Modelling the impact of bias in fecal immunochemical testing on long-term outcomes of colorectal cancer screening.

BACKGROUND: As the impact of unmanaged bias (i.e. systematic source of inaccuracy) in fecal immunochemical test (FIT) analytical performance on long-term colorectal cancer (CRC) outcomes is unknown, we assessed the impact bias in FIT performance in an ongoing FIT-based CRC screening program. METHODS: This study consisted of two parts: cross-sectional observational data analysis to estimate change in short-term outcomes and microsimulation modelling to estimate change in long-term outcomes assuming different levels of bias by assuming 15 % lower up to 15 % higher Hemoglobin detected in the stool compared to observed. Two scenarios were considered: bias occurring 1) one-time only, due to the occasional bias associated with the FIT kits used in 2020 and 2) consistently due to a constant bias associated with the FIT kits used from 2020 onwards. RESULTS: With a hypothetical bias of -15 % to +15 %, we observed a positivity rate ranging from 6.7 % to 7.8 %, and a detection rate for CRC between 0.65 % and 0.68 %. Single biases in FIT performance resulted in less than 0.1 % change in long-term CRC screening outcomes, while consistent biases resulted in a much larger change (up to 1.4 % in CRC cases and CRC-related deaths and up to 2.07 % in total costs). Detecting lower Hemoglobin concentrations resulted in a relatively larger change on long-term CRC outcomes in comparison to positive bias. CONCLUSIONS: Because of the substantial impact of consistent FIT bias, it is important to set evidence-based acceptance criteria of bias on long-term CRC screening outcomes and in particular, the introduction of an asymmetrical or upward shifted tolerance interval for FIT bias.

The molecular genetic make-up of male breast cancer.

Male breast cancer (MBC) is extremely rare and accounts for less than 1% of all breast malignancies. Therefore, clinical management of MBC is currently guided by research on the disease in females. In this study, DNA obtained from 45 formalin-fixed paraffin-embedded (FFPE) MBCs with and 90 MBCs (52 FFPE and 38 fresh-frozen) without matched normal tissues was subjected to massively parallel sequencing targeting all exons of 1943 cancer-related genes. The landscape of mutations and copy number alterations was compared to that of publicly available estrogen receptor (ER)-positive female breast cancers (smFBCs) and correlated to prognosis. From the 135 MBCs, 90% showed ductal histology, 96% were ER-positive, 66% were progesterone receptor (PR)-positive, and 2% HER2-positive, resulting in 50, 46 and 4% luminal A-like, luminal B-like and basal-like cases, respectively. Five patients had Klinefelter syndrome (4%) and 11% of patients harbored pathogenic BRCA2 germline mutations. The genomic landscape of MBC to some extent recapitulated that of smFBC, with recurrent PIK3CA (36%) and GATA3 (15%) somatic mutations, and with 40% of the most frequently amplified genes overlapping between both sexes. TP53 (3%) somatic mutations were significantly less frequent in MBC compared to smFBC, whereas somatic mutations in genes regulating chromatin function and homologous recombination deficiency-related signatures were more prevalent. MDM2 amplifications were frequent (13%), correlated with protein overexpression (P = 0.001) and predicted poor outcome (P = 0.007). In conclusion, despite similarities in the genomic landscape between MBC and smFBC, MBC is a molecularly unique and heterogeneous disease requiring its own clinical trials and treatment guidelines.

Comprehensive Proteomic Profiling-derived Immunohistochemistry-based Prediction Models for BRCA1 and BRCA2 Germline Mutation-related Breast Carcinomas.

