Publisher Correction: Barcoding reveals complex clonal behavior in patient-derived xenografts of metastatic triple negative breast cancer.

The original version of this Article contained an error in Fig. 4. In the left histogram of the right panel of Fig. 4d, several data points were inadvertently deleted from the histogram during the production process. This error has been corrected in both the PDF and HTML versions of the Article. The original, incorrect version of Fig. 4 is presented in the accompanying Publisher Correction.

Barcoding reveals complex clonal behavior in patient-derived xenografts of metastatic triple negative breast cancer.

Primary triple negative breast cancers (TNBC) are prone to dissemination but sub-clonal relationships between tumors and resulting metastases are poorly understood. Here we use cellular barcoding of two treatment-naïve TNBC patient-derived xenografts (PDXs) to track the spatio-temporal fate of thousands of barcoded clones in primary tumors, and their metastases. Tumor resection had a major impact on reducing clonal diversity in secondary sites, indicating that most disseminated tumor cells lacked the capacity to 'seed', hence originated from 'shedders' that did not persist. The few clones that continued to grow after resection i.e. 'seeders', did not correlate in frequency with their parental clones in primary tumors. Cisplatin treatment of one BRCA1-mutated PDX model to non-palpable levels had a surprisingly minor impact on clonal diversity in the relapsed tumor yet purged 50% of distal clones. Therefore, clonal features of shedding, seeding and drug resistance are important factors to consider for the design of therapeutic strategies.

Targeting BCL-2 to enhance vulnerability to therapy in estrogen receptor-positive breast cancer.

The last three decades have seen significant progress in our understanding of the role of the pro-survival protein BCL-2 and its family members in apoptosis and cancer. BCL-2 and other pro-survival family members including Mcl-1 and BCL-XL have been shown to have a key role in keeping pro-apoptotic 'effector' proteins BAK and BAX in check. They also neutralize a group of 'sensor' proteins (such as BIM), which are triggered by cytotoxic stimuli such as chemotherapy. BCL-2 proteins therefore have a central role as guardians against apoptosis, helping cancer cells to evade cell death. More recently, an increasing number of BH3 mimetics, which bind and neutralize BCL-2 and/or its pro-survival relatives, have been developed. The utility of targeting BCL-2 in hematological malignancies has become evident in early-phase studies, with remarkable clinical responses seen in heavily pretreated patients. As BCL-2 is overexpressed in ~75% of breast cancer, there has been growing interest in determining whether this new class of drug could show similar promise in breast cancer. This review summarizes our current understanding of the role of BCL-2 and its family members in mammary gland development and breast cancer, recent progress in the development of new BH3 mimetics as well as their potential for targeting estrogen receptor-positive breast cancer.

Pro-apoptotic Bim suppresses breast tumor cell metastasis and is a target gene of SNAI2.

Evasion of cell death is fundamental to the development of cancer and its metastasis. The role of the BCL-2-mediated (intrinsic) apoptotic program in these processes remains poorly understood. Here we have investigated the relevance of the pro-apoptotic protein BIM to breast cancer progression using the MMTV-Polyoma middle-T (PyMT) transgenic model. BIM deficiency in PyMT females did not affect primary tumor growth, but substantially increased the survival of metastatic cells within the lung. These data reveal a role for BIM in the suppression of breast cancer metastasis. Intriguingly, we observed a striking correlation between the expression of BIM and the epithelial to mesenchymal transition transcription factor SNAI2 at the proliferative edge of the tumors. Overexpression and knockdown studies confirmed that these two genes were coordinately expressed, and chromatin immunoprecipitation analysis further revealed that Bim is a target of SNAI2. Taken together, our findings suggest that SNAI2-driven BIM-induced apoptosis may temper metastasis by governing the survival of disseminating breast tumor cells.

Tumour morphology predicts PALB2 germline mutation status.

