Understanding at the molecular level of the cell biology of tumors has led to significant treatment advances in the past. Despite such advances however, development of therapy resistance and tumor recurrence are still unresolved major challenges. This therefore underscores the need to identify novel tumor targets and develop corresponding therapies to supplement existing biologic and cytotoxic approaches so that a deeper and more sustained treatment responses could be achieved. The complement system is emerging as a potential novel target for cancer therapy. Data accumulated to date show that complement proteins, and in particular C1q and its receptors cC1qR/CR and gC1qR/p33/HABP1, are overexpressed in most cancer cells and together are involved not only in shaping the inflammatory tumor microenvironment, but also in the regulation of angiogenesis, metastasis, and cell proliferation. In addition to the soluble form of C1q that is found in plasma, the C1q molecule is also found anchored on the cell membrane of monocytes, macrophages, dendritic cells, and cancer cells, via a 22aa long leader peptide found only in the A-chain. This orientation leaves its 6 globular heads exposed outwardly and thus available for high affinity binding to a wide range of molecular ligands that enhance tumor cell survival, migration, and proliferation. Similarly, the gC1qR molecule is not only overexpressed in most cancer types but is also released into the microenvironment where it has been shown to be associated with cancer cell proliferation and metastasis by activation of the complement and kinin systems. Co-culture of either T cells or cancer cells with purified C1q or anti-gC1qR has been shown to induce an anti-proliferative response. It is therefore postulated that in the tumor microenvironment, the interaction between C1q expressing cancer cells and gC1qR bearing cytotoxic T cells results in T cell suppression in a manner akin to the PD-L1 and PD-1 interaction.

Abstract Introduction: Colorectal cancer (CRC) is the third most common cancer among African Americans (AA) and when compared to Caucasian Americans (CA), they present more advanced CRC disease and lower survival rates. Our previous findings suggest that this may be related to the differential expression in genes linked to cell recruitment and immune response. Therefore, we aimed to investigate the cellular antitumor activity and mutational profile of colon tumors from AAs. We also examined the secretion of cytokines characteristic of immune responses by different effector T helper cells (Th) subsets in AA and CA patients, as well as cell lines, to see if these differences play a role in the health disparities observed between these populations. Lastly, we observed the expression of the Program Death Ligand 1 (PD-L1) in response to the cytokines IL-17A and TNF-α in a microsatellite-unstable (MSI) AA and a microsatellite-stable (MSS) CA colon cancer cell line. Methods: Using IHC, we evaluated the cell recruitment and activation of T and natural killer cells in AA tumors. For mutational analysis, we utilized the TruSight Tumor 170 RUO kit (Illumina). ELISA assays (RayBiotech) were used to examine the secretion of cytokines linked to Th subsets (Th1, Th2, Th17) and inflammation in plasma from the AA and CA CRC patients, as well as in supernatants from the AA and CA colon cancer cell lines. Western blots were used to observe the expression of PD-L1 in the in vitro models. Results: ELISAs of plasma of CA and AA patients revealed a differential Th cytokines production patterns between early-stages (I, II) and late-stage (III) disease. The MSI AA cell line showed an increase on PD-L1 protein expression in response to IL-17A and TNF-α with an additive effect when combined in equal concentrations. Lastly, the mutational sequencing allowed us to further investigate the potential alterations that are responsible for the differences that we observed in gene expression between AAs and CAs in our RNA and cytokine expression profiling. Conclusions: Our results indicate that the immune profiles of AA patients differ from CA in terms of cytokines' production; AAs expressed elevated IL-17A, whereas CA expressed elevated IFN-γ, the latter indicative of Th1 immunity that has a more favorable prognosis. As such, these differences could be used as biomarkers and to guide therapeutic strategy for these populations. The mutational sequencing will help us to elucidate the impaired tumor immune response in AAs with colon cancer when compared to CAs that we observed in terms of cell recruitment and cytokine secretion in our previous findings. Importantly, our data indicate that IL-17A and TNF-α promote the protein production of PD-L1 in an MSI AA cell line, which may result in the impairment of T cells' antitumor activity. Taken together, the differences in the immunologic profiles in AA when compared to CA suggest a deficiency of the appropriate immune defense mechanisms in this population that may contribute to the cancer health disparities among CRC patients. Citation Format: Jenny E. Paredes, Ping Ji, Jone Garai, Marzia Spagnardi, Maria Munoz-Sagastibelza, Sayed Imtiaz, Gayle Mendez, Mubarak Akadri, Raavi Gupta, Mohamed Alshal, Maksim Agaronov, Henry Talus, Ellen Li, Jovanny Zabaleta, Laura Martello-Rooney, Jennie Williams. Tumor immune response in colon cancer African American patients and its role in cancer disparities [abstract]. In: Proceedings of the Eleventh AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2018 Nov 2-5; New Orleans, LA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl):Abstract nr C118.