Heredity, mostly due to BRCA germline mutations, is involved in 5% to 10% of all breast cancer cases. Potential BRCA germline mutation carriers may be missed following the current eligibility criteria for BRCA genetic testing. The purpose of this study was to, therefore, develop an immunohistochemistry-based model to predict likelihood of underlying BRCA1 and BRCA2 germline mutations in unselected female breast cancer patients. The study group consisted of 100 BRCA1-related, 46 BRCA2-related, and 94 sporadic breast carcinomas. Tumor expression of 44 proteins involved in (BRCA-related) breast carcinogenesis was assessed by immunohistochemistry. A prediction model for BRCA-related versus non-BRCA-related breast cancer was developed using Lasso logistic regression analysis with cross-validation. The model was assessed for its discriminative value and clinical usefulness. The optimal prediction model included 14 predictors (age, cyclinD1, ERα, ERß, FGFR2, FGFR3, FGFR4, GLUT1, IGFR, Ki67, mitotic activity index, MLH1, p120, and TOP2A), showed excellent discriminative performance (area under the receiving operating characteristic curve=0.943; 95% confidence interval=0.909-0.978), and reasonable calibration. To enhance possible implementation, we developed an alternative model only considering more widely available immunostains. This model included 15 predictors (age, BCL2, CK5/6, CK8/18, cyclinD1, E-cadherin, ERα, HER2, Ki67, mitotic activity index , MLH1, p16, PMS2, PR, and vimentin), and still showed very good discriminative performance (area under the receiving operating characteristic curve=0.853; 95% confidence interval=0.795-0.911). We present a well-applicable and accurate tool to predict which breast cancer patients may have an underlying BRCA germline mutation, largely consisting of immunohistochemical markers independent of clinical characteristics. This may improve identification of potential BRCA germline mutation carriers and optimize referral for germline mutation testing.

Characterizing steroid hormone receptor chromatin binding landscapes in male and female breast cancer.

Male breast cancer (MBC) is rare and poorly characterized. Like the female counterpart, most MBCs are hormonally driven, but relapse after hormonal treatment is also noted. The pan-hormonal action of steroid hormonal receptors, including estrogen receptor alpha (ERα), androgen receptor (AR), progesterone receptor (PR), and glucocorticoid receptor (GR) in this understudied tumor type remains wholly unexamined. This study reveals genomic cross-talk of steroid hormone receptor action and interplay in human tumors, here in the context of MBC, in relation to the female disease and patient outcome. Here we report the characterization of human breast tumors of both genders for cistromic make-up of hormonal regulation in human tumors, revealing genome-wide chromatin binding landscapes of ERα, AR, PR, GR, FOXA1, and GATA3 and enhancer-enriched histone mark H3K4me1. We integrate these data with transcriptomics to reveal gender-selective and genomic location-specific hormone receptor actions, which associate with survival in MBC patients.

Expression of HIF-1α in medullary thyroid cancer identifies a subgroup with poor prognosis.

BACKGROUND: Medullary thyroid cancer (MTC) comprises only 4% of all thyroid cancers and originates from the parafollicular C-cells. HIF-1α expression has been implied as an indicator of worse prognosis in various solid tumors. However, whether expression of HIF-1α is a prognosticator in MTC remained unclear. Our aim was to evaluate the prognostic value of HIF-1α in patients with MTC. METHODS: All patients with MTC who were operated on between 1988 and 2014 in five tertiary referral centers in The Netherlands were included. A tissue microarray was constructed in which 111 primary tumors could be analyzed for expression of HIF-1α, CAIX, Glut-1, VEGF and CD31 and correlated with clinicopathologic variables and survival. RESULTS: The mean age of patients was 46.3 years (SD 15.6), 59 (53.2%) were male. Of the 111 primary tumors, 49 (44.1%) were HIF-1α negative and 62 (55.9%) were HIF-1α positive. Positive HIF-1α expression was an independent negative indicator for progression free survival (PFS) in multivariate cox regression analysis (HR 3.1; 95% CI 1.3 - 7.3). Five-years survival decreased from 94.0% to 65.9% for the HIF-1α positive group (p=0.007). Even within the group of patients with TNM-stage IV disease, HIF-1α positivity was associated with a worse prognosis, shown by a decrease in 5-years survival of 88.0% to 49.3% (p=0.020). CONCLUSION: Expression of HIF-1α is strongly correlated with adverse prognosis of MTC. This could open up new ways for targeted systemic therapy of MTC.

Influence of decalcification procedures on immunohistochemistry and molecular pathology in breast cancer.

