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Saraswati Sukumar

Publications and source records attributed to Saraswati Sukumar.

At least 19 recordsLinked to original sources
PubMed2018

Quantitation of DNA Methylation by Quantitative Multiplex Methylation-Specific PCR (QM-MSP) Assay.

The defining feature of the Quantitative Multiplex Methylation-Specific PCR (QM-MSP) method to sensitively quantify DNA methylation is the two-step PCR approach for a multiplexed analysis of a panel of up to 12 genes in clinical samples with minimal quantities of DNA. In the first step, for up to 12 genes tested, one pair of gene-specific primers (forward and reverse) amplifies the methylated and unmethylated copies of the same gene simultaneously and in multiplex, in one PCR reaction. This methylation-independent amplification step produces amplicons of up to 109 copies per μL after 36 cycles of PCR. In the second step, the amplicons of the first reaction (STEP 1) are quantified with a standard curve using real-time PCR and two independent fluorophores to detect methylated/unmethylated DNA of each gene in the same well (e.g., 6FAM and VIC). One methylated copy is detectable in 100,000 reference gene copies. Methylation is reported on a continuous scale. For the gene panel, the highest level of normal DNA methylation above which a sample would be called positive is derived by using Receiver Operating Characteristic (ROC), maximizing assay specificity and sensitivity to distinguish between normal/benign versus tumor DNA. QM-MSP can be applied to clinical samples of fresh or fixed ductal cells, ductal fluid, nipple fluid, fine needle aspirates, core biopsies, and tumor tissue sections.

Breast Neoplasms↗
PubMed2006

Overexpression of glycosylphosphatidylinositol (GPI) transamidase subunits phosphatidylinositol glycan class T and/or GPI anchor attachment 1 induces tumorigenesis and contributes to invasion in human breast cancer.

Based on the oncogenic role of phosphatidylinositol glycan (PIG) class U in human tumors, we explored the role of two additional subunits of the glycosylphosphatidylinositol (GPI) transamidase complex in human breast cancer. We found that PIG class T (PIG-T) and GPI anchor attachment 1 (GPAA1) were overexpressed in breast cancer cell lines and primary tumors. Forced expression of PIG-T and GPAA1 transformed NIH3T3 cells in vitro and increased tumorigenicity and invasion of these cells in vivo. Suppression of PIG-T expression in breast cancer cell lines led to inhibition of anchorage-independent growth. Moreover, we found that PIG-T and GPAA1 expression levels positively correlated with paxillin phosphorylation in invasive breast cancer cell lines. Furthermore, suppression of PIG-T and GPAA1 expression led to a decrease in paxillin phosphorylation with a concomitant decrease in invasion ability. These results suggest that the GPI transamidase complex is composed of a group of proto-oncogenes that individually or as a group contribute to breast cancer growth. This aberrant growth is mediated, at least partially, by phosphorylation of paxillin, contributing to invasion and progression of breast cancer.

Acyltransferases↗
PubMed2006

HOXB7, a homeodomain protein, is overexpressed in breast cancer and confers epithelial-mesenchymal transition.

Epithelial-mesenchymal transition (EMT) is increasingly recognized as a mechanism whereby cells in primary noninvasive tumors acquire properties essential for migration and invasion. Microarray analyses of microdissected epithelial cells from bone metastasis revealed a HOXB7 overexpression that was 3-fold higher than in primary breast carcinomas and 18-fold higher compared with normal breast. This led us to investigate the role of HOXB7 in neoplastic transformation of breast cells. Expression of HOXB7 in both MCF10A and Madin-Darby canine kidney (MDCK) epithelial cells resulted in the acquisition of both phenotypic and molecular attributes typical of EMT. Loss of epithelial proteins, claudin 1 and claudin 7, mislocalization of claudin 4 and E-cadherin, and the expression of mesenchymal proteins, vimentin and alpha-smooth muscle actin, were observed. MDCK cells expressing HOXB7 exhibited properties of migration and invasion. Unlike MDCK vector-transfected cells, MDCK-HOXB7 cells formed highly vascularized tumors in mice. MDCK-HOXB7 cells overexpressed basic fibroblast growth factor (bFGF), had more active forms of both Ras and RhoA proteins, and displayed higher levels of phosphorylation of p44 and p42 mitogen-activated protein kinase (MAPK; extracellular signal-regulated kinases 1 and 2). Effects initiated by HOXB7 were reversed by specific inhibitors of FGF receptor and the Ras-MAPK pathways. These data provide support for a function for HOXB7 in promoting tumor invasion through activation of Ras/Rho pathway by up-regulating bFGF, a known transcriptional target of HOXB7. Reversal of these effects by HOXB7-specific siRNA further suggested that these effects were mediated by HOXB7. Thus, HOXB7 overexpression caused EMT in epithelial cells, accompanied by acquisition of aggressive properties of tumorigenicity, migration, and invasion.

