Low level of Fibrillarin, a ribosome biogenesis factor, is a new independent marker of poor outcome in breast cancer.

BACKGROUND: A current critical need remains in the identification of prognostic and predictive markers in early breast cancer. It appears that a distinctive trait of cancer cells is their addiction to hyperactivation of ribosome biogenesis. Thus, ribosome biogenesis might be an innovative source of biomarkers that remains to be evaluated. METHODS: Here, fibrillarin (FBL) was used as a surrogate marker of ribosome biogenesis due to its essential role in the early steps of ribosome biogenesis and its association with poor prognosis in breast cancer when overexpressed. Using 3,275 non-metastatic primary breast tumors, we analysed FBL mRNA expression levels and protein nucleolar organisation. Usage of TCGA dataset allowed transcriptomic comparison between the different FBL expression levels-related breast tumours. RESULTS: We unexpectedly discovered that in addition to breast tumours expressing high level of FBL, about 10% of the breast tumors express low level of FBL. A correlation between low FBL mRNA level and lack of FBL detection at protein level using immunohistochemistry was observed. Interestingly, multivariate analyses revealed that these low FBL tumors displayed poor outcome compared to current clinical gold standards. Transcriptomic data revealed that FBL expression is proportionally associated with distinct amount of ribosomes, low FBL level being associated with low amount of ribosomes. Moreover, the molecular programs supported by low and high FBL expressing tumors were distinct. CONCLUSION: Altogether, we identified FBL as a powerful ribosome biogenesis-related independent marker of breast cancer outcome. Surprisingly we unveil a dual association of the ribosome biogenesis FBL factor with prognosis. These data suggest that hyper- but also hypo-activation of ribosome biogenesis are molecular traits of distinct tumors.

Erratum: Ribosomal RNA 2' O-methylation as a novel layer of inter-tumour heterogeneity in breast cancer.

[This corrects the article DOI: 10.1093/narcan/zcaa036.].

Cluster analyses of the TCGA and a TMA dataset using the coexpression of HSP27 and CRYAB improves alignment with clinical-pathological parameters of breast cancer and suggests different epichaperome influences for each sHSP.

Our cluster analysis of the Cancer Genome Atlas for co-expression of HSP27 and CRYAB in breast cancer patients identified three patient groups based on their expression level combination (high HSP27 + low CRYAB; low HSP27 + high CRYAB; similar HSP27 + CRYAB). Our analyses also suggest that there is a statistically significant inverse relationship between HSP27 and CRYAB and known clinicopathological markers in breast cancer. Screening an unbiased 248 breast cancer patient tissue microarray (TMA) for the protein expression of HSP27 and phosphorylated HSP27 (HSP27-82pS) with CRYAB also identified three patient groups based on HSP27 and CRYAB expression levels. TMA24 also had recorded clinical-pathological parameters, such as ER and PR receptor status, patient survival, and TP53 mutation status. High HSP27 protein levels were significant with ER and PR expression. HSP27-82pS associated with the best patient survival (Log Rank test). High CRYAB expression in combination with wild-type TP53 was significant for patient survival, but a different patient outcome was observed when mutant TP53 was combined with high CRYAB expression. Our data suggest that HSP27 and CRYAB have different epichaperome influences in breast cancer, but more importantly evidence the value of a cluster analysis that considers their coexpression. Our approach can deliver convergence for archival datasets as well as those from recent treatment and patient cohorts and can align HSP27 and CRYAB expression to important clinical-pathological features of breast cancer.

Ribosomal RNA 2'O-methylation as a novel layer of inter-tumour heterogeneity in breast cancer.

Recent epitranscriptomics studies unravelled that ribosomal RNA (rRNA) 2'O-methylation is an additional layer of gene expression regulation highlighting the ribosome as a novel actor of translation control. However, this major finding lies on evidences coming mainly, if not exclusively, from cellular models. Using the innovative next-generation RiboMeth-seq technology, we established the first rRNA 2'O-methylation landscape in 195 primary human breast tumours. We uncovered the existence of compulsory/stable sites, which show limited inter-patient variability in their 2'O-methylation level, which map on functionally important sites of the human ribosome structure and which are surrounded by variable sites found from the second nucleotide layers. Our data demonstrate that some positions within the rRNA molecules can tolerate absence of 2'O-methylation in tumoral and healthy tissues. We also reveal that rRNA 2'O-methylation exhibits intra- and inter-patient variability in breast tumours. Its level is indeed differentially associated with breast cancer subtype and tumour grade. Altogether, our rRNA 2'O-methylation profiling of a large-scale human sample collection provides the first compelling evidence that ribosome variability occurs in humans and suggests that rRNA 2'O-methylation might represent a relevant element of tumour biology useful in clinic. This novel variability at molecular level offers an additional layer to capture the cancer heterogeneity and associates with specific features of tumour biology thus offering a novel targetable molecular signature in cancer.

