Abdeljabar El Andaloussi

NK Cell-Mediated Regulation of Protective Memory Responses against Intracellular Ehrlichial Pathogens

Ehrlichiae are gram-negative obligate intracellular bacteria that cause potentially fatal human monocytic ehrlichiosis. We previously showed that natural killer (NK) cells play a critical role in host defense against Ehrlichia during primary infection. However, the contribution of NK cells to the memory response against Ehrlichia remains elusive. Primary infection of C57BL/6 mice with Ehrlichia muris provides long-term protection against a second challenge with the highly virulent Ixodes ovatus Ehrlichia (IOE), which ordinarily causes fatal disease in naïve mice. Here, we show that the depletion of NK cells in E. muris-primed mice abrogates the protective memory response against IOE. Approximately, 80% of NK cell-depleted E. muris-primed mice succumbed to lethal IOE infection on days 8–10 after IOE infection, similar to naïve mice infected with the same dose of IOE. The lack of a recall response in NK cell-depleted mice correlated with an increased bacterial burden, extensive liver injury, decreased frequency of Ehrlichia-specific IFN-γ-producing memory CD4+ and CD8+ T-cells, and a low titer of Ehrlichia-specific antibodies. Intraperitoneal infection of mice with E. muris resulted in the production of IL-15, IL-12, and IFN-γ as well as an expansion of activated NKG2D+ NK cells. The adoptive transfer of purified E. muris-primed hepatic and splenic NK cells into Rag2-/-Il2rg-/- recipient mice provided protective immunity against challenge with E. muris. Together, these data suggest that E. muris-induced memory-like NK cells, which contribute to the protective, recall response against Ehrlichia.

Vitamin D and corticotropin-releasing hormone in term and preterm birth: potential contributions to preterm labor and birth outcome

Abstract Background: Poor maternal vitamin D status and elevated circulating corticotropin-releasing hormone (CRH) are associated with preterm birth. It is not known if these risk factors are independent or interrelated. Both are associated with inflammation. Methods: We measured maternal circulating 25-hydroxyvitamin D (25-OH-D) and CRH from 97 samples collected from 15 early-preterm, 31 late-preterm, 21 early-term, and 30 term births. The potential involvement of vitamin D in the regulation of inflammation was evaluated by Q-PCR in human uterine smooth muscle (UTSM) cell line. Results: Maternal 25-OH-D was lowest in early-preterm births (22.9 ± 4.2 ng/ml versus 34.4 ± 1.4 ng/ml; p = .029). Circulating CRH was high in early-preterm births (397 ± 30 pg/ml). Late-preterm (304 ± 13 pg/ml) and early-term births (347 ± 17 pg/ml) were not different from term births (367 ± 19 pg/ml), after accounting for gestational age. Maternal circulating 25-OH-D and CRH were not associated in term births. In preterm births, 25-OH-D below 30 ng/ml was associated with higher CRH. Vitamin D treatment of UTSM significantly reduced mRNA for leptin and IL-6 receptors. Deletion of vitamin D receptor from UTSM promoted the expression of the cox2 inflammatory marker. Conclusion: Early-preterm birth showed a syndrome of high maternal CRH and low vitamin D status.

Immune combinational therapy targeting OX40 and IDO synergistically enhances efficacy of a cancer vaccine