Abstract Colorectal cancer (CRC) is the second most lethal cancer in the United States, but underlying its incidence and mortality is a disproportionate burden in African Americans (AA). Compared to Caucasian Americans (CA), AA patients are 30% more likely to develop and 50% more likely to die from CRC. Socioeconomic factors contribute to racial health disparity. However, recent evidence suggests that biological factors may shape the AA patient predisposition. Clinical assessment has shown that AA CRC patients have a higher rate of chemoresistance, tumor recurrence post-resection, and lower 5-year survival rate post-resection as compared to CA CRC patients. 5-fluorouracil (5-FU) has been the standard of care against CRC for more than 50 years. However, high chemoresistance in AA patients has become apparent. Thus, the formulation of safer and more effective chemotherapeutic agents is paramount. Using the MTT proliferation assay, we evaluated the efficacy of the fluoropyrimidine polymer ‘F10' (a promising chemotherapeutic agent) against 3 novel AA CRC cell lines (SB501, SB521, and CHTN06) and compare its potential effectiveness against 5-FU. Additionally, we assessed the potential differential efficacy of F10 between the three novel AA CRC cell lines and three CA CRC cell lines (e.g., HT29 and a p53+/+ wild type and a p53-/- variant of HCT116). A high potency of F10 in inducing DNA-directed apoptosis was shown to be significant regardless of p53 status. Given that AA CRC patients have a higher rate of unique TP53 polymorphisms linked to chemoresponse and chemoresistance, this finding is important. In 4 out of 6 cell lines, the IC50 value significantly decreased for F10 as compared to 5-FU. HCT116 -/-, HCT116 +/+, HT29, and SB521 exhibited ~12-fold, ~150-fold, ~10-fold, and ~6850-fold decrease in IC50 value for F10 compared to 5-FU, respectively (Two-Sample T-Test: HCT116-/-: p = 0.0043, HCT116 +/+: p = 0.0483, HT29: p = 0.0386, SB521: p = 0.0122). The cell line with wild type p53 exhibited a strong benefit from F10. In general, cell lines with p53 mutations showed benefits, but to a lesser extent; suggesting that both p53-dependent and p53-independent mechanisms are responsible for F10 chemotherapeutic activity. In addition, RNAseq analysis of each cell line was assessed and differential expression compared. Several differences between and within racial cell lines were defined. Notably, in AA some of these were associated with drug absorption (e.g., SLC28A2 and CES1). Additionally, p53 mutations within the AA cell lines were identified. Identifying and understanding genetic variables affecting treatment response is critical in the development of effective chemotherapeutic agents. Overall, validation of F10 as a chemotherapeutic agent is needed to assist in the development of treatment modalities which are effective for all CRC patients regardless of race and ethnicity. Citation Format: Shrey Thaker, William H. Gmeiner, Ping Ji, Jovanny Zabaleta, Tiana Reyes, Matthew DiGiovanni, Xuefeng Wang, Jennie L. Williams. F10 and 5-fluorouracil: Chemotherapeutic potential of F10 in alleviating colon cancer racial health disparity [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1842.

Background:Colorectal cancer is the third most deadly cancer among African Americans (AA). When compared to Caucasian Americans (CA), AA present with more advanced disease and lower survival rates. Here, we investigated if differences in tumor immunology could be contributive to disparities observed between these populations. Methods:We examined gene expression of tumor and non-tumor adjacent tissues from AA and CA by whole transcriptome sequencing, and generated scores for immune cell populations by NanoString. In addition, we utilized "The Cancer Genome Atlas" (TCGA) database from AA and CA as a validation cohort. Finally, we measured the secretion of cytokines characteristic of effector T helper cell (T-h) subsets by ELISA using plasma from each AA and CA participant. Results:Colon tumors from AA patients showed significant fold-change increase in gene expression when compared to CA forFOXP3(6.22 vs. 3.22),IL1B(103 vs. 11.4) andIL8(220 vs. 28.9) (p< 0.05). In contrast, among CA we observed statistically higher gene expression of markers associated with antitumor activity such asGZMB(Granzyme B),IFNGand the immunotherapy targetsPDL1(CD274) andCTLA4(p< 0.05). TCGA data validated our observed higher gene expression ofGZMBandPDL1in CA patients when compared to AA. Notably, our observations on immune cell populations show that AA tumors have significantly higher number of exhausted CD8+ cells (p< 0.01), mast cells (p< 0.02) and increased T regulatory cells when compared to CA. AA colon cancer patients differed from CA in cytokine production patterns in plasma (i.e., reduced IL-12). Conclusions:Our study demonstrates significant differences of the immunological profiles of colon tumors from AA compared to CA that suggest a deficiency of appropriate immune defense mechanisms in terms of gene expression, recruitment of immune cells and systemic secretion of cytokines. As such, these immune differences could be mitigated through population-specific therapeutic approaches.