Distant breast cancer metastases are nowadays routinely biopsied to reassess receptor status and to isolate DNA for sequencing of druggable targets. Bone metastases are the most frequent subgroup. Decalcification procedures may negatively affect antigenicity and DNA quality. We therefore evaluated the effect of several decalcification procedures on receptor status and DNA/RNA quality. In 23 prospectively collected breast tumors, we compared ERα, PR and HER2 status by immunohistochemistry in (non-decalcified) tissue routinely processed for diagnostic purposes and in parallel tissue decalcified in Christensen's buffer with and without microwave, EDTA and Formical-4. Furthermore, HER2 fluorescence in situ hybridization and DNA/RNA quantity and quality were assessed. We found that the percentage of ERα-positive cells were on average lower in EDTA (P=0.049) and Formical-4 (P=0.047) treated cases, compared with controls, and PR expression showed decreased antigenicity after Christensen's buffer treatment (P=0.041). Overall, a good concordance (weighted kappa) was seen for ERα, PR and HER2 immunohistochemistry when comparing the non-decalcified control tissues with the decalcified tissues. For two patients (9%), there was a potential influence on therapeutic decision making with regard to hormonal therapy or HER2-targeted therapy. HER2 fluorescence in situ hybridization interpretation was seriously hampered by Christensen's buffer and Formical-4, and DNA/RNA quantity and quality were decreased after all four decalcification procedures. Validation on paired primary breast tumor specimens and EDTA-treated bone metastases showed that immunohistochemistry and fluorescence in situ hybridization were well assessable and DNA and RNA yield and quality were sufficient. With this, we conclude that common decalcification procedures have only a modest negative influence on hormone and HER2 receptor immunohistochemistry in breast cancer. However, they may seriously affect DNA/RNA-based diagnostic procedures. Overall, EDTA-based decalcification is therefore to be preferred as it best allows fluorescence in situ hybridization and DNA/RNA isolation.

Secretome proteomics reveals candidate non-invasive biomarkers of BRCA1 deficiency in breast cancer.

Breast cancer arising in female BRCA1 mutation carriers is characterized by an aggressive phenotype and early age of onset. We performed tandem mass spectrometry-based proteomics of secretomes and exosome-like extracellular vesicles from BRCA1-deficient and BRCA1-proficient murine breast tumor models to identify extracellular protein biomarkers, which can be used as an adjunct to current diagnostic modalities in patients with BRCA1-deficient breast cancer. We identified 2,107 proteins, of which 215 were highly enriched in the BRCA1-deficient secretome. We demonstrated that BRCA1-deficient secretome proteins could cluster most human BRCA1- and BRCA2-related breast carcinomas at the transcriptome level. Topoisomerase I (TOP1) and P-cadherin (CDH3) expression was investigated by immunohistochemistry on tissue microarrays of a large panel of 253 human breast carcinomas with and without BRCA1/2 mutations. We showed that expression of TOP1 and CDH3 was significantly increased in human BRCA1-related breast carcinomas relative to sporadic cases (p = 0.002 and p < 0.001, respectively). Multiple logistic regression showed that TOP1 (adjusted odds ratio [OR] 3.75; 95% confidence interval [95% CI], 1.85 - 7.71, p < 0.001) as well as CDH3 positivity (adjusted OR 2.45; 95% CI, 1.08 - 5.49, p = 0.032) were associated with BRCA1/2-related breast carcinomas after adjustment for triple-negative phenotype and age. In conclusion, proteome profiling of secretome using murine breast tumor models is a powerful strategy to identify non-invasive candidate biomarkers of BRCA1-deficient breast cancer. We demonstrate that TOP1 and CDH3 are closely associated to BRCA1-deficient breast cancer. These data merit further investigation for early detection of tumors arising in BRCA1 mutation carriers.

p120-Catenin Is Critical for the Development of Invasive Lobular Carcinoma in Mice.

