Rupali Hire

Antiproliferative Activity of Crocin Involves Targeting of Microtubules in Breast Cancer Cells

Crocin, a component of saffron spice, is known to have an anticancer activity. However, the targets of crocin are not known. In this study, crocin was found to inhibit the proliferation of HCC70, HCC1806, HeLa and CCD1059sk cells by targeting microtubules. Crocin depolymerized both the interphase and mitotic microtubules of different cancer cells, inhibited mitosis and induced multipolar spindle formation in these cells. In vitro, crocin inhibited the assembly of pure tubulin as well as the assembly of microtubule-associated protein rich tubulin. Electron microscopic analysis showed that crocin inhibited microtubule assembly while it induced aggregation of tubulin at higher concentrations. Crocin co-eluted with tubulin suggesting that it binds to tubulin. Vinblastine inhibited the binding of crocin to tubulin while podophyllotoxin did not inhibit the crocin binding indicating that crocin binds at the vinblastine site on tubulin. The results suggested that crocin inhibited cell proliferation mainly by disrupting the microtubule network.

Distinct Transcript Isoforms of the Atypical Chemokine Receptor 1 (ACKR1) / Duffy Antigen Receptor for Chemokines (DARC) Gene Are Expressed in Lymphoblasts and Altered Isoform Levels Are Associated with Genetic Ancestry and the Duffy-Null Allele

The Atypical ChemoKine Receptor 1 (ACKR1) gene, better known as Duffy Antigen Receptor for Chemokines (DARC or Duffy), is responsible for the Duffy Blood Group and plays a major role in regulating the circulating homeostatic levels of pro-inflammatory chemokines. Previous studies have shown that one common variant, the Duffy Null (Fy-) allele that is specific to African Ancestry groups, completely removes expression of the gene on erythrocytes; however, these individuals retain endothelial expression. Additional alleles are associated with a myriad of clinical outcomes related to immune responses and inflammation. In addition to allele variants, there are two distinct transcript isoforms of DARC which are expressed from separate promoters, and very little is known about the distinct transcriptional regulation or the distinct functionality of these protein isoforms. Our objective was to determine if the African specific Fy- allele alters the expression pattern of DARC isoforms and therefore could potentially result in a unique signature of the gene products, commonly referred to as antigens. Our work is the first to establish that there is expression of DARC on lymphoblasts. Our data indicates that people of African ancestry have distinct relative levels of DARC isoforms expressed in these cells. We conclude that the expression of both isoforms in combination with alternate alleles yields multiple Duffy antigens in ancestry groups, depending upon the haplotypes across the gene. Importantly, we hypothesize that DARC isoform expression patterns will translate into ancestry-specific inflammatory responses that are correlated with the axis of pro-inflammatory chemokine levels and distinct isoform-specific interactions with these chemokines. Ultimately, this work will increase knowledge of biological mechanisms underlying disparate clinical outcomes of inflammatory-related diseases among ethnic and geographic ancestry groups.

Assessing Heavy Metal and PCB Exposure from Tap Water by Measuring Levels in Plasma from Sporadic Breast Cancer Patients, a Pilot Study

Breast cancer (BrCA) is the most common cancer affecting women around the world. However, it does not arise from the same causative agent among all women. Genetic markers have been associated with heritable or familial breast cancers, which may or may not be confounded by environmental factors, whereas sporadic breast cancer cases are more likely attributable to environmental exposures. Approximately 85% of women diagnosed with BrCA have no family history of the disease. Given this overwhelming bias, more plausible etiologic mechanisms should be investigated to accurately assess a woman’s risk of acquiring breast cancer. It is known that breast cancer risk is highly influenced by exogenous environmental cues altering cancer genes either by genotoxic mechanisms (DNA mutations) or otherwise. Risk assessment should comprehensively incorporate exposures to exogenous factors that are linked to a woman’s individual susceptibility. However, the exact role that some environmental agents (EA) play in tumor formation and/or cancer gene regulation is unclear. In this pilot project, we begin a multi-disciplinary approach to investigate the intersection of environmental exposures, cancer gene response, and BrCA risk. Here, we present data that show environmental exposure to heavy metals and PCBs in drinking water, heavy metal presence in plasma of nine patients with sporadic BrCA, and Toxic Release Inventory and geological data for a metal of concern, uranium, in Northeast Georgia.