Abstract Colorectal cancer (CRC) is the third most common cancer among African Americans (AAs) in the US. When compared to Caucasian Americans (CAs), AAs present with higher incidence and death rates as well as worse prognosis after treatment with 5-Fluorouacil (5-FU). Previous studies have shown a correlation between microsatellite instability (MSI) and response to 5-FU and our recent findings suggest that differences in the tumor immunology and MSI status of AA and CA CRC patients are associated with the observed disparities between these populations. Therefore, we examined if cytokines secretion, protein production and cellular response to 5-FU treatment, differs between colon cancer cell lines from AAs generated in Dr. Williams’ laboratory (SB521, SB501 and CHTN06) and colon cancer cell lines from CA patients (HT29 and HCT116). Methods: We performed whole transcriptome sequencing of colon cancer cell lines (RNAseq), utilizing the NextSeq 500/550 High Output Kit v2.5 (Illumina) and Partek Flow data analysis to correlate gene expression to immune-oncology pathways. ELISA assays (RayBiotech) were used to examine the secretion of cytokines in supernatants from cells treated with interleukin 1β and tumor necrosis factor alpha. We used western blotting for protein detection of the phosphorylated form of c-Jun N-terminal kinases (JNK) as well as cell viability assays for establishing the IC50 of the cell lines to 5-FU. Results: The gene expression results indicated that the immune profiles of AA cell lines differ from CA in genes and cytokines related to cellular anti-tumor activity, including CD8B, IL-1R, IL-1R2, Granzyme B and NFKB. ELISAs of supernatants from CA and AA CRC cell lines revealed a differential cytokines secretion between the two races, namely IL-8. Lastly, the MSI AA cell line showed sensitivity to 5-FU (tenfold less) when compared to the CA cell lines and a distinct protein production pattern. Conclusions: Our gene expression findings demonstrated the differential expression of immunological pathways involved in immune-surveillance and cancer progression in the CRC cell lines between the two races. These results were in accordance with the cytokines’ and protein’s expression patterns observed in the two cohorts of cell lines. Importantly, our data indicates that 5-FU is more efficient in reducing cell viability in the MSI AA cell line than in the two CA cell lines regardless of their MSI status. Altogether, our results illustrate the value of these in vitro models to study 5-FU treatment in AA patients with MSI and MMR mutations and to elucidate the differences in chemotherapy treatment responses between AAs and CAs. In conclusion, we demonstrated distinct immunological profiles and 5-FU sensitivity of AA and CA CRC cell lines. As such, these differences observed could be used to guide new therapeutic strategies. Citation Format: Marzia Spagnardi, Jenny Paredes, Jone Garai, Ping Ji, Ellen Li, Jovanny Zabaleta, Laura Martello-Rooney, Jennie Williams. Tumor biology and cancer health disparity: Gene expression, cytokine secretion, and protein production in African American colon cancer cell lines [abstract]. In: Proceedings of the Twelfth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2019 Sep 20-23; San Francisco, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl_2):Abstract nr B134.