∆Np63/p40 correlates with the location and phenotype of basal/mesenchymal cancer stem-like cells in human ER+ and HER2+ breast cancers.

ΔNp63, also known as p40, regulates stemness of normal mammary gland epithelium and provides stem cell characteristics in basal and HER2-driven murine breast cancer models. Whilst ΔNp63/p40 is a characteristic feature of normal basal cells and basal-type triple-negative breast cancer, some receptor-positive breast cancers express ΔNp63/p40 and its overexpression imparts cancer stem cell-like properties in ER+ cell lines. However, the incidence of ER+ and HER2+ tumours that express ΔNp63/p40 is unclear and the phenotype of ΔNp63/p40+ cells in these tumours remains uncertain. Using immunohistochemistry with p63 isoform-specific antibodies, we identified a ΔNp63/p40+ tumour cell subpopulation in 100 of 173 (58%) non-triple negative breast cancers and the presence of this population associated with improved survival in patients with ER- /HER2+ tumours (p = 0.006). Furthermore, 41% of ER+ /PR+ and/or HER2+ locally metastatic breast cancers expressed ΔNp63/p40, and these cells commonly accounted for <1% of the metastatic tumour cell population that localised to the tumour/stroma interface, exhibited an undifferentiated phenotype and were CD44+ /ALDH- . In vitro studies revealed that MCF7 and T47D (ER+ ) and BT-474 (HER2+ ) breast cancer cell lines similarly contained a small subpopulation of ΔNp63/p40+ cells that increased in mammospheres. In vivo, MCF7 xenografts contained ΔNp63/p40+ cells with a similar phenotype to primary ER+ cancers. Consistent with tumour samples, these cells also showed a distinct location at the tumour/stroma interface, suggesting a role for paracrine factors in the induction or maintenance of ΔNp63/p40. Thus, ΔNp63/p40 is commonly present in a small population of tumour cells with a distinct phenotype and location in ER+ and/or HER2+ human breast cancers.

A comparison of the imaging features of pleomorphic and classical invasive lobular carcinoma.

PURPOSE: Pleomorphic invasive lobular carcinoma (pILC) is a distinct morphological variant of ILC with a poorer prognosis than classical ILC (cILC). The aim of this study was to ascertain whether the conventional imaging appearances of the two entities differ. METHODS: A single-center retrospective review of conventional imaging was undertaken in 150 consecutive patients with histopathologically confirmed ILC (38 pILC; 112 cILC) between April 2010 and July 2015. Mammographic and sonographic findings were evaluated using the BI-RADS lexicon by a radiologist blinded to pathology, and the findings in the two groups were compared. The degree of discrepancy between imaging and pathological sizing in the two groups was evaluated. RESULTS: Lesions were mammographically occult in 11% of pILC and 14% of cILC (p = 0.56). On mammography, skin or trabecular thickening and microcalcification were commoner in pILC than cILC (13% vs. 1%, p < 0.01; 25% vs. 5%, p < 0.01). Architectural distortion was more frequent in cILC than pILC (26% vs. 9%, p = 0.01). On ultrasound, pILC more frequently exhibited mixed echogenicity (28% vs. 13%; p = 0.04), skin thickening, subcutaneous or parenchymal edema (8% vs. 0%; p = 0.02), echogenic surrounding fat (33% vs. 9%; p < 0.01), and posterior acoustic enhancement (10% vs. 1%; p = 0.02) than cILC. CILC was more frequently manifested as a focal area of altered echogenicity (24% vs. 8%; p = 0.04). Mean elastography stiffness was higher for pILC (174.8 vs. 124.6 kPa; p = 0.02). Imaging-pathological size disparity was similar for both subtypes. CONCLUSION: There are differences in the imaging features between pILC and cILC which reflect the more aggressive nature of pILC.