Animals↗
PubMed2006

RUNX3 is frequently inactivated by dual mechanisms of protein mislocalization and promoter hypermethylation in breast cancer.

A tumor suppressor function has been attributed to RUNX3, a member of the RUNX family of transcription factors. Here, we examined alterations in the expression of three members, RUNX1, RUNX2, and RUNX3, and their interacting partner, CBF-beta, in breast cancer. Among them, RUNX3 was consistently underexpressed in breast cancer cell lines and primary tumors. Fifty percent of the breast cancer cell lines (n = 19) showed hypermethylation at the promoter region and displayed significantly lower levels of RUNX3 mRNA expression (P < 0.0001) and protein (P < 0.001). In primary Singaporean breast cancers, 9 of 44 specimens showed undetectable levels of RUNX3 by immunohistochemistry. In 35 of 44 tumors, however, low levels of RUNX3 protein were present. Remarkably, in each case, protein was mislocalized to the cytoplasm. In primary tumors, hypermethylation of RUNX3 was observed in 23 of 44 cases (52%) and was undetectable in matched adjacent normal breast epithelium. Mislocalization of the protein, with or without methylation, seems to account for RUNX3 inactivation in the vast majority of the tumors. In in vitro and in vivo assays, RUNX3 behaved as a growth suppressor in breast cancer cells. Stable expression of RUNX3 in MDA-MB-231 breast cancer cells led to a more cuboidal phenotype, significantly reduced invasiveness in Matrigel invasion assays, and suppressed tumor formation in immunodeficient mice. This study provides biological and mechanistic insights into RUNX3 as the key member of the family that plays a role in breast cancer. Frequent protein mislocalization and methylation could render RUNX3 a valuable marker for early detection and risk assessment.

Antimetabolites, Antineoplastic↗
PubMed2006

Quantitative multiplex methylation-specific PCR analysis doubles detection of tumor cells in breast ductal fluid.

PURPOSE: The challenges of cytology for accurate diagnosis of breast cancer are well recognized. We previously showed that normal and tumor tissue can be distinguished using a technique called quantitative multiplex methylation-specific PCR (QM-MSP). We hypothesized that quantitative analysis of methylated genes will provide enhanced detection of cancer cells present in cytologic specimens. EXPERIMENTAL DESIGN: QM-MSP was done on ductal lavage cells from a set of 37 ductal lavage samples from women undergoing mastectomy (27 with cancer and 3 without). Duct histology information was available for each lavaged duct. QM-MSP data was assessed by measuring cumulative methylation index and by receiver operating characteristic threshold analysis. To determine the baseline level of methylation for each gene in this population, cells from 60 ducts of women at high risk of developing breast cancer were analyzed. RESULTS: QM-MSP findings on a panel of nine genes were correlated to duct histology and ductal lavage cytology. Cytology detected cancer in 33% (7 of 21 ducts) with a specificity of 99% (92 of 93). QM-MSP detected cancer as calculated by cumulative methylation index with a sensitivity of 62% (13 of 21) and specificity of 82% (62 of 76) and by receiver operating characteristic threshold analysis with a sensitivity of 71% (15 of 21) and specificity of 83% (63 of 76). CONCLUSIONS: Compared with cytology, QM-MSP doubled the sensitivity of detection of cancer. This study provides proof of principle by showing the advantages of using methylation analyses to query cytologic specimens and indicates its potential use in diagnosis and in stratifying risk.