Abstract Background: Colorectal cancer is the third most deadly cancer among African Americans (AA). When compared to Caucasian Americans (CA), AA present with more advanced disease and lower survival rates. We have previously demonstrated that colon tumors from AA and CA differ in their immune cell recruitment and their systemic cytokines secretion profiles. Therefore, in this study we investigated if differences in gene expression and mutational profiles could relate to the disparities observed between these populations. Methods: We examined gene expression of colon tumor and non-tumor adjacent tissues from AA (n=20) and CA (n=20) by whole transcriptome sequencing (Illumina); and determined the differential expression of the 170 genes of the Illumina's TruSight Tumor 170 panel (TST170) in AA (n=12) and CA (n=17) colon tumor and non-tumor adjacent tissues. Results: From our gene expression studies, we found that colon tumors from AA patients showed significant fold-change increases in gene expression when compared to CA for FOXP3 (6.97 vs. 3.15), IL1B (122 vs. 14) and IL8 (262 vs. 28) (p < 0.05). In contrast, among CA we observed statistically higher gene expression of markers associated with antitumor activity such as GZMB (Granzyme B), IFNG and immunotherapy targets PDL1 (CD274) and CTLA4 (p < 0.05). Through our mutational profiling we identified 38 genes in AA and 6 in CA differentially expressed between tumor and non-tumor tissues (p < 0.01). Of those, 5 genes associated with several roles in cancer ETV4 (poor prognosis), CCND1 (tumorigenesis), FGFR2 (activation of the RAS-MAPK and the PI3K-AKT pathways) BRCA2 (genome stability), BCL2 (apoptosis regulator) were commonly altered in both cohorts. Conclusions: Our study demonstrates significant differences in the genetic characteristics of colon tumors from AA compared to CA that suggest a deficiency of appropriate immune defense features in terms of gene expression. We also observed divergence in the mutational profiling between the groups, including numerous genes that are indicative of prognosis, treatment and outcomes in colon cancer. As such, these deficits could be mitigated through population-specific therapeutic approaches. Citation Format: Jenny Paredes, Jone Garai, Li Li, Melody Baddoo, Ping Ji, Sayed Imtiaz, Marzia Spagnardi, Mubarak Akadri, Raavi Gupta, Mohamed Alshal, Maksim Agaronov, Henry Talus, Jennie L. Williams, Laura Martello-Rooney, Jovanny Zabaleta. Mutational analysis of colon tumors from African American patients and potential association with cancer disparities [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3618.

Sphingolipids have been implicated in mammalian placental development and function, but their regulation in the placenta remains unclear. Herein we report that alkaline ceramidase 2 (ACER2) plays a key role in sustaining the integrity of the placental vasculature by regulating the homeostasis of sphingolipids in mice. The mouse alkaline ceramidase 2 gene (Acer2) is highly expressed in the placenta between embryonic day (E) 9.5 and E12.5. Acer2 deficiency in both the mother and fetus decreases the placental levels of sphingolipids, including sphingoid bases (sphingosine and dihydrosphingosine) and sphingoid base-1-phosphates (sphingosine-1-phosphate and dihydrosphingosine-1-phosphate) and results in the in utero death of ≈50% of embryos at E12.5 whereas Acer2 deficiency in either the mother or fetus has no such effects. Acer2 deficiency causes hemorrhages from the maternal vasculature in the junctional and/or labyrinthine zones in E12.5 placentas. Moreover, hemorrhagic but not non-hemorrhagic Acer2-deficient placentas exhibit an expansion of parietal trophoblast giant cells with a concomitant decrease in the area of the fetal blood vessel network in the labyrinthine zone, suggesting that Acer2 deficiency results in embryonic lethality due to the atrophy of the fetal blood vessel network in the placenta. Taken together, these results suggest that ACER2 sustains the integrity of the placental vasculature by controlling the homeostasis of sphingolipids in mice.

Colorectal cancer (CRC) is the third most common cancer among African Americans (AA) and when compared to Caucasian Americans (CA), they present with more advanced disease and lower survival rates. Our previous findings suggest that this may be related to the differential expression of genes linked to cell recruitment and immune response. Therefore, we aimed to investigate if differences in the cellular anti-tumor immune activity in AA and CA patients play a role in the disparate cancer progression observed between these populations. Our approach includes examining gene expression and immune cell recruitment at the tumor site and secretion of cytokines characteristic of effector T helper cells (Th) subsets in plasma. Lastly, we observed the expression of several proteins involved in apoptosis, stress, and drug resistance in response to treatment with 5-FU in two microsatellite unstable (MSI) CRC cell lines, one from an AA and one from a CA patient, and a microsatellite stable (MSS) CA colon cancer cell line.Methods: We performed whole transcriptome sequencing in colon tumors, utilizing the NextSeq 500/550 High Output Kit v2.5 (Illumina). ELISA assays (RayBiotech) were used to examine the secretion of cytokines linked to Th subsets (Th1, Th2, Th17) and inflammation in plasma. Using IHC we evaluated the cell recruitment and activation of T and Natural Killer cells in colon tumors and by MetaCore we correlated gene expression to immune-oncology pathways. Western blots were used to evaluate the expression of cleaved caspase 3, phospho-JNK and RRM1 in the in-vitro models.Results: ELISAs of plasma from CA and AA patients revealed a differential Th cytokines production patterns between early stage (I, II) and late stage (III) disease. Our gene expression results indicate that the immune profiles of AA patients differ from CA in the expression of 36 key genes and cytokines related to cellular anti-tumor activity, including FOXP3, Granzyme B and IL-17A, suggestive of more favorable prognosis in the CA tumors. Lastly, the MSI AA cell line showed sensitivity to 5-FU in terms of protein expression when compared to the CA cell lines.Conclusions: Our gene expression findings demonstrated the differential expression of immunological pathways involved in immune-surveillance, cancer progression and antigen presentation in colon tumors from these two ethnicities. These results were in accordance with the systemic cytokines’ expression patterns observed in plasma and cell recruitment to the tumor sites. Importantly, our data indicates that treatment with 5-FU chemotherapy promotes apoptosis and stress in the MSI AA cell line but fails to produce the same effect in the CA cell lines at the same concentrations. Taken together, the differences in the immunological profiles in AA when compared to CA suggests a deficiency of the appropriate immune defense mechanisms in this population that may contribute to the cancer disparities among CRC patients.Citation Format: Jenny Paredes, Jone Garai, Ping Ji, Sayed Imtiaz, Marzia Spagnardi, Maria Munoz-Sagastibelza, Mubarak Akadri, Raavi Gupta, Mohamed Alshal, Maksim Agaronov, Henry Talus, Ellen Li, Jennie Williams, Jovanny Zabaleta, Laura Martello-Rooney. Gene expression and mutational load in colon tumors from African American patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 152.

