In the earliest stages of metastasis, breast cancer cells must reorganize the cytoskeleton to affect cell shape change and promote cell invasion and motility. These events require the cytoskeletal regulators Cdc42 and Rho, their effectors such as N-WASp/WAVE, and direct inducers of actin polymerization such as Arp2/3. Little consideration has been given to molecules that shape the cell membrane. The F-BAR proteins CIP4, TOCA-1, and FBP17 generate membrane curvature and act as scaffolding proteins for activated Cdc42 and N-WASp. We found that expression of CIP4, but not TOCA-1 or FBP17, was increased in invasive breast cancer cell lines in comparison with weakly or noninvasive breast cancer cell lines. Endogenous CIP4 localized to the leading edge of migrating cells and to invadopodia in cells invading gelatin. Because CIP4 serves as a scaffolding protein for Cdc42, Src, and N-WASp, we tested whether loss of CIP4 could result in decreased N-WASp function. Interaction between CIP4 and N-WASp was epidermal growth factor responsive, and CIP4 silencing by small interfering RNA caused decreased tyrosine phosphorylation of N-WASp at a Src-dependent activation site (Y256). CIP4 silencing also impaired the migration and invasion of MDA-MB-231 cells and was associated with decreased formation of invadopodia and gelatin degradation. This study presents a new role for CIP4 in the promotion of migration and invasion of MDA-MB-231 breast cancer cells and establishes the contribution of F-BAR proteins to cancer cell motility and invasion. Cancer Res; 70(21); 8347-56. ©2010 AACR.

BACKGROUND: Src family kinases control multiple cancer cell properties including cell cycle progression, survival, and metastasis. Recent studies suggest that the Src inhibitor dasatinib blocks these critical cancer cell functions. METHODS: Because the molecular mechanism of action of dasatinib in breast cancers has not been investigated, we evaluated the effects of dasatinib as a single agent and in combination with the commonly used chemotherapeutic doxorubicin, on the proliferation, viability, and invasive capacity of breast cancer cells lines earlier categorised as dasatinib-sensitive (MDA-MB-231) and moderately resistant (MCF7 and T47D). We also tested the effects of these drugs on the actin cytoskeleton and associated signalling pathways. RESULTS: The cell lines tested varied widely in sensitivity to growth inhibition (IC 50 ¼ 0.16 -12.3 mM), despite comparable Src kinase inhibition by dasatinib (IC 50 ¼ 17 -37 nM). In the most sensitive cell line, MDA-MB-231, dasatinib treatment induced significant G 1 accumulation with little apoptosis, disrupted cellular morphology, blocked migration, inhibited invasion through Matrigel (Po0.01), and blocked the formation of invadopodia (Po0.001). Importantly, combination treatment with doxorubicin resulted in synergistic growth inhibition in all cell lines and blocked the migration and invasion of the highly metastatic, triple-negative MDA-MB-231 cell line. CONCLUSION: The observed synergy between dasatinib and doxorubicin warrants the re-evaluation of dasatinib as an effective agent in multi-drug regimens for the treatment of invasive breast cancers.

F-BAR proteins are a newly described family of proteins with unknown physiological significance. Because F-BAR proteins, including Cdc42 interacting protein-4 (CIP4), drive membrane deformation and affect endocytosis, we investigated the role of CIP4 in GLUT4 traffic by flow cytometry in GLUT4myc-expressing L6 myoblasts (L6 GLUT4myc). L6 GLUT4myc cells express CIP4a as the predominant F-BAR protein. siRNA knockdown of CIP4 increased insulin-stimulated 14C-deoxyglucose uptake by elevating cell-surface GLUT4. Enhanced surface GLUT4 was due to decreased endocytosis, which correlated with lower transferrin internalization. Immunoprecipitation of endogenous CIP4 revealed that CIP4 interacted with N-WASp and Dynamin-2 in an insulin-dependent manner. FRET confirmed the insulin-dependent, subcellular properties of these interactions. Insulin exposure stimulated specific interactions in plasma membrane and cytosolic compartments, followed by a steady-state response that underlies the coordination of proteins needed for GLUT4 traffic. Our findings reveal a physiological function for F-BAR proteins, supporting a previously unrecognized role for the F-BAR protein CIP4 in GLUT4 endocytosis, and show that interactions between CIP4 and Dynamin-2 and between CIP4 and NWASp are spatially coordinated to promote function.

BackgroundInteractions between the epigenome and structural genomic variation are potentially bi-directional. In one direction, structural variants may cause epigenomic changes in cis. In the other direction, specific local epigenomic states such as DNA hypomethylation associate with local genomic instability.MethodsTo study these interactions, we have developed several tools and exposed them to the scientific community using the Software-as-a-Service model via the Genboree Workbench. One key tool is Breakout, an algorithm for fast and accurate detection of structural variants from mate pair sequencing data.ResultsBy applying Breakout and other Genboree Workbench tools we map breakpoints in breast and prostate cancer cell lines and tumors, discriminate between polymorphic breakpoints of germline origin and those of somatic origin, and analyze both types of breakpoints in the context of the Human Epigenome Atlas, ENCODE databases, and other sources of epigenomic profiles. We confirm previous findings that genomic instability in human germline associates with hypomethylation of DNA, binding sites of Suz12, a key member of the PRC2 Polycomb complex, and with PRC2-associated histone marks H3K27me3 and H3K9me3. Breakpoints in germline and in breast cancer associate with distal regulatory of active gene transcription. Breast cancer cell lines and tumors show distinct patterns of structural mutability depending on their ER, PR, or HER2 status.ConclusionsThe patterns of association that we detected suggest that cell-type specific epigenomes may determine cell-type specific patterns of selective structural mutability of the genome.