Body Fluids↗
PubMed2006

Mutational hotspot in exon 20 of PIK3CA in breast cancer among Singapore Chinese.

The recent identification of somatic mutations in the catalytic region of PIK3 (PIK3CA) in breast cancer and demonstration of their oncogenic function has implicated PIK3CA in mammary carcinogenesis. To investigate possible ethnic differences in patterns of PIK3CA mutations in Singaporean Chinese breast cancer and to characterize these in a panel of cell lines, we sequenced exons 9 and 20 in 80 primary tumors, 19 breast cancer cell lines and 7 normal human mammary epithelial cells (HMECs). Searching for novel hotspots of mutation, we sequenced additional exons (1, 2, 6, 7, 14 and 18) in 20 primary tumors and 6 breast cancer cell lines. We detected 33 point mutations in 31 of 80 (39%) breast cancers, and 11 mutations in 10 of 19 (53%) breast cancer cell lines. No mutations were detected in normal breast tissue adjacent to the tumor, or in the 6 normal HMECs. The exon 20 A3140G (H1047R) substitution was identified most frequently (22/31, 71%) and showed a significant association with patient age (p = 0.043) and stage of the disease (p = 0.025), but not with ER/PR status or histological grade of the tumor. The incidence of point mutations in PIK3CA, the A3140G substitution in particular, in Singapore breast cancers are among the most frequent reported to date for any gene in breast cancer. The results suggest that mutation of PIK3CA might contribute to development of early stage breast cancer and could provide a potent target for early diagnosis and therapy.

Asian People↗
PubMed2006

Polyamine analogues down-regulate estrogen receptor alpha expression in human breast cancer cells.

The critical role of polyamines in cell growth has led to the development of a number of agents that interfere with polyamine metabolism including a novel class of polyamine analogues, oligoamines. Here we demonstrate that oligoamines specifically suppress the mRNA and protein expression of estrogen receptor alpha (ERalpha) and ERalpha target genes in ER-positive human breast cancer cell lines, whereas neither ERbeta nor other steroid hormonal receptors are affected by oligoamines. The constitutive expression of a cytomegalovirus promoter-driven exogenous ERalpha in ER-negative MDA-MB-231 human breast cancer cells was not altered by oligoamines, suggesting that oligoamines specifically suppress ERalpha transcription rather than affect mRNA or protein stability. Further analysis demonstrated that oligoamines disrupted the DNA binding activity of Sp1 transcription factor family members to an ERalpha minimal promoter element containing GC/CA-rich boxes. Treatment of MDA-MB-231 cells with the JNK-specific inhibitor SP600125 or expression of the c-Jun dominant negative inhibitor TAM67 blocked the oligoamine-activated JNK/c-Jun pathway and enhanced oligoamine-inhibited ERalpha expression, suggesting that AP-1 is a positive regulator of ERalpha expression and that oligoamine-activated JNK/AP-1 activity may antagonize the down-regulation of ERalpha induced by oligoamines. Taken together, these results suggest a novel antiestrogenic mechanism for specific polyamine analogues in human breast cancer cells.

Breast Neoplasms↗
PubMed2006

Benzoylphenylurea sulfur analogues with potent antitumor activity.

A novel series of BPU analogues were synthesized and evaluated for antitumor activity. In particular, BPU sulfur analogues 6n and 7d were shown to possess up to 10-fold increased potency, when compared to 1 (NSC-639829), against cancer cell lines. 6n was more effective than 1 in causing apoptosis of MCF-7 cells. When compared to other drugs with a similar mechanism of action, 6n retained significant ability to inhibit tubulin assembly, with an IC(50) of 2.1 microM.

Antineoplastic Agents↗
PubMed2006

Epigenetic suppression of secreted frizzled related protein 1 (SFRP1) expression in human breast cancer.