BACKGROUND: Population-based studies of breast cancer have estimated that at least some PALB2 mutations are associated with high breast cancer risk. For women carrying PALB2 mutations, knowing their carrier status could be useful in directing them towards effective cancer risk management and therapeutic strategies. We sought to determine whether morphological features of breast tumours can predict PALB2 germline mutation status. METHODS: Systematic pathology review was conducted on breast tumours from 28 female carriers of PALB2 mutations (non-carriers of other known high-risk mutations, recruited through various resources with varying ascertainment) and on breast tumours from a population-based sample of 828 Australian women diagnosed before the age of 60 years (which included 40 BRCA1 and 18 BRCA2 mutation carriers). Tumour morphological features of the 28 PALB2 mutation carriers were compared with those of 770 women without high-risk mutations. RESULTS: Tumours arising in PALB2 mutation carriers were associated with minimal sclerosis (odds ratio (OR)=19.7; 95% confidence interval (CI)=6.0-64.6; P=5 × 10(-7)). Minimal sclerosis was also a feature that distinguished PALB2 mutation carriers from BRCA1 (P=0.05) and BRCA2 (P=0.04) mutation carriers. CONCLUSION: This study identified minimal sclerosis to be a predictor of germline PALB2 mutation status. Morphological review can therefore facilitate the identification of women most likely to carry mutations in PALB2.

Targeted prostate cancer screening in men with mutations in BRCA1 and BRCA2 detects aggressive prostate cancer: preliminary analysis of the results of the IMPACT study.

OBJECTIVE: To evaluate the role of targeted prostate cancer screening in men with BRCA1 or BRCA2 mutations, an international study, IMPACT (Identification of Men with a genetic predisposition to ProstAte Cancer: Targeted screening in BRCA1/2 mutation carriers and controls), was established. This is the first multicentre screening study targeted at men with a known genetic predisposition to prostate cancer. A preliminary analysis of the data is reported. PATIENTS AND METHODS: Men aged 40-69 years from families with BRCA1 or BRCA2 mutations were offered annual prostate specific antigen (PSA) testing, and those with PSA > 3 ng/mL, were offered a prostate biopsy. Controls were men age-matched (± 5 years) who were negative for the familial mutation. RESULTS: In total, 300 men were recruited (205 mutation carriers; 89 BRCA1, 116 BRCA2 and 95 controls) over 33 months. At the baseline screen (year 1), 7.0% (21/300) underwent a prostate biopsy. Prostate cancer was diagnosed in ten individuals, a prevalence of 3.3%. The positive predictive value of PSA screening in this cohort was 47·6% (10/21). One prostate cancer was diagnosed at year 2. Of the 11 prostate cancers diagnosed, nine were in mutation carriers, two in controls, and eight were clinically significant. CONCLUSIONS: The present study shows that the positive predictive value of PSA screening in BRCA mutation carriers is high and that screening detects clinically significant prostate cancer. These results support the rationale for continued screening in such men.

Analysis of Brca1-deficient mouse mammary glands reveals reciprocal regulation of Brca1 and c-kit.

Disruption of the breast cancer susceptibility gene Brca1 results in defective lobular-alveolar development in the mammary gland and a predisposition to breast tumourigenesis in humans and in mice. Recent evidence suggests that BRCA1 loss in humans is associated with an expansion of the luminal progenitor cell compartment in the normal breast and tumours with a luminal progenitor-like expression profile. To further investigate the role of BRCA1 in the mammary gland, we examined the consequences of Brca1 loss in mouse mammary epithelial cells in vitro and in vivo. Here, we show that Brca1 loss is associated with defective morphogenesis of SCp2 and HC11 mouse mammary epithelial cell lines and that in the MMTV-Cre Brca1(Co/Co) mouse model of Brca1 loss, there is an accumulation of luminal progenitor (CD61(+)CD29(lo)CD24(+)) cells during pregnancy. By day 1 of lactation, there are marked differences in the expression of 1379 genes, with most significantly altered pathways and networks, including lactation, the immune response and cancer. One of the most differentially expressed genes was the luminal progenitor marker, c-kit. Immunohistochemical analysis revealed that the increase in c-kit levels is associated with an increase in c-kit positivity. Interestingly, an inverse association between Brca1 and c-kit expression was also observed during mammary epithelial differentiation, and small interfering RNA-mediated knockdown of Brca1 resulted in a significant increase in c-kit mRNA levels. We found no evidence that c-kit plays a direct role in regulating differentiation of HC11 cells, suggesting that Brca1-mediated induction of c-kit probably contributes to Brca1-associated tumourigenesis via another cellular process, and that c-kit is likely to be a marker rather than a mediator of defective lobular-alveolar development resulting from Brca1 loss.

