The Xu Lab

The lab is focused on two very critical areas of cancer research: tumor metastasis and tumor-stroma interaction/tumor microenvironment. More recently, through genetic, cell biological and biochemical analyses, the team has obtained first line evidence that ZIP4 has both metal-transporter functions and non-transporter functions. Through genetic, cell biological and biochemical analyses, the lab identified several ZIP4-related regulatory and functional processes not previously known to involve ZIP4. Specifically, the team has shown that ZIP4, along with SOX9, was upregulated by LPA via PPAR? in epithelial ovarian cancer cells.

ZIP4 is required for LPA-induced upregulation of the EOC side population cells, which are an enriched source of cancer stem cells (CSC) and progenitor cells. Using CRISPR-mediated knockout (KO) cell lines for ZIP4, SOX9, and PPAR?, the lab has shown that ZIP4 is functionally involved in CSC-related cellular activities including proliferation, anoikis-resistance, colony-formation, spheroid-formation, drug-resistance, and the EOC side population (SP) in human high-grade serous ovarian cancer (HGSOC) cells. Three new findings were particularly noteworthy.

1. ZIP4 behaved as a bond fide oncogene, being able to transform human ovarian surface epithelial (HOSE) cells and potentially fallopian tube epithelial (FTE) cells.
2. ZIP4+, but not ZIP4– cells were able to initiate tumor and ascites formation with relatively low cell numbers in short time.
3. ZIP4 has both extracellular zinc- dependent and -independent activities and detected the novel ZIP4-Notch3 interaction.

LPA’s Functional Involvement in EOC

The Xu Laboratory’s early and seminal contributions to the cancer field were the discovery that a small lipid factor lysophosphatidic acid (LPA) is functionally involved in epithelial ovarian cancer (EOC). We have pioneered research in the role of lipid-signaling molecules in ovarian and other cancers and identified the first lipid growth factor in EOC ascites. We have shown that LPA stimulates proliferation of EOC cells, and are among the first group of investigators to show LPA’s activity in cell migration and invasion of EOC cells in vitro and tumor metastasis in vivo. Lipids had been mainly considered as cell membrane building blocks. The important cell signaling roles of lysophospholipids, including LPA, have only been significantly recognized in recent decades. However, prior to the lab’s work (published in 1995), the roles and signaling pathways of LPA were mainly studied in model systems, such as NIH3T3 cells. To date, more than 1000 papers have been published on LPA’s roles and/or signaling in various cancers and LPA has been considered as an oncolipid in EOC and other cancers.

1232.
Xu Y., Gaudette DC, Boynton J, Frankel A, Fang X-J, Sharma A, Hurteau J, Casey G, Goodbody AE, Mellors A, Holub BJ Mills G. (1995) Characterization of an Ovarian Cancer Activating Factor(OCAF) in Ascites from Ovarian Cancer Patients. Clinical Cancer Res. 1, 1223-1232. 1995. PMID: 9815916.

1233.
Sengupta S, Xiao Y, and Xu Y. A novel laminin-induced LPA autocrine loop in the migration of ovarian cancer cells. FASEB J. 17, 1570-1572, 2003. PMID: 12824286.

1234.
Kim KS, Sengupta S, Berk M, Kwak YG, Escobar PF, Belinson J, Mok SC, Xu Y. Hypoxia Enhances Lysophosphatidic Acid Responsiveness in Ovarian Cancer Cells and Lysophosphatidic Acid Induces Ovarian Tumor Metastasis In vivo. Cancer Res. 2006 66:7983-90. PMID: 16912173.

Xu, Y. Lysophospholipid Signaling in the Epithelial Ovarian Cancer Tumor Microenvironment. Cancer (Basel). 2018 Jul 9;10(7). pii: E227. doi: 10.3390/cancers10070227. PMID: 29987226. 

LPA/LPC and PLA2 Activity as Cancer Markers

The lab was the first to demonstrate that LPA is a potential marker for the early detection of ovarian cancer and lysophosphatidylcholine (LPC) is a useful marker for colorectal and other cancers. EOC is the most deadly gynecologic disease, mainly due to the lack of highly sensitive and specific methods for early detection and effective treatments for late-stage and recurrent diseases.

In addition, the lab has demonstrated that the enzymatic activities of several phospholipase A2s, critical enzymes regulating the levels of LPC and LPA, are markers for colorectal cancer (CRC), lung, bladder, and pancreatic cancers. The development of early detection markers for EOC and other cancers has the potential to save many lives. Our findings have been confirmed by independent and blinded studies internationally. The group has also developed highly effective electrospray ionization mass spectrometry (ESI-MS)-based lipid analysis and simplified lipid extraction methods to analyze lysophospholipid levels in biological fluids (such as blood, ascites, peritoneal washings, urine, cells, and cell supernatants). These methods are highly sensitive, quantitative and reproducible.

