Germline BRCA1 mutation analysis in Indian breast/ovarian cancer families

Most of the predisposition to hereditary breast and ovarian cancer has been attributed to inherited defects in two tumor suppressor genes BRCA1 and BRCA2. To explore the contribution of BRCA1 mutations to hereditary breast cancer among Indian women, we examined the coding sequence of the BRCA1 gene in 14 breast cancer patients with a positive family history of breast and/or ovarian cancer. Mutation analysis was carried out using conformation sensitive gel electrophoresis (CSGE) followed by sequencing. Three mutations (21%) in the BRCA1 gene were identified. Two of them are novel mutations of which one is a missense mutation in exon 7 near the RING finger domain, while the other is a one base pair deletion in exon 11 which results in protein truncation. The third mutation, 185delAG, has been previously described in Ashkenazi Jewish families. To our knowledge this is the first report of a study of germline BRCA1 mutation analysis in familial breast cancer in India. Our data from 14 different families suggests a lower prevalence but definite involvement of germline mutations in the BRCA1 gene among Indian women with breast cancer and a family history of breast cancer.

A Monoclonal Antibody against Human Notch1 Ligand-Binding Domain Depletes Subpopulation of Putative Breast Cancer Stem-like Cells

Overexpression of Notch receptors and ligands has been associated with various cancers and developmental disorders, making Notch a potential therapeutic target. Here, we report characterization of Notch1 monoclonal antibodies (mAb) with therapeutic potential. The mAbs generated against epidermal growth factor (EGF) repeats 11 to 15 inhibited binding of Jagged1 and Delta-like4 and consequently, signaling in a dose-dependent manner, the antibodies against EGF repeats 11 to 12 being more effective than those against repeats 13 to 15. These data emphasize the role of EGF repeats 11 to 12 in ligand binding. One of the mAbs, 602.101, which specifically recognizes Notch1, inhibited ligand-dependent expression of downstream target genes of Notch such as HES-1, HES-5, and HEY-L in the breast cancer cell line MDA-MB-231. The mAb also decreased cell proliferation and induced apoptotic cell death. Furthermore, exposure to this antibody reduced CD44(Hi)/CD24(Low) subpopulation in MDA-MB-231 cells, suggesting a decrease in the cancer stem-like cell subpopulation. This was confirmed by showing that exposure to the antibody decreased the primary, secondary, and tertiary mammosphere formation efficiency of the cells. Interestingly, effect of the antibody on the putative stem-like cells appeared to be irreversible, because the mammosphere-forming efficiency could not be salvaged even after antibody removal during the secondary sphere formation. The antibody also modulated expression of genes associated with stemness and epithelial-mesenchymal transition. Thus, targeting individual Notch receptors by specific mAbs is a potential therapeutic strategy to reduce the potential breast cancer stem-like cell subpopulation. Mol Cancer Ther; 11(1); 77-86. (C) 2011 AACR.

Distinct Roles of FANCO/RAD51C Protein in DNA Damage Signaling and Repair Implications for Fanconi Anemia and Breast Cancer Susceptibility

RAD51C, a RAD51 paralog, has been implicated in homologous recombination (HR), and germ line mutations in RAD51C are known to cause Fanconi anemia (FA)-like disorder and breast and ovarian cancers. The role of RAD51C in the FA pathway of DNA interstrand cross-link (ICL) repair and as a tumor suppressor is obscure. Here, we report that RAD51C deficiency leads to ICL sensitivity, chromatid-type errors, and G(2)/M accumulation, which are hallmarks of the FA phenotype. We find that RAD51C is dispensable for ICL unhooking and FANCD2 monoubiquitination but is essential for HR, confirming the downstream role of RAD51C in ICL repair. Furthermore, we demonstrate that RAD51C plays a vital role in the HR-mediated repair of DNA lesions associated with replication. Finally, we show that RAD51C participates in ICL and double strand break-induced DNA damage signaling and controls intra-S-phase checkpoint through CHK2 activation. Our analyses with pathological mutants of RAD51C that were identified in FA and breast and ovarian cancers reveal that RAD51C regulates HR and DNA damage signaling distinctly. Together, these results unravel the critical role of RAD51C in the FA pathway of ICL repair and as a tumor suppressor.