Expression of Secreted frizzled related protein 1 (SFRP1), a recently identified tumor suppressor gene encoding a WNT signaling antagonist, has been found to be frequently down-regulated in breast cancer and is associated with disease progression and poor prognosis. Here, we investigated the role of epigenetic silencing of SFRP1 in breast cancer cell lines and primary breast tumors. Through analyses by methylation-specific PCR and bisulfite sequencing, promoter methylation of SFRP1 was detected in 88% (7/8) of breast cancer cell lines, 17% (1/6) of grade 1 of ductal carcinoma in situ (DCIS), 69% (9/13) of grade 2 and 3 of DCIS, 68% (19/28) of invasive ductal carcinomas (IDC) and 33% (6/18) of lobular carcinomas but not in any (0/14) of normal mammoplasty specimens and mammary epithelial organoids examined. Real-time RT-PCR studies indicated that loss or downregulation of SFRP1 expression in tumors is frequently associated with promoter hypermethylation. In addition, breast cancer cell lines with SFRP1 promoter hypermethylation reexpressed SFRP1 mRNA after treatment with 5-azaC, implying that DNA methylation is the predominant epigenetic mechanism for SFRP1 gene silencing. These findings suggest that frequent downregulation of SFRP1 expression in breast cancer can be attributed, in large part, to aberrant promoter hypermethylation in conjunction with or without histone deacetylation. Based on the frequency of tumor-specific hypermethylation in this gene, SFRP1 could provide a valuable marker for breast cancer.

Acetylation↗
PubMed2006

Ductal access for prevention and therapy of mammary tumors.

In cancer patients and in those at high risk, systemic exposure to agents for therapy or prevention is accompanied by undesirable side effects. We hypothesized that it is possible to prevent and treat breast cancer by introducing anticancer agents into the mammary ductal network. Here, we show the efficacy of intraductally administered anticancer agents 4-hydroxytamoxifen and pegylated liposomal doxorubicin (PLD) in the prevention and treatment of breast cancer using the rat N-methyl-N'-nitrosourea-induced and spontaneous HER-2/neu transgenic mouse (neu-N) models of breast cancer. Intraductal administration of PLD to neu-N mice caused regression of established tumors and prevented tumor development more effectively than i.v. injection (P < 0.0001). Intraductal administration resulted in lower circulating levels of PLD compared with i.v. administration, with no evidence of systemic toxicity or long-term histopathologic changes in the mammary gland. Compared with systemic administration, intraductal injection provides direct access to breast lesions with higher local and lower systemic drug exposure. These studies suggest that this approach has potential for application to prevention and neoadjuvant therapy of early breast cancer.

Animals↗
PubMed2006

Two-color quantitative multiplex methylation-specific PCR.

In recent years, several methylation-specific PCR-based techniques have been developed to identify and characterize hypermethylation of CpG dinucleotides with the primary goal of elucidating a better understanding of the role of DNA methylation in important biological processes, such as chromosome X inactivation and carcinogenesis. The specificity of methylation-specific PCR (MSP) techniques relies on amplifying sodium bisulfite-treated DNA with primers specific to predicted sequences of unmethylated and methylated DNA within the gene of interest. In the past, unmethylated and methylated reactions were singleplex and performed in separate wells. In this paper we report a modification of the real-time quantitative multiplex MSP (QM-MSP) technique of Fackler and colleagues that can be applied to any real-time MSP experiment. Although co-amplification with multiple fluorophores is common in standard reverse transcription PCR (RT-PCR), MSP presents unique challenges both mechanistically and operationally that must be overcome in order to successfully co-amplify two methylation-specific targets. In this two-color modification, unmethylated and methylated primer/probe sets are successfully co-amplified in the same reaction using FAM- and VIC-labeled probes. Our modification decreases the cost and time of each real-time experiment by allowing increased throughput of clinical samples and by doubling either the number of genes or the number of samples that can be analyzed per real-time plate.

Breast Neoplasms↗
PubMed2005

Identification of biomarkers for breast cancer in nipple aspiration and ductal lavage fluid.