Loss of E-cadherin expression is causal to the development of invasive lobular breast carcinoma (ILC). E-cadherin loss leads to dismantling of the adherens junction and subsequent translocation of p120-catenin (p120) to the cytosol and nucleus. Although p120 is critical for the metastatic potential of ILC through the regulation of Rock-dependent anoikis resistance, it remains unknown whether p120 also contributes to ILC development. Using genetically engineered mouse models with mammary gland-specific inactivation of E-cadherin, p120 and p53, we demonstrate that ILC formation induced by E-cadherin and p53 loss is severely impaired upon concomitant inactivation of p120. Tumors that developed in the triple-knockout mice were mostly basal sarcomatoid carcinomas that displayed overt nuclear atypia and multinucleation. In line with the strong reduction in ILC incidence in triple-knockout mice compared to E-cadherin and p53 double-knockout mice, no functional redundancy of p120 family members was observed in mouse ILC development, as expression and localization of ARVCF, p0071 or δ-catenin was unaltered in ILCs from triple-knockout mice. In conclusion, we show that loss of p120 in the context of the p53-deficient mouse models is dominant over E-cadherin inactivation and its inactivation promotes the development of basal, epithelial-to-mesenchymal-transition (EMT)-type invasive mammary tumors.

Recruitment of the Mammalian Histone-modifying EMSY Complex to Target Genes Is Regulated by ZNF131.

Recent work from others and us revealed interactions between the Sin3/HDAC complex, the H3K4me3 demethylase KDM5A, GATAD1, and EMSY. Here, we characterize the EMSY/KDM5A/SIN3B complex in detail by quantitative interaction proteomics and ChIP-sequencing. We identify a novel substoichiometric interactor of the complex, transcription factor ZNF131, which recruits EMSY to a large number of active, H3K4me3 marked promoters. Interestingly, using an EMSY knock-out line and subsequent rescue experiments, we show that EMSY is in most cases positively correlated with transcriptional activity of its target genes and stimulates cell proliferation. Finally, by immunohistochemical staining of primary breast tissue microarrays we find that EMSY/KDM5A/SIN3B complex subunits are frequently overexpressed in primary breast cancer cases in a correlative manner. Taken together, these data open venues for exploring the possibility that sporadic breast cancer patients with EMSY amplification might benefit from epigenetic combination therapy targeting both the KDM5A demethylase and histone deacetylases.

Copy number profiling by array comparative genomic hybridization identifies frequently occurring BRCA2-like male breast cancer.

Genomic aberrations can be used to subtype breast cancer. In this study, we investigated DNA copy number (CN) profiles of 69 cases of male breast cancer (MBC) by array comparative genomic hybridization (aCGH) to detect recurrent gains and losses in comparison with female breast cancers (FBC). Further, we classified these profiles as BRCA1-like, BRCA2-like or non-BRCA-like profiles using previous classifiers derived from FBC, and correlated these profiles with pathological characteristics. We observed large CN gains on chromosome arms 1q, 5p, 8q, 10p, 16p, 17q, and chromosomes 20 and X. Large losses were seen on chromosomes/chromosome arms 1p, 6p, 8p, 9, 11q, 13, 14q, 16q, 17p, and 22. The pattern of gains and losses in estrogen receptor positive (ER+) MBC was largely similar to ER+ FBC, except for gains on chromosome X in MBC, which were uncommon in FBC. Out of 69 MBC patients, 15 patients (22%) had a BRCA2-like profile, of which 2 (3%) were also BRCA1-like. One patient (1%) was only BRCA1-like; the remaining 53 (77%) patients were classified as non-BRCA-like. BRCA2-like cases were more often p53 accumulated than non-BRCA-like cases (P = 0.014). In conclusion, the pattern of gains and losses in ER+ MBC was largely similar to that of its ER+ FBC counterpart, except for gains on chromosome X in MBC, which are uncommon in FBC. A significant proportion of MBC has a BRCA2-like aCGH profile, pointing to a potentially hereditary nature, and indicating that they could benefit from a drug regimen targeting BRCA defects as in FBC.

miRNA expression patterns in normal breast tissue and invasive breast cancers of BRCA1 and BRCA2 germ-line mutation carriers.