Enhancing an effector immune response by cancer vaccines has not been clinically successful so far. Although the necessary immune response may be elicited using vaccine-based therapies, this has not been sufficient for a positive clinical outcome; since most cancers can escape immune surveillance via multiple immune suppressive mechanisms induced in the tumor environment. A relatively recent strategy to increase the therapeutic efficacy of tumor vaccination is to combine it with alternative immunotherapeutic approaches. Here we tested if simultaneous targeting of the effector arm of the immune system with an agonist anti-OX40 mAb together with targeting of suppressive mechanisms in the tumor, via inhibition of indoleamine-(2,3)-dioxygenase (IDO), could enhance the anti-tumor activity of vaccine in mouse models of cancer. The OX40 molecule is a co-stimulatory receptor expressed on T-cells that can lead to proliferation and enhancement of T-cell effector function when triggered with an agonistic antibody. However, the tumor secretes a number of suppressive signals as a protective mechanism against its destruction, one of which is IDO. We demonstrate that treatment of TC-1 tumor bearing mice with an anti-OX40 agonist antibody in combination with vaccine leads to the enhancement of antigen-specific T cell responses. The dose of 1 mg/kg anti-OX40 antibody stimulated the effector arm of T cells, which ultimately led to a significant increase in the CD8+/regulatory T-cell (Treg) ratio within tumors. Further, this combination of vaccine and anti-OX40 antibody treatment led to a significant inhibition of tumor growth and to a prolonged survival of tumor bearing mice compared to control, and resulted in complete tumor regression in 20% of treated mice. We found, however, that this anti-tumor activity could be further enhanced through the combination of vaccine, anti-OX40 and 1-methyltryptophan (1-MT), an IDO activity inhibitor. IDO has been shown to be secreted by tumor cells, suppressive dendritic cells and macrophages in tumor environment, and is known to be responsible for suppressing effector cells and inducing Tregs. Our data demonstrate that vaccine/anti-OX40 antibody/1-MT combination leads to a more profound inhibition of tumor growth in mice and complete regression of established tumors in 60% of treated mice. In conclusion, we provide evidence that simultaneous targeting of the effector arm of the immune system, via anti-OX40 antibody, and suppressive mechanisms within the tumor, via 1-MT, in combination with cancer vaccine has a synergistic effect for tumor eradication and is a promising strategy that could potentially enhance the overall efficacy of cancer treatment in patients.

Uterine Fibroids: Bridging Genomic Defects and Chronic Inflammation

Abstract Uterine fibroids (UF; aka leiomyoma, myomas) are the most common benign tumors of female reproductive tract. They are highly prevalent, with 70 to 80% of women burdened by the end of their reproductive years. Fibroids are a leading cause of pelvic pain, abnormal vaginal bleeding, pelvic bulk symptoms, miscarriage, and infertility. They are the leading indication for hysterectomy, and costs exceed 34 billion dollars annually in the United States alone. Recently, somatic mutations in exons 1 and 2 of Med12 gene emerged as common UF driver mutations. Unfortunately, the detailed etiology of UF is not fully realized. Particularly, very little is known about possible dysregulation of inflammatory and immune processes and their possible contribution to UF pathogenesis. The notion on possible impact of altered estrogen and progesterone signaling in UF on inflammatory responses and DNA repair machinery that can conceivably lead to tumor‐specific somatic mutation is indeed an intriguing concept which has some foundation in available observation in other hormonally responsive tissues. This review highlights and summarizes our current knowledge on the convergence of such pathways and their relevance for UF pathogenesis.

Abstract 466: Systemic early immune priming via tumor-secreted cytokines facilitates breast cancer metastasis in syngeneic mouse model