Predictors of the use of complementary and alternative medicine (CAM) by women at high risk for breast cancer.

BACKGROUND: Few data exist regarding the use of complementary and alternative medicine (CAM) by unaffected women at high risk of breast cancer. METHODS: Self-reported CAM use by women from multiple-case breast cancer families was obtained by questionnaire. Factors associated with CAM use were assessed using multiple logistic regression. RESULTS: Of 892 women, 55% (n=489) used CAM, 6% (n=53) specifically to prevent cancer. CAM use was independently associated with tertiary education level (OR 2.56, 95% CI 1.83-3.58, p<0.001), greater physical activity (OR 1.05 per hour of physical activity/week, 95% CI 1.00-1.10, p=0.049), greater anxiety (OR 1.92, 95% CI 1.16-3.16, p=0.01), not currently smoking (OR 0.64, 95% CI 0.42-0.97, p=0.037) and lower perceived BC risk (OR 0.82 per 20 percentage points, 95% CI 0.72-0.94, p=0.005). CONCLUSIONS: The majority of high-risk women use CAM, but mostly for reasons other than cancer prevention. Most predictors of CAM use are consistent with the limited literature for women at high risk for cancer.

Delineating the epithelial hierarchy in the mouse mammary gland.

Reconstitution assays have shown that mouse mammary stem cells reside within the mature mammary gland in vivo. Single cells could be prospectively isolated and shown to regenerate an entire mammary gland that exhibited full developmental capacity. The more recent identification of luminal progenitor populations has indicated that the mammary epithelium is organized in a hierarchical manner. Further definition of epithelial cell types in both mouse and human mammary glands will provide insight into the "cells of origin" in the different subtypes of breast cancer, as well as the nature of cancer-propagating cells. Here, we review the known characteristics of mammary stem and progenitor cells, their steroid receptor status, and the pathways that have thus far been implicated in regulating their self-renewal and differentiation.

Expression of LMO4 and outcome in pancreatic ductal adenocarcinoma.

Identification of a biomarker of prognosis and response to therapy that can be assessed preoperatively would significantly improve overall outcomes for patients with pancreatic cancer. In this study, patients whose tumours exhibited high LMO4 expression had a significant survival advantage following operative resection, whereas the survival of those patients whose tumours had low or no LMO4 expression was not significantly different when resection was compared with operative biopsy alone.

Risk-reducing surgery, screening and chemoprevention practices of BRCA1 and BRCA2 mutation carriers: a prospective cohort study.

This study prospectively evaluated the utilization of cancer risk management strategies in a multi-institutional cohort of BRCA1 and BRCA2 mutation carriers using a self-report questionnaire. Of 142 unaffected female mutation carriers, 70 (49%) had elected to receive their mutation result. Of those who knew their mutation result, 11% underwent bilateral mastectomy (BM), 29% had bilateral oophorectomy (BO), 78% performed regular breast self-examination (BSE), and 80%, 89%, 67%, and 0% had at least annual clinical breast examination (CBE), mammography, transvaginal ultrasound (TVU), and CA125, respectively. A further 20%, 7%, 0%, 21%, and 75%, respectively, reported never having had these tests. For women who elected not to receive their mutation result, 0% underwent BM, 6% underwent BO, and 77%, 42%, 56%, 7%, and 0% had regular BSE, CBE, mammography, TVU, and CA125, respectively. Only one woman used chemoprevention outside a clinical trial. Uptake of prophylactic surgery and screening was associated with knowing one's mutation status (for all behaviors except BSE), age (for BO and CBE) and residence (for mammography). In this cohort, the minority of mutation carriers utilized risk-reducing surgery or chemoprevention and a substantial minority were not undergoing regular cancer-screening tests.