Stanniocalcin 2 expression is associated with a favourable outcome in male breast cancer.

Breast cancer can occur in either gender; however, it is rare in men, accounting for <1% of diagnosed cases. In a previous transcriptomic screen of male breast cancer (MBC) and female breast cancer (FBC) occurrences, we observed that Stanniocalcin 2 (STC2) was overexpressed in the former. The aim of this study was to confirm the expression of STC2 in MBC and to investigate whether this had an impact on patient prognosis. Following an earlier transcriptomic screen, STC2 gene expression was confirmed by RT-qPCR in matched MBC and FBC samples as well as in tumour-associated fibroblasts derived from each gender. Subsequently, STC2 protein expression was examined immunohistochemically in tissue microarrays containing 477 MBC cases. Cumulative survival probabilities were calculated using the Kaplan-Meier method and multivariate survival analysis was performed using the Cox hazard model. Gender-specific STC2 gene expression showed a 5.6-fold upregulation of STC2 transcripts in MBC, also supported by data deposited in Oncomine™. STC2 protein expression was a positive prognostic factor for disease-free survival (DFS; Log-rank; total p = 0.035, HR = 0.49; tumour cells p = 0.017, HR = 0.44; stroma p = 0.030, HR = 0.48) but had no significant impact on overall survival (Log-rank; total p = 0.23, HR = 0.71; tumour cells p = 0.069, HR = 0.59; stroma p = 0.650, HR = 0.87). Importantly, multivariate analysis adjusted for patient age at diagnosis, node staging, tumour size, ER, and PR status revealed that total STC2 expression as well as expression in tumour cells was an independent prognostic factor for DFS (Cox regression; p = 0.018, HR = 0.983; p = 0.015, HR = 0.984, respectively). In conclusion, STC2 expression is abundant in MBC where it is an independent prognostic factor for DFS.

Breast cancer: influence of tumour volume estimation method at MRI on prediction of pathological response to neoadjuvant chemotherapy.

OBJECTIVE: Does method of tumour volume measurement on MRI influence prediction of treatment outcome in patients with primary breast cancer undergoing neoadjuvant chemotherapy (NAC)?. METHOD: The study comprised of 136 women with biopsy-proven breast cancer scheduled for MRI monitoring during NAC treatment. Dynamic contrast-enhanced images were acquired at baseline (pre-NAC) and interim (post three NAC cycles) time points. Functional tumour volumes (FTVs), automatically derived using vendor software and enhancing tumour volumes (ETVs), user-derived using a semi-automated thresholding technique, were calculated at each time point and percentage changes calculated. Response, assessed using residual cancer burden (RCB) score on surgically resected specimens, was compared statistically with volumetric changes and receiver operating characteristic analysis performed. RESULTS: Mean volumetric differences for each RCB response category were (FTV/ETV): pathological complete response (pCR) 95.5/96.8%, RCB-I 69.8/66.7%, RCB-II 64.0/65.5%, RCB-III 25.4/24.0%. Differences were significant between pCR and RCB-II/RCB-III categories (p < 0.040; unpaired t-test) using FTV measures and between pCR and RCB-I/RCB-II/RCB-III categories (p < 0.006; unpaired t-test) when ETV was used. Receiver operating characteristic analysis for pCR identification post-NAC yielded area under the curve for FTV/ETV of 0.834/0.920 respectively. Sensitivity and specificity for FTV was 80.0 and 76.8% for FTV and 81.0 and 91.8% for ETV. CONCLUSION: ETV changes can identify patients likely to achieve a complete response to NAC. Potentially, this could impact patient management regarding the possible avoidance of post-NAC surgery. Advances in Knowledge: Interim changes in ETV are more useful than FTV in predicting final pathological response to NAC. ETV differentiates patients who will achieve a complete response from those who will have residual disease.

Assessing the effectiveness of NICE criteria for stratifying breast cancer risk in a UK cohort.