Metastatic breast cancer is the second leading cause of cancer-related death among women in the United States. Although the genetic and epigenetic differences between the metastatic versus non-metastatic breast tumors have been well studied, early events between tumor and immune system in metastatic process remain poorly understood. In order to determine early events, we utilized murine mammary tumors (4T1 as metastatic, EMT6 or 67NR as non-metastatic) in syngeneic mouse model. The 4T1 tumor contained a higher proportion of cancer stem cell (CSC) population compared to the non-metastatic EMT6 or 67NR clones. Although, both murine tumor cell lines (50K each) grow to same size tumors within 8 weeks, 4T1 tumors develop spontaneous metastasis in 100% of animals most of which do not survive more than 8 weeks due to extensive wide spread metastasis to lung, liver and bone. We observed immune infiltrates in the lungs of 4T1 tumor bearing mice as early as 1 week. We next assessed the cytokine profile of metastatic 4T1 tumor compared to non-metastatic counterparts (EMT6 or 67NR) secretes significantly higher levels of inflammatory cytokines, including the IL6, IL8, RANTES, GCSF, GM-CSF, IL12, CXCL16 and CXCL5. MDSCs are potent suppressor of anti-tumor immunity and a significant impediment to cancer therapy. We therefore hypothesized that the tumor secreted inflammatory cytokines promotes the systemic expansion of MDSCs that down regulate immune surveillance and anti-tumor immunity, thus facilitating tumor progression. We sought to determine whether 4T1 tumors could induce MDSCs in mice. Murine 4T1 or EMT6 tumor cells (at 50K cells each) were implanted into the fat pads of BALB/c mice, then sacrificed (4 mice from each group) at weeks 1, 2, 3 and 4 for subsequent evaluation of the MDSC expansion in bone marrow, spleen, lung and tumors. The MDSC induction and infiltration in bone marrow, spleen, lung and tumors were observed as early as one-week post-implantation of 4T1 tumor compared to the EMT6. Furthermore, the MDSCs isolated from 4T1 tumor bearing animals were more suppressive than that of the EMT6 tumor bearing mice. We determined that non-metastatic EMT6-Luciferase tumor growth and metastasis is robustly enhanced in pre-primed animals (in which metastatic 4T1 cells were pre-implanted in the fat pads and resected after 10 days when tumors were 2mm in size) or by IP injection of inflammatory cytokine rich 4T1 conditioned medium when compared to injection of EMT6-Luci cells into naive animals. Our preliminary findings suggested that 4T1 tumors within 10 days of implantation created a systemic tumor-promoting microenvironment and thus promoted the metastatic spread of EMT6-Luci. Together these studies strongly suggest that metastatic 4T1 tumor with high CSC phenotype generate a permissive systemic microenvironment for successful metastasis via secretion of inflammatory cytokines in syngeneic BALB/c mice. Citation Format: Hasan Korkaya, Eunmi Lee, Maria Ouzounova, Abdeljabar El Andaloussi, Ena Novakovic, Raziye Piranlioglu, Mehmet F. Demirci, Shawn G. Clouthier, Max S. Wicha. Systemic early immune priming via tumor-secreted cytokines facilitates breast cancer metastasis in syngeneic mouse model. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 466. doi:10.1158/1538-7445.AM2015-466

Diet-induced vitamin D deficiency triggers inflammation and DNA damage profile in murine myometrium

Background Previously, we reported a significantly higher prevalence of uterine fibroids (UFs) in African American women. This minority group also commonly suffers from vitamin D deficiency. We have demonstrated that 1,25(OH)2D3 attains a fibroid growth inhibitory impact through its ability to block the G1/S (gap 1/synthesis) phase of the cell cycle. Vitamin D is involved in DNA damage as well as in immune response regulation, anti-inflammation, autoimmunity and cancer. Since most of the prior data on vitamin D and UF were generated in vitro via established cell lines, it was necessary to verify and validate this observation in vivo using a diet-induced vitamin D-deficient mouse model. Materials and Methods Our model of vitamin D lacking function was established using 8-week exposure of C57/BL6 mice to vitamin D-deficient diet provides evidence of different functions accomplished by vitamin D in the regulation of myometrium homeostasis disrupted in the context of uterine fibroid. Results We found that vitamin D deficiency was associated with increased expression of sex steroid receptors in murine myometrium, increased expression of proliferation related genes, the promotion of fibrosis and enhanced inflammation, and promoted immunosuppression through Tregs expansion in murine myometrium. We also showed that a vitamin D deficient diet enhanced DNA damage in murine myometrium. Conclusion Our model mimics the effects in humans that a lack of vitamin D has and propels the study of in vivo interaction between inflammation, genomic instability and cell proliferation in the myometrium.