Introduction of SV40ER and hTERT into mammospheres generates breast cancer cells with stem cell properties

Emerging evidence suggests that cancers arise in stem/progenitor cells. Yet, the requirements for transformation of these primitive cells remains poorly understood. In this study, we have exploited the `mammosphere' system that selects for primitive mammary stem/progenitor cells to explore their potential and requirements for transformation. Introduction of Simian Virus 40 Early Region and hTERT into mammosphere-derived cells led to the generation of NBLE, an immortalized mammary epithelial cell line. The NBLEs largely comprised of bi-potent progenitors with long-term self-renewal and multi-lineage differentiation potential. Clonal and karyotype analyses revealed the existence of heterogeneous population within NBLEs with varied proliferation, differentiation and sphere-forming potential. Significantly, injection of NBLEs into immunocompromised mice resulted in the generation of invasive ductal adenocarcinomas. Further, these cells harbored a sub-population of CD44(+)/CD24(-) fraction that alone had sphere- and tumor-initiating potential and resembled the breast cancer stem cell gene signature. Interestingly, prolonged in vitro culturing led to their further enrichment. The NBLE cells also showed increased expression of stemness and epithelial to mesenchymal transition markers, deregulated self-renewal pathways, activated DNA-damage response and cancer-associated chromosomal aberrations-all of which are likely to have contributed to their tumorigenic transformation. Thus, unlike previous in vitro transformation studies that used adherent, more differentiated human mammary epithelial cells our study demonstrates that the mammosphere-derived, less-differentiated cells undergo tumorigenic conversion with only two genetic elements, without requiring oncogenic Ras. Moreover, the striking phenotypic and molecular resemblance of the NBLE-generated tumors with naturally arising breast adenocarcinomas supports the notion of a primitive breast cell as the origin for this subtype of breast cancer. Finally, the NBLEs represent a heterogeneous population of cells with striking plasticity, capable of differentiation, self-renewal and tumorigenicity, thus offering a unique model system to study the molecular mechanisms involved with these processes. Oncogene (2012) 31, 1896-1909; doi:10.1038/onc.2011.378; published online 29 August 2011

Ultrasound Assisted Optical Tomography: Estimation of phase Shift experienced by photon on transit through US insonified region for detection of breast tumor

A Monte Carlo model of ultrasound modulation of multiply scattered coherent light in a highly scattering media has been carried out for estimating the phase shift experienced by a photon beam on its transit through US insonified region. The phase shift is related to the tissue stiffness, thereby opening an avenue for possible breast tumor detection. When the scattering centers in the tissue medium is exposed to a deterministic forcing with the help of a focused ultrasound (US) beam, due to the fact that US-induced oscillation is almost along particular direction, the direction defined by the transducer axis, the scattering events increase, thereby increasing the phase shift experienced by light that traverses through the medium. The phase shift is found to increase with increase in anisotropy g of the medium. However, as the size of the focused region which is the region of interest (ROI) increases, a large number of scattering events take place within the ROI, the ensemble average of the phase shift (Delta phi) becomes very close to zero. The phase of the individual photon is randomly distributed over 2 pi when the scattered photon path crosses a large number of ultrasound wavelengths in the focused region. This is true at high ultrasound frequency (1 MHz) when mean free path length of photon l(s) is comparable to wavelength of US beam. However, at much lower US frequencies (100 Hz), the wavelength of sound is orders of magnitude larger than l(s), and with a high value of g (g 0.9), there is a distinct measurable phase difference for the photon that traverses through the insonified region. Experiments are carried out for validation of simulation results.

Extracts of Strawberry Fruits Induce Intrinsic Pathway of Apoptosis in Breast Cancer Cells and Inhibits Tumor Progression in Mice

Background: The consumption of berry fruits, including strawberries, has been suggested to have beneficial effects against oxidative stress mediated diseases. Berries contain multiple phenolic compounds and secondary metabolites that contribute to their biological properties. Methodology/Principal Findings: Current study investigates the anticancer activity of the methanolic extract of strawberry (MESB) fruits in leukaemia (CEM) and breast cancer (T47D) cell lines ex vivo, and its cancer therapeutic and chemopreventive potential in mice models. Results of MTT, trypan blue and LDH assays suggested that MESB can induce cytotoxicity in cancer cells, irrespective of origin, in a concentration-and time-dependent manner. Treatment of mice bearing breast adenocarcinoma with MESB blocked the proliferation of tumor cells in a time-dependent manner and resulted in extended life span. Histological and immunohistochemical studies suggest that MESB treatment affected tumor cell proliferation by activating apoptosis and did not result in any side effects. Finally, we show that MESB can induce intrinsic pathway of apoptosis by activating p73 in breast cancer cells, when tumor suppressor gene p53 is mutated. Conclusions/Significance: The present study reveals that strawberry fruits possess both cancer preventive and therapeutic values and we discuss the mechanism by which it is achieved.