Breast cancer risk is a common indication for referral to clinical genetics services. UK National Institute of Health and Care Excellence (NICE) guidelines use family history (FH) to stratify by 10-year risk of breast cancer from age 40. Patients are stratified into population risk (PR, 10-year risk <3%), moderate (MR, 3-8%) and high risk (HR, >8%). Women at increased risk are offered screening at or prior to age 40. To assess the clinical effectiveness of current risk stratification, FH data were obtained for all unaffected women with a FH of breast cancer aged <50, referred to cancer genetics from 2000-2010. Patients were risk stratified by NICE criteria, identifying patients who subsequently developed breast cancer. A total of 1409 women had 15,414 patient years of follow-up. Thirty invasive breast cancers developed, 13 in MR and 13 in HR women. Kaplan-Meier analysis demonstrated no significant difference in the rate of breast cancer development between PR and MR women from ages 40 to 49 (Log rank p = 0.431). There was a significant difference between ages 40 and 49 years between PR and HR women (p = 0.036), but not on exclusion of BRCA mutation carriers (p = 0.136). NICE absolute 10-year risk thresholds between ages 40 and 49 were not met in any risk group, when risk was estimated using the guidelines (PR = 0.82%, MR = 1.68%, HR = 3.56%). Our data suggest that improved criteria are required for risk assessment prior to age 50 and screening resources may be best focussed on those with highly penetrant mutations in cancer risk genes.

Correlation of X-ray diffraction signatures of breast tissue and their histopathological classification.

This pilot study examines the correlation of X-ray diffraction (XRD) measurements with the histopathological analysis of breast tissue. Eight breast cancer samples were investigated. Each sample contained a mixture of normal and cancerous tissues. In total, 522 separate XRD measurements were made at different locations across the samples (8 in total). The resulting XRD spectra were subjected to principal component analysis (PCA) in order to determine if there were any distinguishing features that could be used to identify different tissue components. 99.0% of the variation between the spectra were described by the first two principal components (PC). Comparing the location of points in PC space with the classification determined by histopathology indicated correlation between the shape/magnitude of the XRD spectra and the tissue type. These results are encouraging and suggest that XRD could be used for the intraoperative or postoperative classification of bulk tissue samples.

Characterisation of male breast cancer: a descriptive biomarker study from a large patient series.

Male breast cancer (MBC) is rare. We assembled 446 MBCs on tissue microarrays and assessed clinicopathological information, together with data from 15 published studies, totalling 1984 cases. By immunohistochemistry we investigated 14 biomarkers (ERα, ERß1, ERß2, ERß5, PR, AR, Bcl-2, HER2, p53, E-cadherin, Ki67, survivin, prolactin, FOXA1) for survival impact. The main histological subtype in our cohort and combined analyses was ductal (81%, 83%), grade 2; (40%, 44%), respectively. Cases were predominantly ERα (84%, 82%) and PR positive (74%, 71%), respectively, with HER2 expression being infrequent (2%, 10%), respectively. In our cohort, advanced age (>67) was the strongest predictor of overall (OS) and disease free survival (DFS) (p = 0.00001; p = 0.01, respectively). Node positivity negatively impacted DFS (p = 0.04). FOXA1 p = 0.005) and AR p = 0.009) were both positively prognostic for DFS, remaining upon multivariate analysis. Network analysis showed ERα, AR and FOXA1 significantly correlated. In summary, the principle phenotype of MBC was luminal A, ductal, grade 2. In ERα+ MBC, only AR had prognostic significance, suggesting AR blockade could be employed therapeutically.

A Case-Matched Gender Comparison Transcriptomic Screen Identifies eIF4E and eIF5 as Potential Prognostic Markers in Male Breast Cancer.