Genetic, functional, and histopathological evaluation of two C-terminal BRCA1 missense variants.

BACKGROUND: The vast majority of BRCA1 missense sequence variants remain uncharacterized for their possible effect on protein expression and function, and therefore are unclassified in terms of their pathogenicity. BRCA1 plays diverse cellular roles and it is unlikely that any single functional assay will accurately reflect the total cellular implications of missense mutations in this gene. OBJECTIVE: To elucidate the effect of two BRCA1 variants, 5236G>C (G1706A) and 5242C>A (A1708E) on BRCA1 function, and to survey the relative usefulness of several assays to direct the characterisation of other unclassified variants in BRCA genes. METHODS AND RESULTS: Data from a range of bioinformatic, genetic, and histopathological analyses, and in vitro functional assays indicated that the 1708E variant was associated with the disruption of different cellular functions of BRCA1. In transient transfection experiments in T47D and 293T cells, the 1708E product was mislocalised to the cytoplasm and induced centrosome amplification in 293T cells. The 1708E variant also failed to transactivate transcription of reporter constructs in mammalian transcriptional transactivation assays. In contrast, the 1706A variant displayed a phenotype comparable to wildtype BRCA1 in these assays. Consistent with functional data, tumours from 1708E carriers showed typical BRCA1 pathology, while tumour material from 1706A carriers displayed few histopathological features associated with BRCA1 related tumours. CONCLUSIONS: A comprehensive range of genetic, bioinformatic, and functional analyses have been combined for the characterisation of BRCA1 unclassified sequence variants. Consistent with the functional analyses, the combined odds of causality calculated for the 1706A variant after multifactorial likelihood analysis (1:142) indicates a definitive classification of this variant as "benign". In contrast, functional assays of the 1708E variant indicate that it is pathogenic, possibly through subcellular mislocalisation. However, the combined odds of 262:1 in favour of causality of this variant does not meet the minimal ratio of 1000:1 for classification as pathogenic, and A1708E remains formally designated as unclassified. Our findings highlight the importance of comprehensive genetic information, together with detailed functional analysis for the definitive categorisation of unclassified sequence variants. This combination of analyses may have direct application to the characterisation of other unclassified variants in BRCA1 and BRCA2.

Bfk: a novel weakly proapoptotic member of the Bcl-2 protein family with a BH3 and a BH2 region.

Proteins of the Bcl-2 family are critical regulators of apoptosis. Proapoptotic members, like Bax, contain three of the four Bcl-2 homology regions (BH1-3), while BH3-only proteins, like Bim, possess only the short BH3 motif. Database searches revealed Bfk, an unusual novel member of the Bcl-2 family that contains a BH2 and BH3 region but not BH1 or BH4. Bfk is thus most closely related to Bcl-G(L). It lacks a C-terminal membrane anchor and is cytosolic. Enforced expression of Bfk weakly promoted apoptosis and antagonized Bcl-2's prosurvival function. Like Bcl-G(L), Bfk did not bind to any Bcl-2 family members, even though its BH3 motif can mediate association with prosurvival proteins. Low amounts of Bfk were found in stomach, ovary, bone marrow and spleen, but its level in the mammary gland rose markedly during pregnancy, suggesting that Bfk may play a role in mammary development.

The LIM domain gene LMO4 inhibits differentiation of mammary epithelial cells in vitro and is overexpressed in breast cancer.