Abstract 132: Investigating correlations of DARC under-expression (Duffy null phenotypes) with increased breast cancer lymph node metastasis

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA DARC (aka Duffy), a chemokine receptor, is expressed on erythrocytes and endothelial cells in blood vessels. It has been linked to inhibition of tumor metastasis, and to regulation of plasma levels of some cytokines. The Duffy null (Fy-) phenotype, in which DARC is not expressed on red blood cells, is especially prevalent among people of West African descent. Little is known about how the presence or absence of DARC expression may affect breast cancer metastases and overall survival among African-American and European- American women. Our lab is investigating whether Fy- phenotypes could be associated with increased BrCa metastasis by genotyping the Fy- (null and weak) alleles and assessing DARC gene expression in breast and lymphoblast cells using qPCR and Immunofluorescence (IF). There are two isoforms of DARC, each with the Fy- SNP located in the promoter of one and the 5’UTR of the other. Due to this SNP, it is plausible that the alternate isoform is specific to these cell types and may still be expressed. Here we present data from the preliminary stages of this project; including characterizing Duffy alleles in our cell line models. We have genotyped the 3 known DARC polymorphisms that result in Fy- (and weak) erythrocyte phenotypes in breast cancer and lymphoblast cell lines. Specifically, we validated the genotype of HAPMAP lines from Yoruba, African-American, and CEPH-European descent for the -541T>C point mutation Fy- allele (rs2814778). Our breast cancer (BrCa) lines are derived from tumors of varying molecular subtypes, isolated from African-American women. We show that these BrCa lines are homozygous for the Fy- allele and yet still express the DARC protein. The specific isoform expressed in these cells (DARC-2) may not be the same as used to phenotype the Fy- status in erythrocytes (DARC-1). This suggests that a Fy- genotype may have some tissue specific expression of DARC-2 in epithelial cells. As well, we show that the levels of expression of the two DARC isoforms vary and are distinct among ancestral groups. We also show expression on the RNA level for individuals who have the duffy null genotype, which will be further validated. In conclusion, we hypothesize that Fy- status, as determined in endothelial and erythrocytes in people of African descent, may remove the metastatic protection of DARC. Also, alternate expression of DARC-2 on epithelial cells may increase the rate of metastases in the African population. Currently, we are analyzing clinical samples (blood and breast tissues) from local breast cancer patients for Fy- status and will determine if this correlates with metastatic disease. In addition, biochemical experiments will be conducted to determine whether DARC-related chemokines are in higher prevalence in the Fy- patients, how the isoforms differ in their affinity to the chemokine ligands, and whether this is associated with specific breast cancer subtypes. Citation Format: Andrea Walens, Brianna Bennett, Kauthar Mumin, Michael Lou, Rupali Hire, Michele Monteil, Melissa Davis. Investigating correlations of DARC under-expression (Duffy null phenotypes) with increased breast cancer lymph node metastasis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 132. doi:10.1158/1538-7445.AM2014-132

Abstract 953: Atypical chemokine receptor 1 (ACKR1/DARC) expressing tumors are associated with distinct recruitment of immune cells and increased pro-inflammatory chemokines

Interactions between chemokines and their receptors can improve a host’s anti-tumor response by influencing the targeted migration of immune cells via a chemokine gradient. Atypical Chemokine Receptor 1 (ACKR1/DARC), a genetically diverse transmembrane GPCR, acts as a decoy receptor for a variety of CXC and CC chemokines, including those with pro-malignant and pro-inflammatory effects, such as CCL2 (MCP-1, MCAF, JE) and CXCL8 (IL-8). The purpose of this study is to determine if the migration of tumor-associated immune cells is unique based on epithelial ACKR1 expression on breast cancer cells, and if this association is correlated to an increase in pro-malignant chemokines, better survival odds, and differences in race. Immunohistochemistry techniques were used to determine expression levels of ACKR1 on primary breast tumors, along with relative expression of T-cells, B-cells, dendritic cells, and macrophages. Concentrations of pro-inflammatory chemokines in circulation were determined using a Luminex-based immunoassay and matched patient peripheral blood samples. In silco analyses were performed to determine associations between ACKR1 tumor expression status, race, and survival. Finally, using human breast cancer cell lines and immunofluorescence techniques, co-localization between ACKR1 and selected pro-inflammatory chemokines was investigated. Results from these tests indicate that there is differential expression of immune cell types in tumors expressing ACKR1, and this difference was associated with the migration of B-cells and dendritic cells, which were not detected in ACKR1 negative tumors. Significantly increased circulating CCL2 and CXCL8 chemokine levels we also determined to be positively correlated with ACKR1 expression in primary breast tumors. Survival analyses showed a significantly increased relapse free survival in patients having tumors with high ACKR1 expression, while investigations into racial differences revealed a significant race effect, with Caucasians having higher ACKR1 levels on their tumors than African-Americans. Finally, co-localization between ACKR1 with CCL2 and CXCL8 is observed in cultured human breast cancer cells. Given that the data collected shows a tendency for those tumors positively expressing ACKR1 to have a more favorable prognosis, we suggest that a partial role of ACKR1 on breast tumor cells is to sequester pro-inflammatory chemokines in the tumor microenvironment, indirectly recruiting a distinct subset of tumor-associated immune cells. Citation Format: Brittany D. Jenkins, Rupali Hire, Elizabeth Howerth, Michele Monteil, Rachel Martini, Melissa B. Davis. Atypical chemokine receptor 1 (ACKR1/DARC) expressing tumors are associated with distinct recruitment of immune cells and increased pro-inflammatory chemokines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 953. doi:10.1158/1538-7445.AM2017-953

