Kristi A. Egland

Discovery of the breast cancer gene BASE using a molecular approach to enrich for genes encoding membrane and secreted proteins

To identify unknown membrane proteins that could be used as targets for breast and prostate cancer immunotherapies and secreted proteins to be used as diagnostic markers, a cDNA library was generated from membrane-associated polyribosomal RNA derived from four breast cancer cell lines, one normal breast cell line, and a prostate cancer cell line. The membrane-associated polyribosomal cDNA library was subtracted with RNA from normal brain, liver, lung, kidney, and muscle. Of the 15,581 clones sequenced from the subtracted cDNA library, sequences from 10,506 clones map to known genes, but 5,075 sequences, representing 3,181 unique transcripts, are not associated with known genes. As one example, we experimentally investigated expression of a previously uncharacterized breast cancer gene that encodes a secreted protein designated BASE (breast cancer and salivary gland expression). BASE is expressed in many breast cancers but not in essential normal tissues including the five organs used for subtraction. Further analysis of this library should yield additional gene products of use in the diagnosis or treatment of breast or prostate cancer.

Multiple functions of Sushi Domain Containing 2 (SUSD2) in breast tumorigenesis

Routinely used therapies are not adequate to treat the heterogeneity of breast cancer, and consequently, more therapeutic targets are desperately needed. To identify novel targets, we generated a breast cancer cDNA library enriched for genes that encode membrane and secreted proteins. From this library we identified SUSD2 (Sushi Domain Containing 2), which encodes an 822-amino acid protein containing a transmembrane domain and functional domains inherent to adhesion molecules. Previous studies describe the mouse homolog, Susd2, but there are no studies on the human gene associated with breast cancer. Immunohistochemical analysis of human breast tissues showed weak or no expression of SUSD2 in normal epithelial cells, with the endothelial lining of vessels staining positive for SUSD2. However, staining was observed in pathologic breast lesions and in lobular and ductal carcinomas. SUSD2 interacts with galectin-1 (Gal-1), a 14-kDa secreted protein that is synthesized by carcinoma cells and promotes tumor immune evasion, angiogenesis, and metastasis. Interestingly, we found that localization of Gal-1 on the surface of cells is dependent on the presence of SUSD2. Various phenotype assays indicate that SUSD2 increases the invasion of breast cancer cells and contributes to a potential immune evasion mechanism through induction of apoptosis of Jurkat T cells. Using a syngeneic mouse model, we observed accelerated tumor formation and decreased survival in mice with tumors expressing Susd2. We found significantly fewer CD4 tumor infiltrating lymphocytes in mice with tumors expressing Susd2. Together, our findings provide evidence that SUSD2 may represent a promising therapeutic target for breast cancer. Mol Cancer Res; 11(1); 74–85. ©2012 AACR.

SUSD2 promotes tumor-associated macrophage recruitment by increasing levels of MCP-1 in breast cancer

Tumor-associated macrophages (TAMs) play a role in tumor angiogenesis and are recruited into the tumor microenvironment (TME) by secreted chemokines, including Monocyte Chemoattractant Protein-1 (MCP-1/CCL2). Angiogenesis is required to sustain proliferation and enable metastasis of breast cancer (BCa) cells. Understanding the underlying mechanisms of TAM recruitment would allow for the identification of desperately needed novel drug targets. Sushi Domain Containing 2 (SUSD2), a transmembrane protein on BCa cells, was previously shown to promote tumor angiogenesis in a murine model. To identify the role of SUSD2 in angiogenesis, 175 human breast tumors were surveyed by immunohistochemical analysis for the presence of SUSD2 and macrophages. Tumors with high levels of SUSD2 staining contained 2-fold more TAMs, mainly of the M2 pro-angiogenic phenotype. An in vitro co-culture model system was developed by differentiating SC monocytes into SC M0 macrophages. A 2-fold increase in polarized M2 macrophages was observed when M0 macrophages were incubated with SUSD2-expressing BCa cells compared to cancer cells that do not contain SUSD2. Since MCP-1 is known to recruit macrophages, levels of MCP-1 were compared between SUSD2-expressing MDA-MB-231 and MBA-MB-231-vector control cell lines. MCP-1 RNA, intracellular protein and secreted MCP-1 were all significantly increased compared to the vector control. Knockdown of SUSD2 in SKBR3 resulted in significantly decreased levels of secreted MCP-1. Consistently, increased levels of MCP-1 were observed in Susd2-expressing tumors generated from an in vivo isogeneic mouse model compared to the vector control tumors. Because SUSD2 recruits macrophages into the TME and promotes M2 polarization, inhibiting the function of SUSD2 may be an effective therapy for breast cancer patients.