LMO4 belongs to a family of LIM-only transcriptional regulators, the first two members of which are oncoproteins in acute T cell leukemia. We have explored a role for LMO4, initially described as a human breast tumor autoantigen, in developing mammary epithelium and breast oncogenesis. Lmo4 was expressed predominantly in the lobuloalveoli of the mammary gland during pregnancy. Consistent with a role in proliferation, forced expression of this gene inhibited differentiation of mammary epithelial cells. Overexpression of LMO4 mRNA was observed in 5 of 10 human breast cancer cell lines. Moreover, in situ hybridization analysis of 177 primary invasive breast carcinomas revealed overexpression of LMO4 in 56% of specimens. Immunohistochemistry confirmed overexpression in a high percentage (62%) of tumors. These studies imply a role for LMO4 in maintaining proliferation of mammary epithelium and suggest that deregulation of this gene may contribute to breast tumorigenesis.

Design, production and characterization of FLIN2 and FLIN4: the engineering of intramolecular ldb1:LMO complexes.

The nuclear LIM-only (LMO) transcription factors LMO2 and LMO4 play important roles in both normal and leukemic T-cell development. LIM domains are cysteine/histidine-rich domains that contain two structural zinc ions and that function as protein-protein adaptors; members of the LMO family each contain two closely spaced LIM domains. These LMO proteins all bind with high affinity to the nuclear protein LIM domain binding protein 1 (ldb1). The LMO-ldb1 interaction is mediated through the N-terminal LIM domain (LIM1) of LMO proteins and a 38-residue region towards the C-terminus of ldb1 [ldb1(LID)]. Unfortunately, recombinant forms of LMO2 and LMO4 have limited solubility and stability, effectively preventing structural analysis. Therefore, we have designed and constructed a fusion protein in which ldb1(LID) and LIM1 of LMO2 can form an intramolecular complex. The engineered protein, FLIN2 (fusion of the LIM interacting domain of ldb1 and the N-terminal LIM domain of LMO2) has been expressed and purified in milligram quantities. FLIN2 is monomeric, contains significant levels of secondary structure and yields a sharp and well-dispersed one-dimensional (1)H NMR spectrum. The analogous LMO4 protein, FLIN4, has almost identical properties. These data suggest that we will be able to obtain high-resolution structural information about the LMO-ldb1 interactions.

E2F4 is exported from the nucleus in a CRM1-dependent manner.

E2F is a family of transcription factors required for normal cell cycle control and for cell cycle arrest in G1. E2F4 is the most abundant E2F protein in many cell types. In quiescent cells, it is localized to the nucleus, where it is bound to the retinoblastoma-related protein p130. During entry into the cell cycle, the protein disappears from the nucleus and appears in the cytoplasm. The mechanism by which this change occurs has, in the past, been unclear. We have found that E2F4 is actively exported from the nucleus and that leptomycin B, a specific inhibitor of nuclear export, inhibits this process. E2F4 export is mediated by two hydrophobic export sequences, mutations in either of which result in export failure. Individual export mutants of E2F4, but not a mutant with inactivation of both export signals, can be efficiently excluded from the nucleus by forced coexpression of the nuclear export receptor CRM1. Similarly, CRM1 overexpression can prevent cell cycle arrest induced by the cyclin kinase inhibitor p16(INK4a), an E2F4-dependent process. Taken together, these data suggest that nuclear export contributes to the regulation of E2F4 function, including its ability to regulate exit from G1 in association with a suitable pocket protein.

E2F4 and E2F5 play an essential role in pocket protein-mediated G1 control.

E2F transcription factors are major regulators of cell proliferation. The diversity of the E2F family suggests that individual members perform distinct functions in cell cycle control. E2F4 and E2F5 constitute a defined subset of the family. Until now, there has been little understanding of their individual biochemical and biological functions. Here, we report that simultaneous inactivation of E2F4 and E2F5 in mice results in neonatal lethality, suggesting that they perform overlapping functions during mouse development. Embryonic fibroblasts isolated from these mice proliferated normally and reentered from Go with normal kinetics compared to wild-type cells. However, they failed to arrest in G1 in response to p16INK4a. Thus, E2F4 and E2F5 are dispensable for cell cycle progression but necessary for pocket protein-mediated G1 arrest of cycling cells.