Purpose: Breast cancer affects both genders, but is understudied in men. Although still rare, male breast cancer (MBC) is being diagnosed more frequently. Treatments are wholly informed by clinical studies conducted in women, based on assumptions that underlying biology is similar.Experimental Design: A transcriptomic investigation of male and female breast cancer was performed, confirming transcriptomic data in silico Biomarkers were immunohistochemically assessed in 697 MBCs (n = 477, training; n = 220, validation set) and quantified in pre- and posttreatment samples from an MBC patient receiving everolimus and PI3K/mTOR inhibitor.Results: Gender-specific gene expression patterns were identified. eIF transcripts were upregulated in MBC. eIF4E and eIF5 were negatively prognostic for overall survival alone (log-rank P = 0.013; HR = 1.77, 1.12-2.8 and P = 0.035; HR = 1.68, 1.03-2.74, respectively), or when coexpressed (P = 0.01; HR = 2.66, 1.26-5.63), confirmed in the validation set. This remained upon multivariate Cox regression analysis [eIF4E P = 0.016; HR = 2.38 (1.18-4.8), eIF5 P = 0.022; HR = 2.55 (1.14-5.7); coexpression P = 0.001; HR = 7.04 (2.22-22.26)]. Marked reduction in eIF4E and eIF5 expression was seen post BEZ235/everolimus, with extended survival.Conclusions: Translational initiation pathway inhibition could be of clinical utility in MBC patients overexpressing eIF4E and eIF5. With mTOR inhibitors that target this pathway now in the clinic, these biomarkers may represent new targets for therapeutic intervention, although further independent validation is required. Clin Cancer Res; 23(10); 2575-83. ©2016 AACR.

TP53 drives invasion through expression of its Δ133p53ß variant.

TP53 is conventionally thought to prevent cancer formation and progression to metastasis, while mutant TP53 has transforming activities. However, in the clinic, TP53 mutation status does not accurately predict cancer progression. Here we report, based on clinical analysis corroborated with experimental data, that the p53 isoform Δ133p53ß promotes cancer cell invasion, regardless of TP53 mutation status. Δ133p53ß increases risk of cancer recurrence and death in breast cancer patients. Furthermore Δ133p53ß is critical to define invasiveness in a panel of breast and colon cell lines, expressing WT or mutant TP53. Endogenous mutant Δ133p53ß depletion prevents invasiveness without affecting mutant full-length p53 protein expression. Mechanistically WT and mutant Δ133p53ß induces EMT. Our findings provide explanations to 2 long-lasting and important clinical conundrums: how WT TP53 can promote cancer cell invasion and reciprocally why mutant TP53 gene does not systematically induce cancer progression.

A prospective comparison of ER, PR, Ki67 and gene expression in paired sequential core biopsies of primary, untreated breast cancer.

BACKGROUND: Sequential biopsy of breast cancer is used to assess biomarker effects and drug efficacy. The preoperative "window of opportunity" setting is advantageous to test biomarker changes in response to therapeutic agents in previously untreated primary cancers. This study tested the consistency over time of paired, sequential biomarker measurements on primary, operable breast cancer in the absence of drug therapy. METHODS: Immunohistochemistry was performed for ER, PR and Ki67 on paired preoperative/operative tumor samples taken from untreated patients within 2 weeks of each other. Microarray analysis on mRNA extracted from formalin fixed paraffin embedded cores was performed using Affymetrix based arrays on paired core biopsies analysed using Ingenuity Pathway Analysis (IPA) and Gene Set Analysis (GSA). RESULTS: In 41 core/resection pairs, the recognised trend to lower ER, PR and Ki67 score on resected material was confirmed. Concordance for ER, PR and Ki67 without changing biomarker status (e.g. ER+ to ER-) was 90, 74 and 80 % respectively. However, in 23 paired core samples (diagnostic core v on table core), Ki67 using a cut off of 13.25 % was concordant in 22/23 (96 %) and differences in ER and PR immunohistochemistry by Allred or Quickscore between the pairs did not impact hormone receptor status. IPA and GSA demonstrated substantial gene expression changes between paired cores at the mRNA level, including reduced expression of ER pathway analysis on the second core, despite the absence of drug intervention. CONCLUSIONS: Sequential core biopsies of primary breast cancer (but not core versus resection) was consistent and is appropriate to assess the effects of drug therapy in vivo on ER, PR and Ki67 using immunohistochemistry. Conversely, studies utilising mRNA expression may require non-treatment controls to distinguish therapeutic from biopsy differences.

The Informatics Challenges Facing Biobanks: A Perspective from a United Kingdom Biobanking Network.

