Tumor cell extravasation is a key step during cancer metastasis, yet the precise mechanisms that regulate this dynamic process are unclear. We utilized a high-resolution time-lapse intravital imaging approach to visualize the dynamics of cancer cell extravasation in vivo. During intravascular migration, cancer cells form protrusive structures identified as invadopodia by their enrichment of MT1-MMP, cortactin, Tks4, and importantly Tks5, which localizes exclusively to invadopodia. Cancer cells extend invadopodia through the endothelium into the extravascular stroma prior to their extravasation at endothelial junctions. Genetic or pharmacological inhibition of invadopodia initiation (cortactin), maturation (Tks5), or function (Tks4) resulted in an abrogation of cancer cell extravasation and metastatic colony formation in an experimental mouse lung metastasis model. This provides direct evidence of a functional role for invadopodia during cancer cell extravasation and distant metastasis and reveals an opportunity for therapeutic intervention in this clinically important process.

Kisspeptins (KPs), peptide products of the KISS1 metastasis-suppressor gene, are endogenous ligands for a G protein-coupled receptor (KISS1R). KISS1 acts as a metastasis suppressor in numerous human cancers. However, recent studies have demonstrated that an increase in KISS1 and KISS1R expression in patient breast tumors correlates with higher tumor grade and metastatic potential. We have shown that KP-10 stimulates invasion of estrogen receptor ␣ (ER␣)-negative MDA-MB-231 breast cancer cells via transactivation of the epidermal growth factor receptor (EGFR). Here, we report that either KP-10 treatment of ER␣-negative nonmalignant mammary epithelial MCF10A cells or expression of KISS1R in MCF10A cells induced a mesenchymal phenotype and stimulated invasiveness. Similarly, exogenous expression of KISS1R in ER␣-negative SKBR3 breast cancer cells was sufficient to trigger invasion and induced extravasation in vivo. In contrast, KP-10 failed to transactivate EGFR or stimulate invasiveness in the ER␣-positive MCF7 and T47D breast cancer cells. This suggested that ER␣ negatively regulates KISS1R-dependent breast cancer cell migration, invasion, and EGFR transactivation. In support of this, we found that these KP-10-induced effects were ablated upon exogenous expression of ER␣ in the MDA-MB-231 cells, by down-regulating KISS1R expression. Lastly, we have identified IQGAP1, an actin cytoskeletal binding protein as a novel binding partner of KISS1R, and have shown that KISS1R regulates EGFR transactivation in breast cancer cells in an IQGAP1-dependent manner. Overall, our data strongly suggest that the ER␣ status of mammary cells dictates whether KISS1R may be a novel clinical target for treating breast cancer metastasis. (Endocrinology

The interaction of hyaluronan (HA) with mesenchymal progenitor cells impacts trafficking and fate after tissue colonization during wound repair and these events contribute to diseases such as cancer. How this interaction occurs is poorly understood. Using 10T½ cells as a mesenchymal progenitor model and fluorescent (F-HA) or gold-labeled HA (G-HA) polymers, we studied the role of two HA receptors, RHAMM and CD44, in HA binding and uptake in non-adherent and adherent mesenchymal progenitor (10T½) cells to mimic aspects of cell trafficking and tissue colonization. We show that fluorescent labeled HA (F-HA) binding/uptake was high in non-adherent cells but dropped over time as cells became increasingly adherent. Non-adherent cells displayed both CD44 and RHAMM but only function-blocking anti-RHAMM and not anti-CD44 antibodies significantly reduced F-HA binding/uptake. Adherent cells, which also expressed CD44 and RHAMM, primarily utilized CD44 to bind to F-HA since anti-CD44 but not anti-RHAMM antibodies blocked F-HA uptake. RHAMM overexpression in adherent 10T½ cells led to increased F-HA uptake but this increased binding remained CD44 dependent. Further studies showed that RHAMM-transfection increased CD44 mRNA and protein expression while blocking RHAMM function reduced expression. Collectively, these results suggest that cellular microenvironments in which these receptors function as HA binding proteins differ significantly, and that RHAMM plays at least two roles in F-HA binding by acting as an HA receptor in non-attached cells and by regulating CD44 expression and display in attached cells. Our findings demonstrate adhesion-dependent mechanisms governing HA binding/ uptake that may impact development of new mesenchymal cell-based therapies.

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Metastasis in prostate cancer is caused by genetic reprogramming leading to increased cell motility and the formation of invasive structures which allow cells to invade out of the primary tumour in a process known as intravasation. In order to search for novel genes inhibiting invasive phenotypes responsible for intravasation, a genome-wide high throughput short hairpin RNA screen was used to identify genes which suppress aggressive phenotypes in BPH (Benign Prostatic Hyperplasia) prostate epithelial cells when grown in 3D matrigel culture conditions. Using the The RNAi Consortium (TRC) lentiviral shRNA library that covers the entire human transcriptome (80,000 unique constructs), BPH cells were infected such that each cell would integrate a single shRNA construct that will target mRNA expression of a known human gene. Cells were plated at 4500 cells per cm2 until a 2.5X genome-wide coverage was achieved. In 3D matrigel culture conditions, BPH colonies exhibit normal epithelial (round) colony morphology. Hence, we screened for clonogenic BPH colonies that appeared fibroblastic and spindle-shaped. Collection of these “hits” yield gene identities that suppress intravasation. In this ongoing study, we have achieved 2.5x coverage of the TRC shRNA library in BPH cells. After 11 days of growth, 7 “hits” from the BPH screen that exhibit an invasive phenotype were isolated, and were validated again in 3D matrigel culture. These “hits” have revealed 12 potential invasion suppressors involved in pathways including ER stress, cortactin phosphorylation signalling, cell differentiation and genome stability. These hits are currently being re-validated with other shRNAs specific for each gene. Overall, we provide a high-throughput and high-content screen for discovering novel genes that inhibit prostate cancer progression. These functional genomics screens which focus on altered prostate cell colony phenotypes may open new doors for potential drug treatments and a greater understanding of prostate cancer and BPH. Genome-wide shRNA screen invasion suppressor genesHit #GeneSymbolCellular Function1Interleukin 19IL19Upregulates IL6 and TNFA1Polyhomeotic-like protein 3PHC3Part of polycomb group complex (repressor)2Zinc finger protein 397ZNF397Repressor/activator transcription factor2Transmembrane protein 189-ubiquitin-cojugating enzyme E2 variant 1TMEM189-UBE2V1Cytosolic ubiquitination protein3Nuclear protein localization 4 homologNPLOC4Involved in ER stress (retro-translocation of proteins for degradation)4Flavin adenine dinucleotide synthetase 1FADSConverts FMN to FADH25Selenoprotein SSELSInvolved in ER stress (retro-translocation of proteins for degradation)5Serine/threonine-protein kinase 1OSR1Phosphorylates PAK1 (interacts with cortactin)6Ecotropic viral integration site protein 2BEVI2BInvolved in melanocyte and keratinocyte differentiation6Coiled-coil domain containing 111CCDC111Involved in DNA integrity and genome stability7Serine hydrolase-like 1SERHLPeroxisomal protein7Growth factor-induced 1GFI1Transcriptional repressor; involved in hematopoesis and oncogenesis Citation Format: Sean J. Leith, Susan E. Kuruvilla, Jason Moffat, Ann F. Chambers, Eva A. Turley, Joseph L. Chin, Hon S. Leong. A genome-wide shRNA screen for suppressors of prostate cancer cell invasion. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3432. doi:10.1158/1538-7445.AM2014-3432