Abstract Despite progress in closing the gap, health disparities still persist among African American (AA) colon cancer patients both in incidence and death rates as well as worse prognosis after treatment with 5-fluorouacil (5-FU) when compared to Caucasian American (CA) patients. In addition, AA colon cancer patients have higher incidence of mutations on the mismatch DNA-repair mechanism (MMR) than CA patients, and previous studies have shown a correlation between mutations in MMR and microsatellite instability (MSI) to resistance to 5-FU. Therefore, we aim to examine if differential MSI and MMR play a role in the efficacy of 5-FU treatment in AAs. Methods: We examined the microsatellite status and MMR mutations found in the tumors from AA colon cancer patients at our institution from 2010 to 2017 and utilized the AA tumor-derived colon cancer cell line SB-521, generated in Dr. Williams's laboratory, and the CA colon cancer cell line HT-29 for in vitro studies. In order to assess the response to 5-FU, we used the protein levels of the ribonucleotide reductase catalytic subunit M1 (RRM1) protein as a marker for drug resistance, the cleavage of caspase-3 for apoptosis initiation, c-Jun N-terminal kinases (JNK) phosphorylation as indicator of cellular stress, and histone 3 protein levels as reference of gene methylation changes. Results: Our retrospective data (N=83 patients) demonstrated that up to 20% of our colon cancer patients have MSI and up to 30% showed MMR mutations. As hypothesized, the protein levels of the chosen targets in the two cell lines exhibited differences after treatment with 5-FU. The AA cell line SB-521 appears to be more sensitive to the chemotherapeutic in terms of apoptosis, stress response, and drug resistance when compared to HT-29 cells. Conclusions: Altogether, our results illustrate the range of MSI, MMR, and protein patterns in AA tumors and cell line. The cell line SB-521 is a potential model to study 5-FU treatment for patients with MSI and MMR mutations as it has high MSI, does not express MLH1, has reduced MSH2, and overexpresses MSH6 (MMR proteins). Previous data suggest that lack of a functional MMR mechanism may play a role in the response to this chemotherapeutic. Further studies are needed to elucidate the differences in chemotherapy treatment responses between AAs and CAs and their role in colon cancer health disparities. Citation Format: Jenny E. Paredes, Ping Ji, Maria Munoz-Sagastibelza, Malhaar Agrawal, Jennie Williams, Laura Martello-Rooney. Role of microsatellite stability and mismatch DNA repair mechanism in the response to 5-fluorouacil treatment in African American colon cancer cell lines [abstract]. In: Proceedings of the Tenth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2017 Sep 25-28; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2018;27(7 Suppl):Abstract nr A73.