<div>Abstract<p>In the earliest stages of metastasis, breast cancer cells must reorganize the cytoskeleton to affect cell shape change and promote cell invasion and motility. These events require the cytoskeletal regulators Cdc42 and Rho, their effectors such as N-WASp/WAVE, and direct inducers of actin polymerization such as Arp2/3. Little consideration has been given to molecules that shape the cell membrane. The F-BAR proteins CIP4, TOCA-1, and FBP17 generate membrane curvature and act as scaffolding proteins for activated Cdc42 and N-WASp. We found that expression of CIP4, but not TOCA-1 or FBP17, was increased in invasive breast cancer cell lines in comparison with weakly or noninvasive breast cancer cell lines. Endogenous CIP4 localized to the leading edge of migrating cells and to invadopodia in cells invading gelatin. Because CIP4 serves as a scaffolding protein for Cdc42, Src, and N-WASp, we tested whether loss of CIP4 could result in decreased N-WASp function. Interaction between CIP4 and N-WASp was epidermal growth factor responsive, and CIP4 silencing by small interfering RNA caused decreased tyrosine phosphorylation of N-WASp at a Src-dependent activation site (Y256). CIP4 silencing also impaired the migration and invasion of MDA-MB-231 cells and was associated with decreased formation of invadopodia and gelatin degradation. This study presents a new role for CIP4 in the promotion of migration and invasion of MDA-MB-231 breast cancer cells and establishes the contribution of F-BAR proteins to cancer cell motility and invasion. Cancer Res; 70(21); 8347–56. ©2010 AACR.</p></div>

Supplementary Figure 1 from Cdc42-Interacting Protein 4 Promotes Breast Cancer Cell Invasion and Formation of Invadopodia through Activation of N-WASp

Structural variation arising from genomic instability, a hallmark of cancer, contributes to tumor progression through loss of tumor suppressor genes and dysregulation of cancer drivers. Additionally, genomic rearrangements may result in the formation of chimeras, such as the TMPRSS2-ERG fusion, in a subset of prostate cancers. However, a complete catalog of structural variants (SVs) in prostate cancers has not yet been established. Using a genome-wide, paired-end approach with massively parallel sequencing, we mapped breakpoints and copy number variation (CNVs) in 8 prostate cancer cell lines (LNCaP, PC3, DU145, VCaP, MDA-PC2b, LNCaPC4-2, LAPC4, and 22RV1) and 2 non-tumorigenic immortalized prostate epithelial cell lines (PrEC and RWPE1). Because these are cell lines established without a germline reference, we utilized comparable sequencing data from the 1000 Genomes Project to remove structural rearrangements that are present in the normal population. In addition, we removed from the set of prostate cancer cell line variants all events that were found in either of the normal cell lines as a method of correcting for cell culture artifacts. These two approaches removed between 40 and 65% of the structural variant calls from the prostate cancer cell lines. After normalization, RWPE1 was the least rearranged cell line (129 variants) and VCaP was the most heavily rearranged (784 variants). More than 40% of these structural variants fall within 2kb of a known gene, a remarkable enrichment over the 1.5% coding rate of the human genome (approximately 1 gene per 100kb). Structural variants from these 8 cell lines nominated 436 recurrently disrupted genes, in which we find significant enrichment of known transcription factor motifs (NFAT, FOXO4, and others), epigenetic marks (H3K27me3), and cancer related pathways (differentiation and cell-cell adhesion). An integrative approach was employed to nominate potential cancer driver genes from the SV- and CNV-affected genes through extensive comparison with available gene expression, aCGH, and sequencing data from human prostate tumors, and network centrality modeling. As expected, known drivers of prostate cancer, such as PTEN, MYC, and AR were significantly disrupted across multiple data types and therefore ranked highly by our Nomination Index. However, we have also identified several genes that have not previously been implicated in prostate cancer and may represent novel drivers of the disease. Citation Format: Christina Stewart Pichot, Cristian Coarfa, Mark P. Hamilton, Sean M. Hartig, Caroline Schoenherr, Adrian V. Lee, Aleksander Milosavljevic, Sean E. McGuire. Integrative nomination of prostate cancer driver genes from paired-end sequencing [abstract]. In: Proceedings of the AACR Special Conference on Advances in Prostate Cancer Research; 2012 Feb 6-9; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(4 Suppl):Abstract nr B35.

Supplementary Figure 4 from Cdc42-Interacting Protein 4 Promotes Breast Cancer Cell Invasion and Formation of Invadopodia through Activation of N-WASp