Triple negative breast cancers are an aggressive subtype of breast cancer, characterized by the lack of estrogen receptor, progesterone receptor and Her2 expression. Triple negative breast cancers are non-responsive to conventional anti-hormonal and Her2 targeted therapies, making it necessary to identify new molecular targets for therapy. The chemokine CCL2 is overexpressed in invasive breast cancers, and regulates breast cancer progression through multiple mechanisms. With few approaches to target CCL2 activity, its value as a therapeutic target is unclear. In these studies, we developed a novel gene silencing approach that involves complexing siRNAs to TAT cell penetrating peptides (Ca-TAT) through non-covalent calcium cross-linking. Ca-TAT/siRNA complexes penetrated 3D collagen cultures of breast cancer cells and inhibited CCL2 expression more effectively than conventional antibody neutralization. Ca-TAT/siRNA complexes targeting CCL2 were delivered to mice bearing MDA-MB-231 breast tumor xenografts. In vivo CCL2 gene silencing inhibited primary tumor growth and metastasis, associated with a reduction in cancer stem cell renewal and recruitment of M2 macrophages. These studies are the first to demonstrate that targeting CCL2 expression in vivo may be a viable therapeutic approach to treating triple negative breast cancer.

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Ductal carcinoma (DCIS) is the most common form of breast cancer, with 50,000 cases diagnosed every year in the United States. Overtreatment and undertreatment remain significant clinical challenges in patient care. Identifying key mechanisms associated with DCIS progression could uncover new biomarkers to better predict patient prognosis and improve guided treatment. Chemokines are small soluble molecules that regulate cellular homing through molecular gradients. CCL2-mediated recruitment of CCR2 macrophages are a well-established mechanism for metastatic progression. Although the CCL2/CCR2 pathway is a therapeutic target of interest, little is known about the role of CCR2 expression in breast cancer. Here, using a mammary intraductal injection (MIND) model to mimic DCIS formation, the role of CCR2 was explored in minimally invasive SUM225 and highly invasive DCIS.com breast cancer cells. CCR2 overexpression increased SUM225 breast cancer survival and invasion associated with accumulation of CCL2 expressing fibroblasts. CCR2-deficient DCIS.com breast cancer cells formed fewer invasive lesions with fewer CCL2 fibroblasts. Cografting CCL2-deficient fibroblasts with DCIS.com breast cancer cells in the subrenal capsule model inhibited tumor invasion and survival associated with decreased expression of aldehyde dehydrogenase (ALDH1), a proinvasive factor, and decreased expression of HTRA2, a proapoptotic serine protease. Through data mining analysis, high expression of CCR2 and ALDH1 and low HTRA2 expression were correlated with poor prognosis of breast cancer patients. This study demonstrates that CCR2 overexpression in breast cancer drives early-stage breast cancer progression through stromal-dependent expression of CCL2 with important insight into prognosis and treatment of DCIS. .

Cranial neural crest cells (CNCCs) delaminate from embryonic neural folds and migrate to pharyngeal arches, which give rise to most mid-facial structures. CNCC dysfunction plays a prominent role in the etiology of orofacial clefts, a frequent birth malformation. Heterozygous mutations in SPECC1L have been identified in patients with atypical and syndromic clefts. Here, we report that in SPECC1L-knockdown cultured cells, staining of canonical adherens junction (AJ) components, β-catenin and E-cadherin, was increased, and electron micrographs revealed an apico-basal diffusion of AJs. To understand the role of SPECC1L in craniofacial morphogenesis, we generated a mouse model of Specc1l deficiency. Homozygous mutants were embryonic lethal and showed impaired neural tube closure and CNCC delamination. Staining of AJ proteins was increased in the mutant neural folds. This AJ defect is consistent with impaired CNCC delamination, which requires AJ dissolution. Further, PI3K-AKT signaling was reduced and apoptosis was increased in Specc1l mutants. In vitro, moderate inhibition of PI3K-AKT signaling in wildtype cells was sufficient to cause AJ alterations. Importantly, AJ changes induced by SPECC1L-knockdown were rescued by activating the PI3K-AKT pathway. Together, these data indicate SPECC1L as a novel modulator of PI3K-AKT signaling and AJ biology, required for neural tube closure and CNCC delamination.

SPECC1L mutations have been identified in patients with rare atypical orofacial clefts and with syndromic cleft lip and/or palate (CL/P). These mutations cluster in the second coiled-coil and calponin homology domains of SPECC1L and severely affect the ability of SPECC1L to associate with microtubules. We previously showed that gene-trap knockout of Specc1l in mouse results in early embryonic lethality. We now present a truncation mutant mouse allele, Specc1lΔC510, that results in perinatal lethality. Specc1lΔC510/ΔC510 homozygotes showed abnormal palate rugae but did not show cleft palate. However, when crossed with a gene-trap allele, Specc1lcGT/ΔC510 compound heterozygotes showed a palate elevation delay with incompletely penetrant cleft palate. Specc1lcGT/ΔC510 embryos exhibit transient oral epithelial adhesions at E13.5, which may delay shelf elevation. Consistent with oral adhesions, we show periderm layer abnormalities, including ectopic apical expression of adherens junction markers, similar to Irf6 hypomorphic mutants and Arhgap29 heterozygotes. Indeed, SPECC1L expression is drastically reduced in Irf6 mutant palatal shelves. Finally, we wanted to determine if SPECC1L deficiency also contributed to non-syndromic (ns) CL/P. We sequenced 62 Caucasian, 89 Filipino, 90 Ethiopian, 90 Nigerian and 95 Japanese patients with nsCL/P and identified three rare coding variants (p.Ala86Thr, p.Met91Iso and p.Arg546Gln) in six individuals. These variants reside outside of SPECC1L coiled-coil domains and result in milder functional defects than variants associated with syndromic clefting. Together, our data indicate that palate elevation is sensitive to deficiency of SPECC1L dosage and function and that SPECC1L cytoskeletal protein functions downstream of IRF6 in palatogenesis.