The lab has significantly contributed to the lipidomic field and has published many individual and collaborative papers using this cutting-edge technology. More than 10 papers published around the world have confirmed that blood LPA is a marker for EOC, including PMID: 19051824 and two very recent papers published: a large-scale study including 980 OC patients, 872 benign controls and 668 healthy controls (PMID26174150), and a comparative study showing that LPA shows greater value in the diagnosis of ovarian cancer compared to CA-125 and may be employed as a biological index to diagnose ovarian cancer (PMID:26148603).

723.
Xu, Y., Shen, Z., Wiper, DW, Wu. M., Morton, RE., Elson, P., Kennedy, AW., Belinson, J., Markman, M. and G. Casey (1998) Lysophosphatidic acid as a potential biomarker for ovarian and other gynecologic cancers. JAMA 280(8): 719-723. PMID: 9728644.

724.
Sutphen R, Xu Y., Wilbanks GD, Fiorica J, Grendys EC Jr, LaPolla JP, Arango H, Hoffman MS, Martino M, Wakeley K, Griffin D, Blanco RW, Cantor AB, Xiao YJ, Krischer JP. Lysophospholipids are potential biomarkers of ovarian cancer. Cancer Epidemiol Biomarkers Prev 2004 13, 1185-1191, 2004. PMID: 15247129.

725.
Zhenwen Zhao, Yijin Xiao, Paul Elson, Haiyan Tan, Sarah J. Plummer, Michael Berk, Phyu P. Aung, Ian C. Lavery, Jean P. Achkar, Li Li, Graham Casey, and Xu, Y. Plasma lysophosphatidylcholine levels: potential biomarkers for colorectal cancer. J Clin Oncol. 25, 2696-701. 2007. PMID: 17602074. 2007 Jul 1.

Hui Cai, E. Gabriela Chiorean, Michael V. Chiorean, Douglas K. Rex, Bruce W. Robb, Noah M. Hahn, Ziyue Liu, Patrick J. Loehrer, Marietta L. Harrison, and Xu Y. Elevated phospholipase A2 activities in plasma samples from multiple cancers. PloSOne, 2013;8(2):e57081. doi: 10.1371/journal.pone.0057081. Epub 2013 Feb 22, PMID:23451150. 

Mechanistic Studies of Bioactive Lipid Signaling

The lab has identified several novel signaling pathways, mechanisms, and targets of bioactive lipids in EOC and other cancers. These lipids include LPA, sphingosine-1-phosphate (S1P), LPC, and sphinosylphosphorylchoine (SPC).

2006.
Xiaoxian Zhao, Dongmei Wang, Zhenwen Zhao, Yingyi Xiao, Saubhik Sengupta, Yijin Xiao, Renliang Zhang, Kirsten Lauber, Sebastian Wesselborg, Li Feng, Tyler M. Rose, Yue Shen, Junjie Zhang, Glenn Prestwich and Xu Y. Caspase-3 dependent activation of calcium-independent phospholipase A2 enhances cell migration in non-apoptotic ovarian cancer cells. Biol. Chem. 281(39):29357-68, 2006. PMID: 16882668.

2007.
Saubhik Sengupta, Kim KS, Berk MP, Oates R, Escobar P, Belinson J, Li W, Lindner DJ, Williams B, Xu Y.  Lysophosphatidic acid down regulates tissue inhibitor of metalloproteinases (TIMPs), which are negatively involved in LPA-induced cell invasion. 26, 2894-901. 2007. PMID: 17130843.

2008.
Hui Cai and Xu Y. The role of LPA and YAP signaling in long-term migration of human ovarian cancer cells. Cell Communication and Signaling, 2013 Apr 24;11(1):31. doi: 10.1186/1478-811X-11-31. PMID: 23618389.

Qipeng Fan, Qingchun Cai, and Xu, Y. FOXM1 is a downstream target of LPA and YAP oncogenic signaling pathways in high grade serous ovarian cancer. Oncotarget, 2015 Jun 13. [Epub ahead of print]; PMID:26299613. 

Tumor Metastasis and Tumor Microenvironment

The lab has also contributed significantly to tumor metastasis and the tumor microenvironment field, including testing the gene functions in both tumor and host cells and demonstrating co-targeting efficacies both in vitro and in vivo. 

2006.
Juan Ren, Yi-jin Xiao, Lisam Shanjukumar Singh, Xiaoxian Zhao, Zhenwen Zhao, Li Feng, Tyler M. Rose, Glenn D. Prestwich, Xu Y, LPA is constitutively produced by human peritoneal mesothelial cells and enhances adhesion, migration and invasion of ovarian cancer cells. Cancer Res. 66, 3006-3014, 2006. PMID: 16540649.

2007.
Li H, Zhao Z, Wei G, Yan L, Wang D, Zhang H, Sandusky GE, Turk J, Xu Y. Group VIA phospholipase A2 in both host and tumor cells is involved in ovarian cancer development. FASEB J. 2010 Jun 8. [Epub ahead of print] PubMed PMID: 20530749.