miRNA deregulation has been found to promote carcinogenesis. Little is known about miRNA deregulation in hereditary breast tumors as no miRNA expression profiling studies have been performed in normal breast tissue of BRCA1 and BRCA2 mutation carriers. miRNA profiles of 17 BRCA1- and 9 BRCA2-associated breast carcinomas were analyzed using microarrays. Normal breast tissues from BRCA1 and BRCA2 mutation carriers (both n = 5) and non-mutation carriers (n = 10) were also included. Candidate miRNAs were validated by qRT-PCR. Breast carcinomas showed extensive miRNA alteration compared to normal breast tissues in BRCA1 and BRCA2 mutation carriers. Moreover, normal breast tissue from BRCA1 mutation carriers already showed miRNA alterations compared to non-mutation carriers. Chromosomal distribution analysis showed several hotspots containing down- or up-regulated miRNAs. Pathway analysis yielded many similarities between the BRCA1 and BRCA2 axes with miRNAs involved in cell cycle regulation, proliferation and apoptosis. Lesser known pathways were also affected, including cellular movement and protein trafficking. This study provides a comprehensive insight into the potential role of miRNA deregulation in BRCA1/2-associated breast carcinogenesis. The observed extensive miRNA deregulation is likely the result of genome-wide effects of chromosomal instability caused by impaired BRCA1 or BRCA2 function. This study's results also suggest the existence of common pathways driving breast carcinogenesis in both BRCA1 and BRCA2 germ-line mutation carriers.

Targeting DDX3 with a small molecule inhibitor for lung cancer therapy.

Lung cancer is the most common malignancy worldwide and is a focus for developing targeted therapies due to its refractory nature to current treatment. We identified a RNA helicase, DDX3, which is overexpressed in many cancer types including lung cancer and is associated with lower survival in lung cancer patients. We designed a first-in-class small molecule inhibitor, RK-33, which binds to DDX3 and abrogates its activity. Inhibition of DDX3 by RK-33 caused G1 cell cycle arrest, induced apoptosis, and promoted radiation sensitization in DDX3-overexpressing cells. Importantly, RK-33 in combination with radiation induced tumor regression in multiple mouse models of lung cancer. Mechanistically, loss of DDX3 function either by shRNA or by RK-33 impaired Wnt signaling through disruption of the DDX3-ß-catenin axis and inhibited non-homologous end joining-the major DNA repair pathway in mammalian somatic cells. Overall, inhibition of DDX3 by RK-33 promotes tumor regression, thus providing a compelling argument to develop DDX3 inhibitors for lung cancer therapy.

Nodal metastasis and survival in oral cancer: Association with protein expression of SLPI, not with LCN2, TACSTD2, or THBS2.

BACKGROUND: Gene expression profiling revealed a strong signature predicting lymph node metastases in oral squamous cell carcinoma (OSCC). Four of the most predictive genes are secretory leukocyte protease inhibitor (SLPI), lipocalin-2 (LCN2), thrombospondin-2 (THBS2), and tumor-associated calcium signal transducer 2 (TACSTD2). This study correlates their protein expression with lymph node metastases, overall survival (OS), and disease-specific survival (DSS). METHODS: Two hundred twelve patients with OSCC were included for protein expression analysis by immunohistochemistry. RESULTS: SLPI expression correlates with lymph node metastases in the whole cohort, not in a subgroup of cT1 to 2N0. SLPI expression correlates with OS (hazard ratio [HR] = 0.61) and DSS (HR = 0.47) in multivariate analysis. LCN2, THBS2, and TACSTD2 show no correlation with lymph node metastases, OS, or DSS. CONCLUSION: Although SLPI expression correlates with lymph node metastases, it has no additional value in determining lymph node metastases in early oral cancer. However, it is an independent predictor for both OS and DSS and therefore a relevant prognostic biomarker in OSCC.

Hypoxia-inducible factor 1-α in chronic gastrointestinal ischemia.