Intravenous Infusion of Nucleated Peripheral Blood Cells Restores Fertility in Mice with Chemotherapy-Induced Premature Ovarian Failure

Cancer treatment with specific chemotherapeutic agents has been well documented to have an adverse impact on female fertility leading to premature ovarian failure (POF). The objective of this study is to investigate if chemotherapeutic induced POF can be reversed by the infusion of autologous nucleated peripheral blood cells (PBMC). To reach our goal, mice were treated with a single intraperitoneal injections of busulfan and cyclophosphamide to induce POF. This was followed by transfusion of PBMC. The ovarian morphology and functional parameters were monitored by radioimmunoassay, real-time PCR, immunofluorescence and immunohistochemistry analysis. Our study showed that chemotherapy (CTX) protracted estrous cycle period and repressed E2 production. In addition, CTX decreased the expressions of steroidogenesis markers, CYP-17 synthesis, StAR (steroidogenic acute regulatory protein), and Connexin-43 protein expression in the ovarian follicles. We also observed reduced numbers and sizes of the primordial and primary follicles in CTX-treated mice compared to untreated controls (p < 0.05). When both CTX and untreated control groups were stimulated with gonadotrophin, the control group produced ten times more ova than the CTX group. Finally, the treatment of premature ovarian failure induced by CTX with autologous PBMC transfusion resulted in over-expression and a statistically significant increase in several stem cell markers and restoration of fertility. Infusion with PBMC in CTX further decreased the estrous cycle length by 2.5 times (p < 0.01). We found that transfusion of autologous PBMC to mice with chemotherapy induced POF was very effective at restoring fertility. These results are similar to other studies using bone marrow derived mesenchymal stem cells.

Defective expression of ATG4D abrogates autophagy and promotes growth in human uterine fibroids

Uterine fibroids (UF) are the most common pelvic tumors in women of reproductive-age and they usually cause heavy menstrual bleeding, pain and infertility. Autophagy is a collection of processes that enables the cells to digest and recycle their cytoplasmic contents, such as toxic protein aggregates, defunct or disused organelles and invading microorganisms. Dysregulation in autophagy process were described in neoplasms; however, the contribution of autophagy to the pathogenesis of UF remains unknown. In this study, we demonstrate that autophagy is deregulated in human UF as evidenced by significant accumulation of autophagosome in human UF cells compared to normal myometrium cells. Analysis of the autophagy markers revealed an enhanced initiation of the autophagy in UF tissues compared to their adjacent myometrial tissues (MyoF). However, autophagosome maturation and flux was blocked in UF tissues, as marked by accumulation of LC3-B and P62 protein. This block was associated with defective expression of autophagy-related protein 4 (ATG4) in the UF tissues compared to MyoF in ~90% of patient samples. Silencing of ATG4D in normal human myometrial cells resulted in defective autophagy flux, enhanced cell proliferation and increased extracellular matrix production, which phenocopy UF cell line. This study indicates that impairment of autophagy flux secondary to defective expression of ATG4D expression is a new mechanistic aberration that contributes to UF pathogenesis. Targeting autophagy pathway could provide novel medical therapeutic approach for non-surgical treatment of UF.

Abstract 2956: Chemical library screen identifies compounds that target S100A8/S100A9 complex and MDSC accumulation

We previously demonstrated that MDSC subsets accumulated in primary tumor and distant organs regulate tumor plasticity. The mouse transcriptome analysis of in vitro co-cultures and samples from syngeneic mouse model revealed that granulocytic subset of myeloid-derived suppressor cells (gMDSCs) from metastatic 4T1 tumor bearing mice regulate several hundred genes in tumor cells upon co-culture. The top genes are S100A8, S100A9, MMP8, FPR1, CCL3, and TGFβ2 which also predicted poor survival in human solid tumors including breast cancer. Therefore, we called these 6 genes as “metastatic gene signature”. It has been reported that S100A8/S100A9 heterotetramer, called calprotectin, play a key role in inflammation-associated cancer progression. To investigate the role of calprotectin (S100A8/A9) in tumor-mediated immunosuppression and metastasis, we first utilized Tasquinimod, a small molecule targeting S100A9 only. However, Tasquinimod treatment of 4T1 tumor-bearing mice had a moderate anti-tumor activity which may be due to a limited activity on granulocytic MDSC accumulation. In addition, we determined that there was a significant upregulation of S100A8 in MDSCs from Tasquinimod treated mice. This data suggested that inhibiting only S100A9 leads to activation of S100A8 and thus may be ineffective targeting of MDSCs. We therefore run a computational screen of the NCI chemical library against the crystal structure of calprotectin (S100A8/S100A9) and identified 40 lead compounds. We then performed in vitro screening assay to identify compounds that inhibit MDSC induction. We identified 3 compounds that significantly suppressed gMDSC differentiation. We are currently performing in vivo studies with these 3 candidate drugs in our murine breast cancer model and will present our findings at the AACR meeting. We believe that this study will provide a novel drug targeting S100A8/S100A9 heterotetramer, a key molecule in MDSC induction and its regulatory functions during tumor progression. Citation Format: Eunmi Lee, Maria Ouzounova, Raziye Piranlioglu, Abdeljabar El Andaloussi, Sena Arbag, Gang Zhou, Hasan Korkaya. Chemical library screen identifies compounds that target S100A8/S100A9 complex and MDSC accumulation [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 2956. doi:10.1158/1538-7445.AM2017-2956