Cytotoxic and pro-apoptotic effects of Abrus precatorius L. on human metastatic breast cancer cell line, MDA-MB-231

Abrus precatorius is highly regarded as a universal panacea in the herbal medicine with diverse pharmacological activity spectra. This experimental study on the mechanism of the anticancer activity of A. precatorius leaf extracts, may offer new evidence for A. precatorius in the treatment of breast cancer in clinical practice. Cell death was determined by using MTT assay. Further analyses were carried out by doing DNA laddering, PARP cleavage, FACS, semi-quantitative RT-PCR and detection of cellular reactive oxygen species (ROS) by DCFDA assay. A. precatorius showed very striking inhibition on MDA-MB-231 cells. MTT assay showed more than 75 % inhibition of the cells and treated cells indicated visible laddering pattern with thick compact band. PARP cleavage produced 89 kDa cleavage product which was associated with apoptosis. Flow cytometer exhibited a sub-G0/G1 peak as an indicative of apoptosis. mRNA expression level of apoptosis-related genes p21 and p53 was markedly increased in cells treated with the extract as compared to control. The up-regulation of p21 and p53 may be the molecular mechanisms by which A. precatorius extract which induces apoptosis. An increase in the concentration of A. precatorius extract does not generate ROS, instead it reduces ROS formation in MDA-MB-231 cells, as evident from the shift in fluorescence below untreated control. This is the first report showing that A. precatorius leaf extract exhibits a growth inhibitory effect by induction of apoptosis in MDA-MB-231 cells. Our results contribute towards validation of the A. precatorius extract as a potentially effective chemopreventive or therapeutic agent against breast cancer.

Regulation of protumorigenic pathways by Insulin like growth factor binding protein2 and its association along with beta-catenin in breast cancer lymph node metastasis

Background: Insulin like growth factor binding proteins modulate the mitogenic and pro survival effects of IGF. Elevated expression of IGFBP2 is associated with progression of tumors that include prostate, ovarian, glioma among others. Though implicated in the progression of breast cancer, the molecular mechanisms involved in IGFBP2 actions are not well defined. This study investigates the molecular targets and biological pathways targeted by IGFBP2 in breast cancer. Methods: Transcriptome analysis of breast tumor cells (BT474) with stable knockdown of IGFBP2 and breast tumors having differential expression of IGFBP2 by immunohistochemistry was performed using microarray. Differential gene expression was established using R-Bioconductor package. For validation, gene expression was determined by qPCR. Inhibitors of IGF1R and integrin pathway were utilized to study the mechanism of regulation of beta-catenin. Immunohistochemical and immunocytochemical staining was performed on breast tumors and experimental cells, respectively for beta-catenin and IGFBP2 expression. Results: Knockdown of IGFBP2 resulted in differential expression of 2067 up regulated and 2002 down regulated genes in breast cancer cells. Down regulated genes principally belong to cell cycle, DNA replication, repair, p53 signaling, oxidative phosphorylation, Wnt signaling. Whole genome expression analysis of breast tumors with or without IGFBP2 expression indicated changes in genes belonging to Focal adhesion, Map kinase and Wnt signaling pathways. Interestingly, IGFBP2 knockdown clones showed reduced expression of beta-catenin compared to control cells which was restored upon IGFBP2 re-expression. The regulation of beta-catenin by IGFBP2 was found to be IGF1R and integrin pathway dependent. Furthermore, IGFBP2 and beta-catenin are co-ordinately overexpressed in breast tumors and correlate with lymph node metastasis. Conclusion: This study highlights regulation of beta-catenin by IGFBP2 in breast cancer cells and most importantly, combined expression of IGFBP2 and beta-catenin is associated with lymph node metastasis of breast tumors.

Bmi1 regulates self-renewal and epithelial to mesenchymal transition in breast cancer cells through Nanog

Background: The Bmi1 polycomb ring finger oncogene, a transcriptional repressor belonging to the Polycomb group of proteins plays an important role in the regulation of stem cell self-renewal and is elevated in several cancers. In the current study, we have explored the role of Bmi1 in regulating the stemness and drug resistance of breast cancer cells. Methods: Using real time PCR and immunohistochemistry primary breast tissues were analyzed. Retro-and lentiviruses were utilized to overexpress and knockdown Bmi1, RT-PCR and Western blot was performed to evaluate mRNA and protein expression. Stemness properties were analyzed by flow cytometry and sphere-formation and tumor formation was determined by mouse xenograft experiments. Dual luciferase assay was employed to assess promoter activity and MTT assay was used to analyze drug response. Results: We found Bmi1 overexpression in 64% of grade III invasive ductal breast adenocarcinomas compared to normal breast tissues. Bmi1 overexpression in immortalized and transformed breast epithelial cells increased their sphere-forming efficiency, induced epithelial to mesenchymal transition ( EMT) with an increase in the expression of stemness-related genes. Knockdown of Bmi1 in tumorigenic breast cells induced epithelial morphology, reduced expression of stemness-related genes, decreased the IC50 values of doxorubicin and abrogated tumor-formation. Bmi1-high tumors showed elevated Nanog expression whereas the tumors with lower Bmi1 showed reduced Nanog levels. Overexpression of Bmi1 increased Nanog levels whereas knockdown of Bmi1 reduced its expression. Dual luciferase promoter-reporter assay revealed Bmi1 positively regulated the Nanog and NF kappa B promoter activity. RT-PCR analysis showed that Bmi1 overexpression activated the NF kappa B pathway whereas Bmi1 knockdown reduced the expression of NF kappa B target genes, suggesting that Bmi1 might regulate Nanog expression through the NF kappa B pathway. Conclusions: Our study showed that Bmi1 is overexpressed in several high-grade, invasive ductal breast adenocarcinomas, thus supporting its role as a prognostic marker. While Bmi1 overexpression increased self-renewal and promoted EMT, its knockdown reversed EMT, reduced stemness, and rendered cells drug sensitive, thus highlighting a crucial role for Bmi1 in regulating the stemness and drug response of breast cancer cells. Bmi1 may control self-renewal through the regulation of Nanog expression via the NF kappa B pathway.