The incidence and mortality rates of colorectal carcinoma (CRC) are higher among African Americans (AAs) compared with Caucasian Americans (CAs). To assess the molecular properties associated with racial health disparity, three cell lines derived from colorectal tumors of three AA subjects were established. Cellular and molecular characterization of the cell lines designated CHTN06, SB501 and SB521 was performed using standard technologies, including immunofluorescence, electron microscopy, karyotyping, reverse transcription-polymerase chain reaction, ELISA and immunoblot analysis. The histology and morphology of CHTN06 xenografts were examined by hematoxylin and eosin staining. A total of three AA CRC cell lines derived from primary tumors were established and characterized. These cell lines were successfully cultured without immortalization and were found to be tumorigenic as mouse xenografts. In the present study, immunoblotting and immunofluorescence confirmed the expression of proteins known to be dysregulated in CRC, such as p53, DNA mismatch repair proteins and villin-1. Oncogenic miRNAs (i.e., miR-17, miR-21, miR-182, miR-210 and miR-222) were overexpressed in the AA CRC lines compared with the CA CRC lines (HT-29, HCT116 and SW480). Additionally, the AA CRC cell lines exhibited a differential inflammatory profile compared with HT-29 (CA CRC cell line); specifically noted was IL-8 secretion in response to inflammatory stimuli. In conclusion, three novel cell lines derived from AA CRC tissues were generated. These cell lines were characterized as epithelial in nature and exhibited differential expression of several miRNAs and inflammatory responses compared with commercially available cell lines of CA origin. The CRC cell lines CHTN06, SB501 and SB521 represent novel tools that may be used to provide diverse in vitro and in vivo models for studying CRC and racial health disparity.

The complement protein C1q and its receptors, gC1qR and cC1qR, are expressed on various normal as well as malignant cells and can play both proand anti-proliferative roles. These opposing roles, in turn, depend on the localization of these molecules i.e. surface versus secreted. The present studies were designed to examine the functions of the soluble (secreted) and membrane forms of both C1qRs and C1q using the SkBr3 Her2+ cancer cell line as a model for breast cancer. Our results show that SkBr3 cells express not only gC1qR and cC1qR, but also C1q. Importantly, co-culture of SkBr3 cells with either purified C1q or the C1q globular head (gh) modules, ghA, ghB and ghC, resulted in a significant inhibition of cell growth with ghA and ghC showing stronger effects than ghB. Conversely, co-culture of SkBr3 cells with either anti-C1q or an antibody recognizing the gC1qR site on ghA, resulted in complete inhibition of cell growth indicating that like gC1qR, membrane-bound C1q is also pro-proliferative. Co-culture of SkbR3 with anti-cC1qR was also found to inhibit cell proliferation. In contrast, addition of purified recombinant gC1qR to SkBr3 cells enhanced cell growth and reduced cell death even under nutrient-depleted conditions suggesting that it is a pro-proliferative autocrine signal. Although SkBr3 cells do not secrete C1q during normal cell proliferation, they release gC1qR. Interestingly, the anti-proliferative effect of exogenously added C1q was inhibited when added to SkBr3 cells in gC1qR-rich medium lending credence to the postulate that secreted gC1qR not only provides an autocrine signal for proliferation but also serves as a molecular checkpoint in the tumor cell microenvironment by denying C1q access to the cell surface. These observations demonstrate that cell surfaceexpressed C1q and C1qRs are involved in breast cancer cell proliferation, and that soluble gC1qR appears to serve as an autocrine growth signal. Thus, the C1q receptor–C1q axis may provide potential novel targets for therapeutic intervention in breast cancer.

Abstract Colorectal cancer (CRC) is the third most common cancer among African Americans (AA) and when compared to Caucasian Americans (CA), they present more advanced CRC disease and lower survival rates. Recent findings suggest that this may be related to the differential expression in genes linked to inflammation and immune response. Therefore, we aimed to investigate if tumors from AA colon cancer patients diverge in their immunologic profile from CA and if the immune response of a CRC cell line derived from an AA tumor will differ from a CA CRC cell line. Additionally, we are recording the genetic profiles of colon tumors and outcomes from AA patients at our institution. Methods: Using DESeq2 we evaluated the differential gene expression pattern by whole transcriptome sequencing (Illumina) of 10 CRC tissues (and matching adjacent non-tumor tissue) from both AA and CA individuals. We focused on genes involved in immune checkpoints and inflammation. We also examined the secretion of Interleukin 8 (IL-8) in plasma from our AA CRC patients. For the in vitro experiments, we used the AA tumor-derived colon cancer cell line SB-521, generated in Dr. Williams' laboratory, and the CA colon cancer cell line HT-29 to determine if the cell lines expressed the Programmed death-ligand 1 (PD-L1). Lastly, we analyzed the microsatellite (MSI) status and MMR mutations in tumors from AA colon cancer patients at our institution and correlated their genetic analysis to response to chemotherapies and survival. Results: The genomic data revealed that AA and CA tumors had a significant difference of expression in a total of 221 genes. Remarkably, some of these genes included PD1, IL1B, IL17A, IL10, IL5, CD80 and FOXP3. The cytokine IL-8 concentration detected by ELISA in plasma of these patients revealed a differential expression between early stages (I, II) and late stages (III, IV). As hypothesized, the MSI and AA tumor-derived cell line SB-521 expressed PD-L1 and showed an increase in protein levels in response to TNF-α treatment (the CA cell line HT-29 did not express PD-L1). Lastly, our retrospective data (N=200 patients) demonstrated that up to 20% of our AA colon cancer patients have MSI and/or MMR mutations. Conclusions: Altogether, our results suggest that the immune profiles of the tumors from AA patients differ from CA and these differences could be used as biomarkers and to guide therapeutic strategy for these populations. Also, since the AA cell line presented distinct inflammatory patterns and when compared to the HT-29 CA cell line, it is a potential model to study MSI and PD-L1 in AA. Hence, we aim to supplement our preliminary data on AA patients with MSI and MMR mutations at Downstate and to elucidate what other genomic differences exist and cytokines' secretion patterns observed. In conclusion, we will address the immune and molecular biology of CRC tumors in AA through genomic and in vitro studies, and generate patient's data on AA diagnosed with colon cancer. Citation Format: Jenny E. Paredes, Ping Ji, Maria Munoz-Sagastibelza, Sayed Imtiaz, Kaylene Barrera, Raavi Gupta, Maksim Agaronov, Henry Talus, Jovanny Zabaleta, Jennie Williams, Laura Martello-Rooney. Immune checkpoints and inflammation in colon tumors from African Americans [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 646.