Abstract 4493: The co-chaperone UNC45A controls cancer cell proliferation through Nek7 and centrosomal separation

Recent findings have shown that the Heat Shock Protein 90 (Hsp90) co-chaperone UNC45A is overexpressed in ovarian and breast cancers. Previously, we have shown that UNC45A is a centrosomal protein essential for cervical tumor cell growth through activation of the checkpoint kinase 1 (ChK1). In this report, we further examined the role of UNC45A in breast tumorigenesis using a variety of biochemical and cell biology techniques and animal models. We confirmed that UNC45A is highly overexpressed in human breast-infiltrating ductal carcinomas as compared to adjacent normal tissues. Silencing UNC45A in vitro blocked the proliferation of all breast cancer subtypes and drastically reduced tumor growth of the triple negative MDA-MB-231 cell line implanted in mammary fat pads of NOD/SCID mice. However, loss of UNC45A did not affect the proliferation of normal mammary cells. Remarkably, UNC45A becomes more nuclear in human cancer tissues and cancer cell lines as compared to normal tissues and non-transformed Hs578Bst and HME mammary cell lines, respectively. This suggests an important nuclear function for UNC45A during tumorigenesis. Microarray analysis of mRNA from Hs578T cells showed that loss of UNC45A alters the expression of 121 genes, involved in cancer and cellular development and growth networks. Relevant to cell proliferation, we found that Nek7 gene was significantly repressed upon silencing UNC45A, which was validated by RTqPCR and Western blot analyses in multiple breast cancer cell lines. Nek7 is a member of the NIMA (never in mitosis, gene A) family of serine/threonine kinases. It plays a key role in centrosomal separation during mitosis. This correlates neatly with our observation that loss of UNC45A causes a centrosomal separation defect, cell proliferation arrest and death of breast cancer cell lines. ChIP experiments showed that UNC45A binds to the promoter of the Nek7 gene, suggesting direct transcriptional regulation. Interestingly, the UNC45A sequence contains four LxxLL motifs, which are thought to be signatures for co-activator binding to nuclear receptors. Furthermore, computational analysis identified two glucocorticoid response elements (GRE) consensus sequences in the Nek7 promoter, suggesting its transcriptional regulation by the glucocorticoid receptor (GR). This hypothesis was further strengthened by a significant decrease in the mRNA and protein levels of Nek7 upon silencing GR. Thus, our data suggest that UNC45A functions as a GR co-activator to control Nek7 gene transcription. Consistent with this, immunoprecipitation experiments confirmed that UNC45A and GR form endogenous complexes, and treatment of Hs578T and MCF7 cell lines with dexamethasone upregulates Nek7 mRNA and protein levels. In conclusion our data strongly support the premise that UNC45A promotes Nek7 transcription through activation of GR, and thus controls centrosomal separation and cancer cell proliferation. Citation Format: Yasmeen Jilani, Nada H. Eisa, Kashish Kainth, Sumin Lu, Nehal M. Elsherbiny, Laila A. Eissa, Mamdouh M. Elshishtawy, Hasan Korkaya, Abdeljabar El Andaloussi, Ahmed Chadli. The co-chaperone UNC45A controls cancer cell proliferation through Nek7 and centrosomal separation [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 4493. doi:10.1158/1538-7445.AM2017-4493