PURPOSE: To establish a comprehensive proteomic approach for biomarker discovery and validation in breast fluid. EXPERIMENTAL DESIGN: A total of 95 specimens from three institutions were used including 10 nipple aspiration fluid (5 stage I/II cancerous breasts and 5 age-matched healthy controls), 42 ductal lavage fluid from 14 patients with unilateral stage I/II cancer (25 from 9 cancerous breasts and 17 from 7 contralateral breasts), and 42 ductal lavage fluid from 14 high-risk women (multiple ducts repeated lavage). Differentially expressed protein/peptides were discovered by proteomic analysis of training sample, using ProteinChip arrays and surface-enhanced laser desorption ionization (SELDI) time-of-flight mass spectrometry, and validated on independently collected testing samples. After protein identification, ELISA was done to confirm the SELDI findings. RESULTS: We were able to obtain reproducible protein profiles using minimal amount of protein (1 mug) by applying an optimized chip protocol and SELDI. We were able to select cancer-associated biomarkers despite large individual variability by applying both unsupervised and supervised cluster analysis. Furthermore, we were able to train and test candidate biomarkers on independently collected samples and identified one component of a multimarker panel as human neutrophil peptides 1 to 3. CONCLUSIONS: Breast fluid is a rich source of breast cancer biomarkers. In combination with high-throughput novel proteomic profiling technology and multicenter study design, markers that are highly specific to breast cancer can be discovered and validated. Our observations also suggest that persistent elevation of human neutrophil peptide in high-risk women may imply early onset of cancer not yet detectable by current detection method. Proof of this hypothesis requires follow-up on a larger study population.

Adenocarcinoma↗
PubMed2005

Tumor-specific changes in mtDNA content in human cancer.

Mitochondrial DNA (mtDNA) alterations are associated with various cancer types, suggesting that the mitochondrial genome may be a critical contributing factor in carcinogenesis. mtDNA alterations have been suggested as a potentially sensitive and specific biomarker for several cancer types. We examined mtDNA content in 25 pairs of normal and tumor breast tissue samples, 37 papillary thyroid carcinoma (PTC), 21 benign thyroid neoplasms and in 20 paired normal and PTC samples. Our results showed that mtDNA content was reduced in 80% of the breast tumors relative to their corresponding normal. mtDNA was increased in papillary thyroid carcinomas, however, when compared to the corresponding normal DNA taken from the same individual. Also, mtDNA content was increased in none-paired PTC samples compared to the normal controls. Our findings indicate that changes in mtDNA content during carcinogenesis may be regulated in a tumor specific manner. Additionally, changes in mtDNA levels did not correlate with tumor grade and metastasis, suggesting that these alterations may occur in the early stages of tumorigenesis. Our findings suggest that mtDNA content can be used as a molecular diagnostic tool to help identify genetic abnormalities in human tumors.

Breast Neoplasms↗
PubMed2005

Role of p53/p21(Waf1/Cip1) in the regulation of polyamine analogue-induced growth inhibition and cell death in human breast cancer cells.

Intracellular polyamines are absolutely required for cell proliferation and many tumors have abnormal requirements for polyamines. Therefore, the polyamine metabolic pathway represents a rational target for antineoplastic intervention. A number of polyamine analogues act as potent modulators of cellular polyamine metabolism and exhibit encouraging effects against tumor growth in both cell culture and animal studies. In this study we demonstrate that specific polyamine analogues exhibit differential inhibitory action against growth of human breast cancer MCF-7 cells. Treatment of MCF-7 cells with oligoamine analogues and the symmetrically substituted bis(alkyl)-substituted analogue, BENSpm, produced a G1 cell cycle arrest, while the unsymmetrically substituted bis(alkyl)-substituted analogue, CHENSpm, induced a G2/M cell cycle arrest. All four compounds significantly upregulated p53 and p21 expression in MCF-7 cells. Stable transfection of small interfering RNA (siRNA) targeting p53 blocked the expression of p21 induced by the polyamine analogues and significantly reduced polyamine analogue-induced growth inhibition and apoptosis, suggesting that polyamine analogue-induced p21 expression occurs through p53-dependent mechanisms. The effects of analogue exposure on cyclins and cyclin dependent kinases varied with the specific agent used. Expression of p53 siRNA reversed only BENSpm-modulated the cell cycle arrest, suggesting that regulation of cell cycle arrest by p53/p21 induced by polyamine analogues occurs through agent-specific mechanisms. Understanding the mechanism of p53-mediated cellular responses to polyamine analogue may help to improve the therapeutic efficacy of polyamine analogues in human breast cancer.