Abstract Despite progress in closing the gap, health disparities still persist among African American (AA) colon cancer patients both in incidence and death rates. Previous studies from our group reported that colon tumors from AAs displayed hypermethylation of DNA regions in inflammatory genes such as NELL1, GDF1, ARHGEF4, and ITGA4 when compared to Caucasian Americans (CA). To assess potential differences in the inflammatory response, we utilized two AA colon cancer cell lines generated in our laboratory and compared them with the commercially available CAs colon cancer cell lines, Caco-2 and HT-29. Recent experiments by our group with the anti-inflammatory drugs Ibuprofen, Sulindac and Aspirin, showed significantly higher IC50 values in proliferation assays for AA cell lines compared to CA cell lines. Therefore, we evaluated the anti-inflammatory effects of these drugs by determining secretion of inflammatory cytokines and MAPKs activation in response to the pro-inflammatory cytokine TNF-alpha. The same parameters were tested for the anti-inflammatory cytokine IL-10. As hypothesized, our results in the CA cell lines demonstrated down-regulation of MAPKs activation in response to TNF-alpha after pre-treatment with Ibuprofen. On the contrary, Ibuprofen concentrations as high as the IC50 values for the CA cell lines were not able to induce down-regulation of MAPKs in the AA cell lines. Interestingly, IL-10 treatment was more effective in the AA compared to the CA cell lines, suggesting that the main anti-inflammatory cytokine of the colon will be more effective in controlling inflammation in AA colon tumors. Further studies are needed to elucidate the major differences in inflammatory responses between AA and CA colon cancer cell lines and their potential role in CRC health disparities. As it has been documented that AA colon cancer patients are less responsive to the chemotherapeutics Fluorouracil and Capecitabine, in future studies we will evaluate the effect of these therapeutic agents on AA and CA cell lines in terms of inflammation, cell viability, apoptosis and invasion. Citation Format: Jenny E. Paredes Sanchez, Ping Ji, Maria Munoz-Sagastibelza, Laura Martello-Rooney, Jennie Williams. Inflammatory patterns exhibited by African American colon tumor-derived cell lines [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 2692. doi:10.1158/1538-7445.AM2017-2692

Background Incidence and mortality rates of colorectal carcinoma (CRC) are higher in African Americans (AAs) than in Caucasian Americans (CAs). Deficient micronutrient intake due to dietary restrictions in racial/ethnic populations can alter genetic and molecular profiles leading to dysregulated methylation patterns and the inheritance of somatic to germline mutations. Materials and Methods Total DNA and RNA samples of paired tumor and adjacent normal colon tissues were prepared from AA and CA CRC specimens. Reduced Representation Bisulfite Sequencing (RRBS) and RNA sequencing were employed to evaluate total genome methylation of 5’-regulatory regions and dysregulation of gene expression, respectively. Robust analysis was conducted using a trimming-and-retrieving scheme for RRBS library mapping in conjunction with the BStool toolkit. Results DNA from the tumor of AA CRC patients, compared to adjacent normal tissues, contained 1,588 hypermethylated and 100 hypomethylated differentially methylated regions (DMRs). Whereas, 109 hypermethylated and 4 hypomethylated DMRs were observed in DNA from the tumor of CA CRC patients; representing a 14.6-fold and 25-fold change, respectively. Specifically; CHL1, 4 anti-inflammatory genes (i.e., NELL1, GDF1, ARHGEF4, and ITGA4), and 7 miRNAs (of which miR-9-3p and miR-124-3p have been implicated in CRC) were hypermethylated in DNA samples from AA patients with CRC. From the same sample set, RNAseq analysis revealed 108 downregulated genes (including 14 ribosomal proteins) and 34 upregulated genes (including POLR2B and CYP1B1 [targets of miR-124-3p]) in AA patients with CRC versus CA patients. Conclusion DNA methylation profile and/or products of its downstream targets could serve as biomarker(s) addressing racial health disparity.