Abstract B39: Distinct recruitment of tumor-associated immune cells correlates with increased pro-malignant chemokines in tumors expressing epithelial Atypical Chemokine Receptor 1 (ACKR1/DARC)

The tumor microenvironment is a complex heterogeneous mixture of cancer cells, immune cells, and many other cell types contributing to a myriad of clinical outcomes for breast cancer (BrCa) patients. Chemokine receptors play an important role in maintaining the homeostasis of pro-inflammatory chemokines, and, in turn, help direct the migration of tumor-associated immune cells. Atypical chemokine receptors, such as the Atypical Chemokine Receptor 1 (ACKR1/DARC), act to sequester chemokine activity and control leukocyte migration during tumor-associated inflammation. ACKR1 is unique in that its associated gene carries a fixed mutation in African populations, causing the receptor to not be expressed on red blood cells as a response to endemic malaria in Africa. It is well-known that African-Americans in the United States tend to develop more aggressive BrCa subtypes, and as a result, experience more deaths per year from BrCa related causes. The purpose of this study is to characterize this disparity by investigating the distinct migration of tumor-associated immune cells between ACKR1 positive and negative tumors. We also wish to determine any correlations between ACKR1 and pro-inflammatory chemokines in various BrCa cell types. Using immunohistochemistry, relative expression levels were determined for ACKR1, cytotoxic T-cells, B-cells, dendritic cells, and macrophages in primary breast tumor samples. The levels of pro-inflammatory chemokines in circulation were determined using a Luminex immuno-assay on peripheral blood samples. Localization of ACKR1-associated chemokines was also investigated using immunofluorescence. Initial results from our study cohort indicate differential expression of immune cells on tumors expressing ACKR1, leading to a unique tumor microenvironment. We observed that ACKR1 positive tumors recruited B-cells and dendritic cells, whereas, ACKR1 negative tumors did not. Also, we found a positive correlation between ACKR1 expression in tumor tissue, and expression of CCL2 (MCP-1) and CXCL8 (IL-8) in the peripheral blood of our cancer patients. Finally, a strong co-localization of ACKR1 with CCL2 and CXCL8 was observed in cultured mammalian breast cancer cells. Overall, our pilot data suggests that the presence of ACKR1 on tumor cells changes the tumor microenvironment by recruiting a distinct subset of immune cells and pro-inflammatory chemokines to the area of inflammation. Citation Format: Brittany D. Jenkins, Rachel N. Martini, Rupali Hire, Michele A. Monteil, Melissa B. Davis. Distinct recruitment of tumor-associated immune cells correlates with increased pro-malignant chemokines in tumors expressing epithelial Atypical Chemokine Receptor 1 (ACKR1/DARC). [abstract]. In: Proceedings of the Ninth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2016 Sep 25-28; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2017;26(2 Suppl):Abstract nr B39.

Abstract B25: Triple-negative breast tumors exhibit distinctive patterns of expression and subcellular localization of Insulin-like Growth Factor Binding Protein 6

African-American women of all ages have higher breast cancer mortality rates than Caucasian women, despite lower breast cancer incidence among African-American women over 40. While differences in socioeconomic status and access to healthcare may play a role in this disparity, the disproportionately high occurrence of aggressive, triple-negative breast tumors in African-American women suggests a biological link between ancestry and breast cancer prognosis. Interestingly, both African ancestry and breast tumor aggressiveness are associated with distinct transcript and protein expression levels of Insulin-like growth factor-II (IGF-II) and its binding partner, Insulin-like growth factor binding protein 6 (IGFBP6), and we have previously observed higher levels of IGFBP6 transcript and protein expression in African-American and Yoruba (African) HapMap cell lines when compared to Amish cell lines. To gain a better understanding of potential mechanisms underlying these associations, we have investigated the expression and subcellular localization of these proteins and their transcripts using qPCR and immunofluorescence (IF). Our qPCR data indicate lower IGFBP6 transcript expression in breast cancer cells relative to normal cells, with lower transcript levels in the ER positive breast cancer cells than in triple negative cells. Conversely, our IF data revealed higher levels of IGFBP6 protein in ER positive breast cancer cells than in triple negative breast cancer cells, suggesting that breast tumor subtypes may be associated with unique patterns of transcript and protein regulation in these genes. We also observed a distinctive pattern of IGFBP6 subcellular localization in the triple-negative cells in which aggregates of this protein are localized at the edge of the nucleus, and co-localized with IGF-II. We hypothesize that IGF-II will be more dispersed throughout the nucleus following si-RNA knockdown of IGFBP6. Taken together, these data suggest the possibility of a subtype-specific IGF-II/IGFBP6 complex in triple negative breast cancer. Further studies will assess how this complex may promote tumor aggression in triple-negative breast tumors by inhibiting IGFBP69s IGF-independent, pro-apoptotic qualities within the nucleus and/or prolonging the half-life of IGF-II, thus promoting cancer cell growth. Citation Format: DeJuana Ford, Rupali Hire, Elizabeth Howerth, Briana Bennett, Krissean Lea, Jillian Hood, Melissa Davis. Triple-negative breast tumors exhibit distinctive patterns of expression and subcellular localization of Insulin-like Growth Factor Binding Protein 6. [abstract]. In: Proceedings of the Eighth AACR Conference on The Science of Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; Nov 13-16, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2016;25(3 Suppl):Abstract nr B25.