Chronic gastrointestinal ischemia (CGI) is the result of decreased mucosal perfusion. Typical histological characteristics are lacking which hamper its early diagnosis. Hypoxia-inducible factor-1α (HIF-1α) is expressed under acute hypoxia. We investigated HIF-1α expression in chronic ischemic and inflammatory conditions of the human gastrointestinal (GI) tract. Immunohistochemical expression of HIF-1α was analyzed in 61 patients, including patients with CGI, Helicobacter pylori gastritis, ischemic colitis (IC), infectious colitis, and inflammatory bowel disease (IBD), and 22 controls. HIF-1α expression in >10 % of the cells was regarded as positive staining, and expression <10 % of the cells was considered as negative staining. In the upper GI tract, HIF-1α expression was found in 5/20 CGI patients, but not in controls (p = 0.08). The sensitivity and specificity of HIF-1α expression for diagnosing CGI were 25 and 84%, respectively. In the lower GI tract, HIF-1α was expressed in all patients with IC and infectious colitis and in a majority of IBD patients as well as in 7/12 controls. The sensitivity and specificity of HIF-1α for diagnosing IC were 100 and 51%, respectively. HIF-1α expression was more often (p = 0.02) observed in patients with histological signs of inflammation in the lower GI tract. HIF-1α is expressed in acute and chronic ischemic tissue, but also in normal colon tissue and inflammatory disorders.

Distinct claudin expression characterizes BRCA1-related breast cancer.

AIMS: Members of the claudin family are involved in cancer progression and are differentially expressed in subtypes of breast cancer. Breast cancers in BRCA1 germ line mutation carriers have distinct clinicopathological characteristics. Biomarkers that discriminate between BRCA1-related and sporadic breast cancer cases are needed to improve early identification of mutation carriers. In this study we evaluated protein expression of five major claudins in BRCA1-related breast cancers in comparison with sporadic controls. METHODS AND RESULTS: Forty breast cancers in BRCA1 mutation carriers and 40 age-matched sporadic breast cancers were immunohistochemically stained for claudins 1, 3, 4, 6 and 7. Total intratumoural expression levels were compared to those in the surrounding normal tissue. In addition, subcellular claudin expression was scored. Higher overexpression rates were observed for all five claudins in BRCA1-related breast cancers when compared to sporadic controls. In multivariate analysis, overexpression of claudin 3, 4, and 7 was mainly dependent on ER-status, whereas overexpression of claudin 6 and high membranous expression of claudin 1 were independent of other characteristics. CONCLUSIONS: BRCA1-related breast cancers are characterized by frequent overexpression of claudins. Especially claudin 1 and 6 expression may help to discriminate mutation carriers from sporadic breast cancer cases.

Genomic evolution from primary breast carcinoma to distant metastasis: Few copy number changes of breast cancer related genes.

Cancer initiation and progression is characterized by (epi)genetic aberrations. However, little is known about the changes that occur during breast cancer metastasis. In the present study, multiplex ligation-dependent probe amplification was used to compare copy numbers of 21 established oncogenes and tumor suppressor genes between 55 primary breast cancer samples and corresponding distant metastases. Distant breast cancer metastases generally showed similar gene copy number aberrations compared to their corresponding primary tumors. The few genes that showed differences between primary tumor and metastasis (PRDM14, MED1, CCNE1, TRAF4, MTDH, CDH1) have been implicated in the development of therapy resistance.

Analysis of the genomic response of human prostate cancer cells to histone deacetylase inhibitors.

Histone deacetylases (HDACs) have emerged as important targets for cancer treatment. HDAC-inhibitors (HDACis) are well tolerated in patients and have been approved for the treatment of patients with cutaneous T-cell lymphoma (CTCL). To improve the clinical benefit of HDACis in solid tumors, combination strategies with HDACis could be employed. In this study, we applied Analysis of Functional Annotation (AFA) to provide a comprehensive list of genes and pathways affected upon HDACi-treatment in prostate cancer cells. This approach provides an unbiased and objective approach to high throughput data mining. By performing AFA on gene expression data from prostate cancer cell lines DU-145 (an HDACi-sensitive cell line) and PC3 (a relatively HDACi-resistant cell line) treated with HDACis valproic acid or vorinostat, we identified biological processes that are affected by HDACis and are therefore potential treatment targets for combination therapy. Our analysis revealed that HDAC-inhibition resulted among others in upregulation of major histocompatibility complex (MHC) genes and deregulation of the mitotic spindle checkpoint by downregulation of genes involved in mitosis. These findings were confirmed by AFA on publicly available data sets from HDACi-treated prostate cancer cells. In total, we analyzed 375 microarrays with HDACi treated and non-treated (control) prostate cancer cells. All results from this extensive analysis are provided as an online research source (available at the journal's website and at http://luigimarchionni.org/HDACIs.html). By publishing this data, we aim to enhance our understanding of the cellular changes after HDAC-inhibition, and to identify novel potential combination strategies with HDACis for the treatment of prostate cancer patients.