Colorectal cancer (CRC) incidence and mortality are higher in African Americans (AAs) than in Caucasian Americans (CAs) and microRNAs (miRNAs) have been found to be dysregulated in colonic and other neoplasias. The aim of this exploratory study was to identify candidate miRNAs that could contribute to potential biological differences between AA and CA colon cancers. Total RNA was isolated from tumor and paired adjacent normal colon tissue from 30 AA and 31 CA colon cancer patients archived at Stony Brook University (SBU) and Washington University (WU)-St. Louis Medical Center. miRNA profiles were determined by probing human genome-wide miRNA arrays with RNA isolated from each sample. Using repeated measures analysis of variance (RANOVA), miRNAs were selected that exhibited significant (p<0.05) interactions between race and tumor or significant (fold change >1.5, p<0.05) main effects of race and/or tumor. Quantitative polymerase chain reaction (q-PCR) was used to confirm miRNAs identified by microarray analysis. Candidate miRNA targets were analyzed using immunohistochemistry. RANOVA results indicated that miR-182, miR152, miR-204, miR-222 and miR-202 exhibited significant race and tumor main effects. Of these miRNAs, q-PCR analysis confirmed that miR-182 was upregulated in AA vs. CA tumors and exhibited significant race:tumor interaction. Immunohistochemical analysis revealed that the levels of FOXO1 and FOXO3A, two potential miR-182 targets, are reduced in AA tumors. miRNAs may play a role in the differences between AA and CA colon cancer. Specifically, differences in miRNA expression levels of miR-182 may contribute to decreased survival in AA colon cancer patients.

We report the synthesis and characterisation of mixed-metal binuclear ruthenium(II)-vanadium(IV) complexes, which were used as potential photodynamic therapeutic agents for melanoma cell growth inhibition. The novel complexes, [Ru(pbt)2(phen2DTT)](PF6)2·1.5H2O 1 (where phen2DTT = 1,4-bis(1,10-phenanthrolin-5-ylsulfanyl)butane-2,3-diol and pbt = 2-(2'-pyridyl)benzothiazole) and [Ru(pbt)2(tpphz)](PF6)2·3H2O 2 (where tpphz = tetrapyrido[3,2-a:2',3'-c:3'',2''-h:2''',3'''-j]phenazine) were synthesised and characterised. Compound 1 was reacted with [VO(sal-L-tryp)(H2O)] (where sal-L-tryp = N-salicylidene-L-tryptophanate) to produce [Ru(pbt)2(phen2DTT)VO(sal-L-tryp)](PF6)2·5H2O 4; while [VO(sal-L-tryp)(H2O)] was reacted with compound 2 to produce [Ru(pbt)2(tpphz)VO(sal-L-tryp)](PF6)2·6H2O 3. All complexes were characterised by elemental analysis, HRMS, ESI MS, UV-visible absorption, ESR spectroscopy, and cyclic voltammetry, where appropriate. In vitro cell toxicity studies (with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay) via dark and light reaction conditions were carried out with sodium diaqua-4,4',4'',4''' tetrasulfophthalocyaninecobaltate(II) (Na4[Co(tspc)(H2O)2]), [VO(sal-L-tryp)(phen)]·H2O, and the chloride salts of complexes 3 and 4. Such studies involved A431, human epidermoid carcinoma cells; human amelanotic malignant melanoma cells; and HFF, non-cancerous human skin fibroblast cells. Both chloride salts of complexes 3 and 4 were found to be more toxic to melanoma cells than to non-cancerous fibroblast cells, and preferentially led to apoptosis of the melanoma cells over non-cancerous skin cells. The anti-cancer property of the chloride salts of complexes 3 and 4 was further enhanced when treated cells were exposed to light, while no such effect was observed on non-cancerous skin fibroblast cells. ESR and (51)V NMR spectroscopic studies were also used to assess the stability of the chloride salts of complexes 3 and 4 in aqueous media at pH 7.19. This research illustrates the potential for using mixed-metal binuclear ruthenium(II)-vanadium(IV) complexes to fight skin cancer.