Apoptosis↗
PubMed2005

Somatic mutation and gain of copy number of PIK3CA in human breast cancer.

INTRODUCTION: Phosphatidylinositol 3-kinases (PI3Ks) are a group of lipid kinases that regulate signaling pathways involved in cell proliferation, adhesion, survival, and motility. Even though PIK3CA amplification and somatic mutation have been reported previously in various kinds of human cancers, the genetic change in PIK3CA in human breast cancer has not been clearly identified. METHODS: Fifteen breast cancer cell lines and 92 primary breast tumors (33 with matched normal tissue) were used to check somatic mutation and gene copy number of PIK3CA. For the somatic mutation study, we specifically checked exons 1, 9, and 20, which have been reported to be hot spots in colon cancer. For the analysis of the gene copy number, we used quantitative real-time PCR and fluorescence in situ hybridization. We also treated several breast cancer cells with the PIK3CA inhibitor LY294002 and compared the apoptosis status in cells with and without PIK3CA mutation. RESULTS: We identified a 20.6% (19 of 92) and 33.3% (5 of 15) PIK3CA somatic mutation frequency in primary breast tumors and cell lines, respectively. We also found that 8.7% (8 of 92) of the tumors harbored a gain of PIK3CA gene copy number. Only four cases in this study contained both an increase in the gene copy number and a somatic mutation. In addition, mutation of PIK3CA correlated with the status of Akt phosphorylation in some breast cancer cells and inhibition of PIK3CA-induced increased apoptosis in breast cancer cells with PIK3CA mutation. CONCLUSION: Somatic mutation rather than a gain of gene copy number of PIK3CA is the frequent genetic alteration that contributes to human breast cancer progression. The frequent and clustered mutations within PIK3CA make it an attractive molecular marker for early detection and a promising therapeutic target in breast cancer.

Apoptosis↗
PubMed2005

Identification of transcriptional targets of HOXA5.

The homeobox gene HOXA5 encodes a transcription factor that has been shown to play important roles in embryogenesis, hematopoiesis, and tumorigenesis. In order to decipher downstream signaling pathways of HOXA5, we utilized oligonucleotide microarray analysis to identify genes that are differentially expressed in HOXA5-induced cells compared with uninduced cells. Comparative analysis of gene expression changes after 9 h of HOXA5 induction in Hs578T breast cancer cells identified 306 genes whose expression was modulated at least 2-fold. Ten of these 306 genes were also up-regulated by at least 2-fold at 6 h post-induction. The expression of all of these 10 genes was confirmed by semiquantitative reverse transcription-PCR. Among these 10 genes, which are most likely to be direct targets of HOXA5, we initiated an investigation into the pleiotrophin gene by first cloning its promoter. Transient transfection assays indicated that HOXA5 can specifically activate the pleiotrophin promoter. Promoter deletion, chromatin immunoprecipitation assay, and gel-shift assays were performed to show that HOXA5 can directly bind to one binding site on the pleiotrophin promoter. These data strongly suggest that microarray analysis can successfully identify many potential direct downstream genes of HOXA5. Further functional analysis of these targets will allow us to better understand the diverse functions of HOXA5 in embryonic development and tumorigenesis.

Apoptosis↗
PubMed2005

Of Snail, mice, and women.

Mechanisms for breast cancer recurrence and metastases are poorly understood. New evidence from a transgenic mouse mammary tumor model suggests that the transcriptional repressor, Snail, may play a role in recurrence by downregulating E-cadherin and inducing an epithelial-to-mesenchymal transition. Preliminary information from expression microarray data sets from primary human breast cancers suggests that high levels of Snail are correlated with poor clinical outcome for women with early breast cancer. The identification of a molecular pathway involved in mammary tumor recurrence in a mouse model offers both opportunity and challenge to confirm, extend, and exploit these findings in the clinic.

Animals↗