Raphael Sandaltzopoulos, PhD, MBA Lab. of Gene Expression, Molecular Diagnosis and Modern Therapeutics, MBG, DUTH BIOMARKERS DISCOVERY: Searching for ovarian cancer biomarkers Raphael Sandaltzopoulos, PhD, MBA Professor in Molecular Biology rmsandal@mbg.duth.gr

Identification of Biomarkers in ovarian cancer

Ovarian Cancer 2010 Estimated US Cancer Deaths The fifth most common type of cancer in women and the leading cause of death from gynecological malignancies. The high mortality rate of ovarian cancer is due to the lack of early clear symptoms. At the time of diagnosis the tumor is usually metastatic. Mutations in a plethora of genes (e.g. p53, BRCA1, PTEN) have been implicated in ovarian carcinogenesis.

TIL- TIL+ MIX ADDER (Amplification of Double-stranded cDNA End Restriction Fragments) An advanced Differential Display methodology incorporating elements of the RFLP technique.

Significant Gene Ontology terms (GO) and the corresponding genes as identified using DAVID v6.7 (N: number of genes) Category Markers of immune responses 16 Transcription Factors/Gene expression 33 Kinases/ Phosphatases 4 Extracellular space proteins 13 TMEM132D GPC6 CXCL9 MEIS 1 SMARCE1 TTF1

GPC6 is over-expressed in TIL+ Epithelial Ovarian Cancer GPC6: Glypican 6 Glycosylphosphatidylinositol-anchored heparan sulfate proteoglycan. The glypicans have been implicated in the control of cell growth and division. GPC 6 is a putative cell surface co-receptor for growth factors, extracellular matrix proteins, proteases and anti-proteases. GPC6 may be secreted upon digestion. It is involved in Notch, Wnt and Hedgehog signaling pathways.

TMEM132D is over-expressed in TIL+ Epithelial Ovarian Cancer Single-pass type I transmembrane protein. May serve as a cell-surface marker for oligodendrocyte differentiation. Implicated in anxiety disorders.

Kaplan-Meier survival function of GPC6/TMEM132D levels (2 groups)

CXCL9 is over-expressed in TIL+ Epithelial Ovarian Cancer

Ιn vitro chemotaxis assay CD8+ T-Lymphocytes Incubate and count cells at bottom Ovarian tumor cell line

Transfection of CXCL9 induces chemotaxis of CD8+ T-lymphocytes 1. Isolate CD8+ T-lymphocytes from healthy donors using anti-CD8 magnetic Dynabeads. 2. Place lymphocytes in upper chamber. 3. Place conditioned medium (from transfected SKOV cells supernatant) in lower chamber. 4. Incubate and then count lymphocytes that have migrated to lower chamber through the membrane.

SMARCE1 is over-expressed in TIL+ Epithelial Ovarian Cancer SMARCE1 (BAF57): SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily e, member 1. SMARCE1 is part of the large ATP-dependent chromatin remodeling complex SWI/SNF. It posesses an HMG domain and interacts with 4-way junction DNA. Coordinates SWI/SNF function with cell-cycle. Its deregulation predisposes to metastasis. Induces apoptosis by stimulating expression of the cylindromatosis tumor suppressor gene.

Overexpression of SMARCE1 induces CD8+ T-cell chemotaxis as efficiently as CXCL9 in a transwell assay

SMARCE1 transfection induces secretion of three chemokines but not CXCL9

SMARCE1 expression correlates with MIP1β and RANTES mRNA in tumor samples

MEIS1 is over-expressed in TIL+ Epithelial Ovarian Cancer Three amino-acid loop extension (TALE) homeobox protein. Homodimerizes and heterodimerizes with PBX1 (also homeobox protein). Involved in hemopoiesis and angiogenesis.

Correlation of MEIS1 mRNA levels with CXCL9 in tumors CXCL9 gene promoter bears MEIS1 recognition elements Hypothesis: Is CXCL9 transcription regulated by MEIS1?  Overexpression of MEIS1 should also trigger chemotaxis.

MEIS1 induces CD8+ T-lymphocyte chemotaxis in vitro If CXCL9 expression depends on MEIS1, then they should ΝΟΤ have additive effect!

Additive effect of MEIS1 and CXCL9 on CD8+ T-cell chemotaxis Our data suggest that the MEIS1-dependent chemotaxis pathway is not related to the CXCL9-dependent chemotaxis pathway!

MEIS1 induces the secretion of six chemokines, but not CXCL9

Correlation of MEIS1 mRNA levels with MIP1B and PARC in ovarian tumors Does MEIS1 transcription factor directly regulate MIP1B and PARC? Can we detect MEIS1 binding on their promoters?

Overexpression and purification of recombinant MEIS1 in E. coli

Binding of recombinant MEIS1 on PARC/CCL18 promoter in vitro (EMSA) Specific probe Scrambled probe

Binding of MEIS1 on PARC/CCL18 promoter in vivo (ChIP) Anti-RNA Pol II DNA ladder Neg. control IgG control Input (1%) GAPDH Binding of MEIS1 on PARC/CCL18 promoter in vivo (ChIP) TSS -100 -200 -300 -400 AAATATCTGTCATGTGA AGCCCCTCAATCAACAA ACCCTAAACCTTTCAAG PARC/CCL18 promoter

Binding of MEIS1 on MIP1b/CCL4 promoter in vivo (ChIP) TSS -600 -400 -200 ATCTTGATTCAAATCTT GGAAAGACTGACATGAA MEIS1 binds to one of the two promoter regions that harbor MEIS1 cognate elements.

CONCLUSIONS We have identified genes associated with CD8+ T-cell infiltration in early stage ovarian cancer by a differential display assay. We have validated TTF1, CPG6, TMEM132D, SMARCE1 and MEIS1 as CD8 predictors. High levels of CPG6 and TMEM132D correlate with better prognosis. SMARCE1 and MEIS1 induce CD8+ T lymphocytes chemotaxis in an in vitro transwell system. SMARCE1 induces the secretion of chemokines CCL5, CCL4 (and IL8?). MEIS1 induces the secretion of PARC/CCL18, MIP1β/CCL4, RANTES/CCL5, GROa, IL8, NAP2/CXCL7. MEIS1 binds to CCL18, CCL4 and CCL7 promoters.