of human ovarian surface epithelial cells. Together, these data suggest that DLX5 plays a significant role in the pathogenesis of some ovarian cancers.
Ovarian Cancer; DLX5; AKT; IRS-2; MET
Based on the Cognitive-Social Health Information Processing model, we identified cognitive profiles of women at risk for breast and ovarian cancer. Prior to genetic counseling, participants (N = 171) completed a study questionnaire concerning their cognitive and affective responses to to being at genetic risk. Using cluster analysis, four cognitive profiles were generated: (a) high perceived risk/low coping; (b) low value of screening/high expectancy of cancer; (c) moderate perceived risk/moderate efficacy of prevention/low informativeness of test result; and (d) high efficacy of prevention/high coping. The majority of women in clusters one, two and three were unaffected, whereas cluster four consisted almost entirely of affected women. Women in cluster one had the highest number of affected relatives and experienced higher levels of distress than women in the other three clusters. These results highlight the need to consider the psychological profile of women undergoing genetic testing when designing counseling interventions and messages.
breast and ovarian cancer risk; cluster analysis; cognitive profiles; genetic testing; psychological distress