In vitro and in vivo evaluation of effect of excipients in local delivery of paclitaxel using microporous infusion balloon catheters

Drug-infusion balloons are one of the currently used local drug delivery devices for preventing restenosis after endovascular treatments. An antiproliferative drug (paclitaxel, PAT) is infused through the balloon using a cremophor-based formulation to control restenosis. However, the major limitations of this approach are poor in vivo drug uptake and a limit in the amount of PAT delivered because of cremophor toxicity. In this study, we have investigated the use of different excipients for effectively infusing PAT out of the balloon for improved drug uptake in the tissue. The excipients include nanoparticle albumin-bound PAT (nab-PAT, a nanobiomaterial used in cancer therapy), urea (a hydrophilic agent used for faster drug transfer), iodixanol (a contrast agent used for coronary angiography), and cremophor-PAT (the most commonly used PAT formulation). An in vitro drug release, smooth muscle cell (SMC) response, endothelial cell (EC) response, and in vivo drug uptake were investigated for all the different excipients of PAT infused through the balloon. The nab-PAT was as effective as cremophor in infusing out of the balloon and inhibiting SMC growth. Also, nab-PAT showed a significantly greater amount of in vivo PAT uptake than that of cremophor-PAT. Urea and iodixanol were not effective in delivering a clinically relevant dose of PAT due to the poor solubility of PAT in these excipients. Urea eradicated all the SMCs and ECs, suggesting a toxic effect, which impedes its use in balloon-based therapy. Thus, this study demonstrated that nab-PAT is an effective formulation to locally deliver PAT through infusion balloons.

Abstract 301A: A novel epithelial ovarian cancer protein, SUSD2, inhibits platelet activation and binding to tumor cells

Over 30% of ovarian cancer (OvCa) patients present with thrombocytosis (elevated plasma platelet count above 450,000 per cubic millimeter) at the time of diagnosis. These patients exhibit shorter survival times and a higher likelihood of advanced stage disease. Platelets have also been shown to enhance metastasis and promote tumor cell survival by coating tumor cells which provides immune escape and protection from chemotherapeutic agents. While previous studies have shown that platelets exhibit differential OvCa cell binding and facilitate cancer cell growth, the mechanism and molecules involved have yet to be elucidated. SUSD2 (SUShi Domain containing 2), an 822 amino acid type I transmembrane protein, is highly abundant in ovarian tumors and normal endothelial cells that line the blood vessels. Endothelial cells do not normally adhere to platelets; therefore, we hypothesized that SUSD2 inhibits the binding of platelets to cancer cells, which subsequently decreases platelet activation. To investigate the role of SUSD2 in platelet binding, SUSD2 knock-down (SUSD2neg) and non-targeting (SUSD2pos) OVCAR3 cell lines were generated. SUSD2pos and SUSD2neg OVCAR3 cell lines were cultured and grown to confluency. Platelets were isolated, dyed with Calcein-AM, washed and added to the cancer cells. After a 15-minute incubation, the co-culture was washed to remove non-adherent platelets. We demonstrated that platelets were bound to the OVCAR3-SUSD2neg an average of 35% more compared to OVCAR3-SUSD2pos cells. In order to identify the receptor on the platelets mediating adhesion to the tumor cells, we used the drug eptifibatide to block GPIIb/IIIa, an integrin receptor on the platelet surface that primarily binds to fibrinogen. Using the same methods described above, platelets were incubated with the inhibitor before being co-incubated with the cancer cells. Treating the platelets with eptifibatide decreased platelet binding by an average of 35% to both cancer cell lines, indicating that platelet binding to tumor cells is partially, yet equally, mediated by GPIIb/IIIa regardless of the presence of SUSD2. The role of this receptor was further explored by performing flow cytometry on the supernatant collected from co-culturing platelets with the cancer cells. The conditioned media contains the platelets that were not bound to the cancer cells. We measured the activation of the unbound platelets using the anti-PAC-1 antibody that binds exclusively to the activated conformation of GPIIb/IIIa. Incubation with the OVCAR-3-SUSD2neg cells activated the unbound platelets three-fold more compared to the OVCAR3-SUSD2pos cells. These data indicate that SUSD2 expression may mediate the activation state of GPIIb/IIIa, and this, in turn, may affect adhesion mediated by other factors. Citation Format: Tyson Lager, Megan Thacker, Charissa Etrheim, Kristi A. Egland, Mark K. Larson, Jennifer A. A. Gubbels. A novel epithelial ovarian cancer protein, SUSD2, inhibits platelet activation and binding to tumor cells. [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 301A. doi:10.1158/1538-7445.AM2015-301A