Loss of p120-catenin induces metastatic progression of breast cancer by inducing anoikis resistance and augmenting growth factor receptor signaling.

Metastatic breast cancer remains the chief cause of cancer-related death among women in the Western world. Although loss of cell-cell adhesion is key to breast cancer progression, little is known about the underlying mechanisms that drive tumor invasion and metastasis. Here, we show that somatic loss of p120-catenin (p120) in a conditional mouse model of noninvasive mammary carcinoma results in formation of stromal-dense tumors that resemble human metaplastic breast cancer and metastasize to lungs and lymph nodes. Loss of p120 in anchorage-dependent breast cancer cell lines strongly promoted anoikis resistance through hypersensitization of growth factor receptor (GFR) signaling. Interestingly, p120 deletion also induced secretion of inflammatory cytokines, a feature that likely underlies the formation of the prometastatic microenvironment in p120-negative mammary carcinomas. Our results establish a preclinical platform to develop tailored intervention regimens that target GFR signals to treat p120-negative metastatic breast cancers.

Regulation of E2F1 by the von Hippel-Lindau tumour suppressor protein predicts survival in renal cell cancer patients.

Biallelic mutations of the von Hippel-Lindau (VHL) gene are the most common cause of sporadic and inherited renal cell carcinoma (RCC). Loss of VHL has been reported to affect cell proliferation by deregulating cell cycle-associated proteins. We report that the VHL gene product (pVHL) inhibits E2F1 expression at both mRNA and protein level in zebrafish and human RCC cells, while loss of VHL increases E2F1 expression in patient kidney tumour tissue and RCC cells, resulting in a delay of cell cycle progression. RCCs from von Hippel-Lindau patients with known germline VHL mutations express significantly more E2F1 compared to sporadic RCCs with either clear-cell (cc) or non-cc histology. Analysis of 138 primary RCCs reveals that E2F1 expression is significantly higher in tumours with a diameter ≤7 cm and with a favourable American Joint Committee on Cancer (AJCC) stage. The expression of E2F1 in RCC significantly correlates with p27 expression, suggesting that increased expression of E2F1 in RCC induces tumour cell senescence via p27. Cox regression analysis shows significant prediction of E2F1 expression for disease-free survival and overall survival, implying that E2F1 expression in kidney tumour is a novel prognostic factor for patients with RCC.

Expression of the RNA helicase DDX3 and the hypoxia response in breast cancer.

AIMS: DDX3 is an RNA helicase that has antiapoptotic properties, and promotes proliferation and transformation. In addition, DDX3 was shown to be a direct downstream target of HIF-1α (the master regulatory of the hypoxia response) in breast cancer cell lines. However, the relation between DDX3 and hypoxia has not been addressed in human tumors. In this paper, we studied the relation between DDX3 and the hypoxic responsive proteins in human breast cancer. METHODS AND RESULTS: DDX3 expression was investigated by immunohistochemistry in breast cancer in comparison with hypoxia related proteins HIF-1α, GLUT1, CAIX, EGFR, HER2, Akt1, FOXO4, p53, ERα, COMMD1, FER kinase, PIN1, E-cadherin, p21, p27, Transferrin receptor, FOXO3A, c-Met and Notch1. DDX3 was overexpressed in 127 of 366 breast cancer patients, and was correlated with overexpression of HIF-1α and its downstream genes CAIX and GLUT1. Moreover, DDX3 expression correlated with hypoxia-related proteins EGFR, HER2, FOXO4, ERα and c-Met in a HIF-1α dependent fashion, and with COMMD1, FER kinase, Akt1, E-cadherin, TfR and FOXO3A independent of HIF-1α. CONCLUSIONS: In invasive breast cancer, expression of DDX3 was correlated with overexpression of HIF-1α and many other hypoxia related proteins, pointing to a distinct role for DDX3 under hypoxic conditions and supporting the oncogenic role of DDX3 which could have clinical implication for current development of DDX3 inhibitors.