The challenges facing biobanks are changing from simple collections of materials to quality-assured fit-for-purpose clinically annotated samples. As a result, informatics awareness and capabilities of a biobank are now intrinsically related to quality. A biobank may be considered a data repository, in the form of raw data (the unprocessed samples), data surrounding the samples (processing and storage conditions), supplementary data (such as clinical annotations), and an increasing ethical requirement for biobanks to have a mechanism for researchers to return their data. The informatics capabilities of a biobank are no longer simply knowing sample locations; instead the capabilities will become a distinguishing factor in the ability of a biobank to provide appropriate samples. There is an increasing requirement for biobanking systems (whether in-house or commercially sourced) to ensure the informatics systems stay apace with the changes being experienced by the biobanking community. In turn, there is a requirement for the biobanks to have a clear informatics policy and directive that is embedded into the wider decision making process. As an example, the Breast Cancer Campaign Tissue Bank in the UK was a collaboration between four individual and diverse biobanks in the UK, and an informatics platform has been developed to address the challenges of running a distributed network. From developing such a system there are key observations about what can or cannot be achieved by informatics in isolation. This article will highlight some of the lessons learned during this development process.

Comparing computer-generated and pathologist-generated tumour segmentations for immunohistochemical scoring of breast tissue microarrays.

BACKGROUND: Tissue microarrays (TMAs) have become a valuable resource for biomarker expression in translational research. Immunohistochemical (IHC) assessment of TMAs is the principal method for analysing large numbers of patient samples, but manual IHC assessment of TMAs remains a challenging and laborious task. With advances in image analysis, computer-generated analyses of TMAs have the potential to lessen the burden of expert pathologist review. METHODS: In current commercial software computerised oestrogen receptor (ER) scoring relies on tumour localisation in the form of hand-drawn annotations. In this study, tumour localisation for ER scoring was evaluated comparing computer-generated segmentation masks with those of two specialist breast pathologists. Automatically and manually obtained segmentation masks were used to obtain IHC scores for thirty-two ER-stained invasive breast cancer TMA samples using FDA-approved IHC scoring software. RESULTS: Although pixel-level comparisons showed lower agreement between automated and manual segmentation masks (κ=0.81) than between pathologists' masks (κ=0.91), this had little impact on computed IHC scores (Allred; =0.91, Quickscore; =0.92). CONCLUSIONS: The proposed automated system provides consistent measurements thus ensuring standardisation, and shows promise for increasing IHC analysis of nuclear staining in TMAs from large clinical trials.

Expression profiling of nuclear receptors in breast cancer identifies TLX as a mediator of growth and invasion in triple-negative breast cancer.

The Nuclear Receptor (NR) superfamily of transcription factors comprises 48 members, several of which have been implicated in breast cancer. Most important is estrogen receptor-α (ERα), which is a key therapeutic target. ERα action is facilitated by co-operativity with other NR and there is evidence that ERα function may be recapitulated by other NRs in ERα-negative breast cancer. In order to examine the inter-relationships between nuclear receptors, and to obtain evidence for previously unsuspected roles for any NRs, we undertook quantitative RT-PCR and bioinformatics analysis to examine their expression in breast cancer. While most NRs were expressed, bioinformatic analyses differentiated tumours into distinct prognostic groups that were validated by analyzing public microarray data sets. Although ERα and progesterone receptor were dominant in distinguishing prognostic groups, other NR strengthened these groups. Clustering analysis identified several family members with potential importance in breast cancer. Specifically, RORγ is identified as being co-expressed with ERα, whilst several NRs are preferentially expressed in ERα-negative disease, with TLX expression being prognostic in this subtype. Functional studies demonstrated the importance of TLX in regulating growth and invasion in ERα-negative breast cancer cells.

MAGE-A Cancer/Testis Antigens Inhibit MDM2 Ubiquitylation Function and Promote Increased Levels of MDM4.