Status of HER-2 gene amplification in breast cancers from Native American women

In the United States, one in eight women will be diagnosed with breast cancer during her lifetime. The American Cancer Society estimates that in 2008, approximately 182,460 new female breast cancer cases will be diagnosed and 40,480 women will die from this disease [1]. However, breast cancer incidence and mortality rates in females vary across ethnic and racial groups. The National Cancer Institute’s Surveillance, Epidemiology, and End Results (SEER) program indicates that incidence rates for breast cancer among Native American women are significantly lower than the Caucasian population [2]. Yet, Native American women tend to present to their physician with larger, more advanced-stage tumors that lead to lower survival rates, when compared to their Caucasian counterparts [3][sn1]. Moreover, mortality rates for Native American women are not decreasing as they are for the Caucasian population [1,4]. It has been particularly difficult to study the molecular basis of breast cancer in the Native American people because the population is small and has limited access to healthcare. Therefore, this report describes a small cohort study of 60 breast cancer samples from Native American patients and determines the status of HER-2 amplification.

Identification of Novel Cancer Target Antigens Utilizing EST and Genome Sequence Databases

Completion of the human genome sequence has opened up an enormous opportunity to researchers all over the world. The Human Genome Project, which includes the expressed sequence tags (ESTs) database and the genome sequence database, provides a huge source of data that can be used to study and identify molecular targets for a wide range of diseases, including cancer. Major efforts must now be devoted to develop strategies by which these databases will be mined efficiently to identify the hidden therapeutic treasures. We have utilized the EST and genome databases, different bioinformatics tools, and several experimental methods to identify tissue-specific genes for prostate and breast cancer. The genes identified can be used as novel targets for the diagnosis and treatment of prostate and breast cancer.

Sushi Domain Containing 2 (SUSD2) inhibits platelet activation and binding to high-grade serous ovarian carcinoma cells

Abstract Platelets play a central role in primary hemostasis affecting tumor survival and metastases. Tumors induce platelets to aggregate and bind to the cancer cells, resulting in protection from immune surveillance and often leading to thrombocytosis. In ovarian cancer (OvCa), one-third of patients present with thrombocytosis, a diagnosis that correlates with shorter survival. SUSD2 (SUShi Domain containing 2), a type I transmembrane protein, shown to inhibit metastatic processes in high-grade serous ovarian carcinoma (HGSOC), is expressed on endothelial cells and thus may influence platelet reactivity. As such, we hypothesized that SUSD2 levels in ovarian cancer-derived cell lines influence platelet activation. We incubated OvCa non-targeting (NT) and SUSD2 knockdown (KD) cell lines with labeled platelets and quantified platelet binding, as well as GPIIb/IIIa integrin activation. The role of GPIIb/IIIa in tumor cell/platelet interaction was also examined by measuring cell–cell adhesion in the presence of eptifibatide. We found that platelets exposed to OvCa cells with low SUSD2 expression display increased tumor cell-platelet binding along with an increase in GPIIb/IIIa receptor activation. As such, platelet activation and binding to HGSOC cells was inversely correlated with the presence of SUSD2. This represents one of the first tumor proteins known to provide differential platelet interaction based on protein status.

Abstract POSTER-BIOL-1345: SUSD2 inhibits spheroids from breaching the mesothelium: a mechanism for increased longevity of patients with SUSD2-expressing high-grade ovarian serous carcinoma

The cause of death among the majority of epithelial ovarian cancer (EOC) patients involves metastasis and subsequent invasion of cancer cells into organs enclosed and adjacent to the abdominal cavity, which can lead to deadly health complications such as bowel obstruction. Thus, it is important to identify the factors that contribute to metastasis and invasion including those that facilitate adhesion to the mesothelium. The anatomical location of the ovary and the fallopian tube within the peritoneal cavity facilitates metastasis of ovarian cancer without involvement of the vasculature. EOC cells exfoliate from the fallopian tube or ovary, disseminate within the peritoneal cavity as free-floating single cells or spheroids and invade mesothelium-covered organs including the bowel. To identify potential immunotherapy targets for epithelial cancers, we generated a cDNA library enriched with genes encoding membrane and secreted proteins that are highly expressed in cancer with minimal expression in normal essential tissues. From this library we identified Sushi Domain Containing 2 (SUSD2), a type I transmembrane protein that localizes to the cell surface and contains several functional domains inherent to adhesion molecules. To determine the expression pattern of SUSD2 in normal and carcinoma ovarian tissues, an immunohistochemical analysis was performed on ovarian tissue samples from patients using an anti-SUSD2 antibody. Weak to no staining was observed in normal epithelial cells. In contrast, various intensities of positive staining for SUSD2 were observed in several types of EOC, including serous, mucinous, endometrioid and transitional adenocarcinomas. A high-grade serous ovarian carcinoma (HGSOC) tissue microarray containing samples from 128 patients was stained with an anti-SUSD2 antibody. A pathologist scored the intensity of SUSD2 staining, and results were correlated with patient outcome. A Kaplan-Meier curve indicated a significant separation for patients with weak SUSD2 staining, median survival 31.7 months, versus patients with strong SUSD2 staining, median survival 49.1 months (p-value = 0.0083). This data suggests that low SUSD2 levels in HGSOC tumors are associated with a poorer prognosis for the patient. To investigate the role of SUSD2 in HGSOC, stable SUSD2 knock-down (KD) and non-targeting (NT) OVCAR3 cell lines were generated. Boyden chamber and wound healing assays were used to assess the effect of SUSD2 on migration. OVCAR3 SUSD2-KD cells migrated at significantly higher rates than the OVCAR3-NT cells suggesting that the presence of SUSD2 in OVCAR3 cells inhibits cell migration. In addition, attenuation of SUSD2 levels in OVCAR3 cells significantly increased mesothelial clearance. Altogether, our findings indicate that SUSD2 negatively impacts the ability of OVCAR3 cells to breach the mesothelium, which defines a mechanism for the increased longevity of patients with SUSD2-expressing HGSOC. Citation Format: Jordan N. Sheets, Marcin Iwanicki, Joyce Liu, Ronny Drapkin, Kristi A. Egland. SUSD2 inhibits spheroids from breaching the mesothelium: a mechanism for increased longevity of patients with SUSD2-expressing high-grade ovarian serous carcinoma [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr POSTER-BIOL-1345.