Coordinate Hyperactivation of Notch1 and Ras/MAPK Pathways Correlates with Poor Patient Survival: Novel Therapeutic Strategy for Aggressive Breast Cancers

Aberrant activation of Notch and Ras pathways has been detected in breast cancers. A synergy between these two pathways has also been shown in breast cell transformation in culture. Yet, the clinical relevance of Notch-Ras cooperation in breast cancer progression remains unexplored. In this study, we show that coordinate hyperactivation of Notch1 and Ras/MAPK pathways in breast cancer patient specimens, as assessed by IHC for cleaved Notch1 and pErk1/2, respectively, correlated with early relapse to vital organs and poor overall survival. Interestingly, majority of such Notch1 (high)Erk(high) cases encompassed the highly aggressive triple-negative breast cancers (TNBC), and were enriched in stem cell markers. We further show that combinatorial inhibition of Notch1 and Ras/MAPK pathways, using a novel mAb against Notch1 and a MEK inhibitor, respectively, led to a significant reduction in proliferation and survival of breast cancer cells compared with individual inhibition. Combined inhibition also abrogated sphere-forming potential, and depleted the putative cancer stem-like cell subpopulation. Most importantly, combinatorial inhibition of Notch1 and Ras/MAPK pathways completely blocked tumor growth in a panel of breast cancer xenografts, including the TNBCs. Thus, our study identifies coordinate hyperactivation of Notch1 and Ras/MAPK pathways as novel biomarkers for poor breast cancer outcome. Furthermore, based on our preclinical data, we propose combinatorial targeting of these two pathways as a treatment strategy for highly aggressive breast cancers, particularly the TNBCs that currently lack any targeted therapeutic module. (C) 2014 AACR.

Rrp1B gene polymorphism (1307T > C) in metastatic progression of breast cancer

Rrp1B (ribosomal RNA processing1 homolog B) is a novel candidate metastasis modifier gene in breast cancer. Functional gene assays demonstrated that a physical and functional interaction existing between Rrp1b and metastasis modifier gene SIPA1 causes reduction in the tumor growth and metastatic potential. Ectopic expression of Rrp1B modulates various metastasis predictive extra cellular matrix (ECM) genes associated with tumor suppression. The aim of this study is to determine the functional significance of single nucleotide polymorphism (SNP) in human Rrp1B gene (1307 T > C; rs9306160) with breast cancer development and progression. The study consists of 493 breast cancer cases recruited from Nizam's Institute of Medical Sciences, Hyderabad, and 558 age-matched healthy female controls from rural and urban areas. Genomic DNA was isolated by non-enzymatic method. Genotyping was done by amplification refractory mutation system (ARMS-PCR) method. Genotypes were reconfirmed by sequencing and results were analyzed statistically. We have performed Insilco analysis to know the RNA secondary structure by using online tool m fold. The TT genotype and T allele frequencies of Rrp1B1307 T > C polymorphism were significantly elevated in breast cancer (chi (2); p = < 0.008) cases compared to controls under different genetic models. The presence of T allele had conferred 1.75-fold risk for breast cancer development (OR = 1.75; 95 % CI = 1.15-2.67). The frequency of TT genotype of Rrp1b 1307T > C polymorphism was significantly elevated in obese patients (chi (2); p = 0.008) and patients with advanced disease (chi (2); p = 0.01) and with increased tumor size (chi (2); p = 0.01). Moreover, elevated frequency of T allele was also associated with positive lymph node status (chi (2); p = 0.04) and Her2 negative receptor status (chi (2); p = 0.006). Presence of Rrp1b1307TT genotype and T allele confer strong risk for breast cancer development and progression.