Melanoma antigen A (MAGE-A) proteins comprise a structurally and biochemically similar sub-family of Cancer/Testis antigens that are expressed in many cancer types and are thought to contribute actively to malignancy. MAGE-A proteins are established regulators of certain cancer-associated transcription factors, including p53, and are activators of several RING finger-dependent ubiquitin E3 ligases. Here, we show that MAGE-A2 associates with MDM2, a ubiquitin E3 ligase that mediates ubiquitylation of more than 20 substrates including mainly p53, MDM2 itself, and MDM4, a potent p53 inhibitor and MDM2 partner that is structurally related to MDM2. We find that MAGE-A2 interacts with MDM2 via the N-terminal p53-binding pocket and the RING finger domain of MDM2 that is required for homo/hetero-dimerization and for E2 ligase interaction. Consistent with these data, we show that MAGE-A2 is a potent inhibitor of the E3 ubiquitin ligase activity of MDM2, yet it does not have any significant effect on p53 turnover mediated by MDM2. Strikingly, however, increased MAGE-A2 expression leads to reduced ubiquitylation and increased levels of MDM4. Similarly, silencing of endogenous MAGE-A expression diminishes MDM4 levels in a manner that can be rescued by the proteasomal inhibitor, bortezomid, and permits increased MDM2/MDM4 association. These data suggest that MAGE-A proteins can: (i) uncouple the ubiquitin ligase and degradation functions of MDM2; (ii) act as potent inhibitors of E3 ligase function; and (iii) regulate the turnover of MDM4. We also find an association between the presence of MAGE-A and increased MDM4 levels in primary breast cancer, suggesting that MAGE-A-dependent control of MDM4 levels has relevance to cancer clinically.

Evidence for biological effects of metformin in operable breast cancer: biomarker analysis in a pre-operative window of opportunity randomized trial.

Metformin has therapeutic potential against breast cancer, but the mechanisms of action in vivo remain uncertain. This study examined biomarker effects of metformin in primary breast cancer in a preoperative window of opportunity trial. Non-diabetic women with operable invasive breast cancer were randomized to receive open label pre-operative metformin (500 mg daily for 1 week then 1 g twice daily for a further week) or as controls, not receiving metformin. Patients in both arms had a core biopsy pre-randomisation and again at the time of surgery. Immunohistochemistry for phospho-AMPK (pAMPK), phospho-Akt (pAkt), insulin receptor, cleaved caspase-3, and Ki67 was performed on formalin-fixed paraffin-embedded cores, scored blinded to treatment and analysed by paired t test. In metformin-treated patients, significant up-regulation of pAMPK (paired t test, p = 0.04) and down-regulation of pAkt (paired t test, p = 0.043) were demonstrated compared to the control group. Insulin receptor and serum insulin remained similar following metformin treatment compared with a rise in insulin receptor and insulin in controls. Significant falls in Ki67 and cleaved caspase-3 (paired t test, p = 0.044) were seen in the metformin-treated patients but not in the control group. Changes were independent of body mass index. These biomarker data suggest mechanisms for metformin action in vivo in breast cancer patients via up-regulation of tumor pAMPK, down-regulation of pAkt, and suppression of insulin responses reflecting cytostatic rather than cytotoxic mechanisms.

Mutant p53 accumulation in human breast cancer is not an intrinsic property or dependent on structural or functional disruption but is regulated by exogenous stress and receptor status.

Many human cancers contain missense TP53 mutations that result in p53 protein accumulation. Although generally considered as a single class of mutations that abrogate wild-type function, individual TP53 mutations may have specific properties and prognostic effects. Tumours that contain missense TP53 mutations show variable p53 stabilization patterns, which may reflect the specific mutation and/or aspects of tumour biology. We used immunohistochemistry on cell lines and human breast cancers with known TP53 missense mutations and assessed the effects of each mutation with four structure-function prediction methods. Cell lines with missense TP53 mutations show variable percentages of cells with p53 stabilization under normal growth conditions, ranging from approximately 50% to almost 100%. Stabilization is not related to structural or functional disruption, but agents that stabilize wild-type p53 increase the percentages of cells showing missense mutant p53 accumulation in cell lines with heterogeneous stabilization. The same heterogeneity of p53 stabilization occurs in primary breast cancers, independent of the effect of the mutation on structural properties or functional disruption. Heterogeneous accumulation is more common in steroid receptor-positive or HER2-positive breast cancers and cell lines than in triple-negative samples. Immunohistochemcal staining patterns associate with Mdm2 levels, proliferation, grade and overall survival, whilst the type of mutation reflects downstream target activity. Inhibiting Mdm2 activity increases the extent of p53 stabilization in some, but not all, breast cancer cell lines. The data indicate that missense mutant p53 stabilization is a complex and variable process in human breast cancers that associates with disease characteristics but is unrelated to structural or functional properties. That agents which stabilize wild-type p53 also stabilize mutant p53 has implications for patients with heterogeneous mutant p53 accumulation, where therapy may activate mutant p53 oncogenic function.