Abstract A71: Expression of Sushi Domain Containing 2 (SUSD2) in high-grade ovarian serous carcinoma correlated with increased longevity of patients

Despite research in therapeutic strategies, ovarian cancer (OvCa) remains the most lethal gynecologic malignancy. The anatomical location of the ovaries within the peritoneal cavity facilitates metastasis by allowing tumor cells sloughed from the main tumor on the ovary to bind to the peritoneum or organs within the peritoneal cavity. These tumor cells are released from the main tumor either as single cells or as spheroids, the latter of which have been shown to be more effective at metastasizing and invading into the mesothelial lining. To identify novel tumor markers and therapeutic targets for epithelial cancers, a cDNA library enriched for cancer genes that encode membrane and secreted proteins was generated. The 18th most abundant gene represented in this library was Sushi Domain Containing 2 (SUSD2) , which encodes an 822 amino acid membrane surface protein. To begin defining the role of SUSD2 in OvCa, stable SUSD2 -expressing and vector control A2780 cell lines were generated. To examine whether SUSD2 affects the growth of OvCa cells, cellular proliferation assays were performed. There was no significant difference in growth rates with SUSD2 over-expression compared to the vector control cell lines. A Boyden chamber assay was performed to analyze the effect of SUSD2 on cell migration. A2780-SUSD2 cell lines and vector-only controls were plated on membranes and allowed to migrate toward a chemoattractant in a lower chamber. SUSD2 over-expression significantly increased migration by greater than 2-fold. In addition, a scratch wound-healing assay was performed, and after 24 hours the A2780-SUSD2 cell line had 2-fold more cells within the wound compared to the A2780-vector control cells (P<0.05). Consistently, agarose beads containing either A2780-SUSD2 or vector cells were grown in chemoattractant containing media. The distance the escaped cells traveled from the bead was measured. A2780-SUSD2 cells traveled farther away from the bead compared to the vector control (717 μm compared to 125 μm, respectively, P<0.05). Spheroid-forming ability was analyzed, and A2780-SUSD2 cells formed smaller and looser spheroids (length and width average of 933 μm) compared to A2780-vector control cells (length and width average of 1237 μm, P=0.025). To correlate OvCa patient outcome with the presence of SUSD2 in the corresponding tumor, a tissue microarray containing high-grade ovarian serous carcinoma core samples from 128 patients was stained with an anti-SUSD2 antibody. The intensity of SUSD2 staining was scored by a pathologist, and results were correlated with patient outcome. A Kaplan-Meier curve indicated a significant separation for patients with low SUSD2 levels, median survival 31.7 months, versus patients with strong SUSD2 staining, median survival 49.1 months (P=0.0083). This data suggests that low SUSD2 levels in OvCa tumors are associated with a poorer prognosis for the patient. Our in vitro assays indicate that expression of SUSD2 in A2780 cells increased the ability of the cells to migrate and caused the formation of smaller spheroids. Because spheroids are better able to resist chemotherapy and invade the mesothelium, the decreased spheroid size of SUSD2 -expressing cells may allow for a better patient prognosis. Citation Format: Jennifer A. A. Gubbels, Elizabeth M. Hultgren, Jessica Johnson, Emily Johnson, Jeffrey Sachs, Michelle S. Hirsch, Joyce Liu, Ronny Drapkin, Kristi A. Egland. Expression of Sushi Domain Containing 2 (SUSD2) in high-grade ovarian serous carcinoma correlated with increased longevity of patients. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr A71.