2008.
Hui Li, Zhenwen Zhao, Caryl Antalis, Zhanzhong Zhao, Robert Emerson, Gang Wei, Sheng Zhang, Zhong-Yin Zhang, and Xu Y. Combinational therapy of an inhibitor of Group VIA phospholipase A2 with paclitaxel is highly effective in blocking ovarian cancer development. Am J Pathol. 2011 Jul;179(1):452-61. Epub 2011 May 17. PMID: 21703423.

Cai Q, Zhao Z, Antalis C, Yan L, Del Priore G, Hamed AH, Stehman FB, Schilder JM, Xu Y. Elevated and secreted phospholipase A2 activities as new potential therapeutic targets in human epithelial ovarian cancer. FASEB J. 2012 Jul 5. [Epub ahead of print]  PMID: 22767227. 

EOC Aggressiveness, Drug-Resistance, and Cancer Stem Cell (CSC) Properties

Recently, the lab has been focusing on delineating the genes involved in cancer aggressiveness, drug-resistance, and CSC. Identification of driver genes/proteins in EOC tumorigenic processes is critical to increasing the cure rate for EOC, which has not been improved for several decades. While the TCAG data generated from large-scale integrated genomic analyses have set the genetic landscape of EOC, the driver genes, functional players, and critical regulators for each step of EOC development are still largely unknown and need to be functionally determined. In addition, although many in vitro studies have been conducted to identify functional genes involved in EOC, in vivo approaches are more clinically relevant, due to the pivotal contributions of the host system (tumor microenvironment).

With these rationales in mind, the lab has developed a highly aggressive EOC cell line (ID8-P1 or P1) from ID8-P0 (without in vivo passage, or P0) through in vivo passage in C57BL6 mice. ID8 syngeneic mouse EOC line was obtained through spontaneous transformation of normal ovarian surface epithelial cells from C57BL6 mice by repetitive passage in vitro. The time to tumor/ascites formation and mouse morbidity was reduced from ~90 days for ID8-P0 cells to ~ 30 days in P1 cells. The lab has shown that the human EOC cell line shares functional and signaling similarities to those in the ID8-P0 and –P1 pair (refs. see below). In addition, the group has generated a pair of in vivo-passaged human EOC cells from the serous EOC cell line Caov3, which share many similarities to the ID8-P0 and –P1 pair (unpublished data).

The lab has taken advantage of the development of high-throughput sequencing technologies and innovative bioinformatics analyses in recent years to obtain more complete and systematic data on mRNA expression changes, splicing pattern changes, and alteration in non-coding RNAs in the pair of ID8-P0 and -P1 cells (to be published elsewhere). The duplicates displayed highly consistent results. The lab has confirmed the results using RT-qPCR, Western blot analyses, and/or ELISA assays in ID8 and human EOC cells for more than 40 genes identified to be up-regulated in P1 vs. P0 cells in the RNAseq data (to be published elsewhere).

ZIP4 is the top gene altered (upregulation 182 fold in ID8-P1 vs. ID8-P0 cells). In particular, the genes in recent publications (see below), those shown in the preliminary data, and genes to be studied in this proposal are significantly up-regulated in ID8-P1 vs. ID8-P0 cells as detected in the RNAseq data. This list includes (numbers are fold increase) C-Kit (CD117), 44.3; Epcam, 4.9; Notch3, 11.2 (but not Notch1, which expressed at very low levels in both ID8-P1 vs. ID8-P0 cells, as well as PE01 and PE04 cells); Aldh1, 8.8; Sox9, 2.6; Cd44, 1.4, Notch target genes [85]: Hes1, 2.1; Cdkn1a,2.6; Dtx1, 24.5; and Cflar,1.4. All of these changes have a P value < 10-4. Ppar? is also a Notch target gene, which was 2.65-fold upregulated (P=0.01). These results indicate that high-quality RNAseq data are invaluable in guiding the functional and mechanistic studies.

1. Qingchun Cai1*, Libo Yan1, and Xu Y Anoikis resistance is a critical feature of highly aggressive ovarian cancer cells. Oncogene. 2014 Aug 18;0. doi: 10.1038/onc.2014.264. [Epub ahead of print]; PMID: 25132267.
2. Qingchun Cai and Xu, Y. Microenvironment reprograms the circuits in tumor cells. Molecular & Cellular Oncology. 20152:1, e969634, DOI: 10.4161/23723548.2014.969634.
3. Qipeng Fan, Qingchun Cai, and Xu Y. LPA Regulates SOX9 in Ovarian Cancer Cells Journal of Obstetrics and Gynecology: Received Date: 30 March, 2017; Accepted Date: 3 June, 2017; Published Date: 23 June, 2017.
4. Qipeng Fan, Qingchun Cai, Pengfei Li, Wenyan Wang, Jing Wang, Emily Gerry, Tian-Li Wang, Ie-Ming Shih, Kenneth P. Nephew, and Xu Y. “The novel ZIP4 regulation and its role in ovarian cancer” [https://www.ncbi.nlm.nih.gov/pubmed/?term=29163813] 2017 Sep 30;8(52):90090-90107. PMID:29163813.