Abstract A63: The role of DARC in the immunological tumor microenvironment of breast cancer

The Duffy Antigen Receptor for Chemokines (DARC or ACKR1) gene regulates circulating levels of inflammatory chemokines and major alleles of the gene are associated with a myriad of clinical outcomes. There are two unique isoforms of DARC which are expressed from distinct separate promoters, but little is known about the expression or regulation of these isoforms. Using quantitative RT-PCR and Immunohistochemical studies of lymphoblastic cells from relevant ancestry groups, we recently found that a DARC polymorphism, the Duffy Null (Fy-) allele found almost exclusively in African ancestry groups, is associated with altered regulation of these isoforms. Our data indicates that people of African ancestry who carry the Fy- allele will have an altered expression pattern of DARC isoforms. Thusly, we investigated whether the Fy- allele is associated with distinct recruitment of monocytes to breast tumor epithelia, regulated by DARC modulated chemokines. This may translate into a suite of distinct inflammatory responses correlated with specific chemokine action and may explain the disparate clinical outcomes of breast cancer in this population. Citation Format: Melissa B. Davis, Michele Monteil, Rupali Hire, Elizabeth W. Howerth. The role of DARC in the immunological tumor microenvironment of breast cancer. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr A63.

Abstract C66: Insulin-like growth factor binding protein-6 is differentially methylated in African American, African, and Amish HapMap cell lines

African American women, while having lower lifetime incidence of breast cancer (BrCa) than Caucasian women, have higher BrCa mortality rates, partially due to a higher incidence of the more aggressive, basal-like molecular subtype of BrCa. Correlations of this subtype within African ancestral groups suggest a potential link to African Ancestry and basal-like BrCa. While socioeconomic factors may also play a role in higher mortality rates, there is mounting evidence that the differential epigenetic regulation of cancer genes may also contribute to this disparity. To address this, our lab is investigating the epigenetic regulation of genes in the insulin-like growth factor pathway using qPCR, Immunohistochemistry (IHC) and Bisulfite-DNA Sequencing. This presentation specifically highlights novel data on the expression of insulin-like growth factor binding protein-6 (IGFBP-6), an IGF-II regulator and agent of cell proliferation, which has been shown previously to be epigenetically modified in cancer cells. Our qPCR and IHC data demonstrates higher IGFBP-6 transcription and protein expression in basal-like BrCa cells when compared to normal breast and ER+ BrCa cells. To address potential ancestry-specific expression, we also evaluated IGFBP-6 expression in African and African-American HapMap lymphoblast cell lines compared to CEPH-European (Amish) cell lines. Both qPCR and IHC data indicate higher overall expression of this gene in African lineages. Lastly, we have measured the differential methylation in two regions of the IGFBP-6 CpG island among African, African-American, and Amish cell lines. We show that IGFBP-6 methylation correlates with the expression patterns. Differential methylation could be due to Ancestral-Specific polymorphisms in this region, altering potential methylation sites. We conclude that the distinct levels of IGFBP-6 expression and promoter methylation in African and African-American cell lines may correspond to the increased incidence of basal-like BrCa in these populations, due to prolonged half-life and activity of the IGF-II growth signal. Our future studies include evaluation of whether this epigenetically-mediated up-regulation of IGFBP-6 directly leads to the excessive proliferation of basal-like tumors. Citation Format: DeJuana Ford, Briana Bennett, Myrielis Rivera, Rupali Hire, Melissa Davis. Insulin-like growth factor binding protein-6 is differentially methylated in African American, African, and Amish HapMap cell lines. [abstract]. In: Proceedings of the Sixth AACR Conference: The Science of Cancer Health Disparities; Dec 6–9, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2014;23(11 Suppl):Abstract nr C66. doi:10.1158/1538-7755.DISP13-C66