Targeting of SUMOylation leads to cBAF complex stabilization and disruption of the SS18::SSX transcriptome in Synovial Sarcoma.

Synovial Sarcoma (SS) is driven by the SS18::SSX fusion oncoprotein and is ultimately refractory to therapeutic approaches. SS18::SSX alters ATP-dependent chromatin remodeling BAF (mammalian SWI/SNF) complexes, leading to the degradation of canonical (cBAF) complex and amplified presence of an SS18::SSX-containing non-canonical BAF (ncBAF or GBAF) that drives an SS-specific transcription program and tumorigenesis. We demonstrate that SS18::SSX activates the SUMOylation program and SSs are sensitive to the small molecule SAE1/2 inhibitor, TAK-981. Mechanistically, TAK-981 de-SUMOylates the cBAF subunit SMARCE1, stabilizing and restoring cBAF on chromatin, shifting away from SS18::SSX-ncBAF-driven transcription, associated with DNA damage and cell death and resulting in tumor inhibition across both human and mouse SS tumor models. TAK-981 synergized with cytotoxic chemotherapy through increased DNA damage, leading to tumor regression. Targeting the SUMOylation pathway in SS restores cBAF complexes and blocks the SS18::SSX-ncBAF transcriptome, identifying a therapeutic vulnerability in SS, positioning the in-clinic TAK-981 to treat SS.

Targeting of SUMOylation leads to cBAF complex stabilization and disruption of the SS18::SSX transcriptome in Synovial Sarcoma.

Synovial Sarcoma (SS) is driven by the SS18::SSX fusion oncoprotein. and is ultimately refractory to therapeutic approaches. SS18::SSX alters ATP-dependent chromatin remodeling BAF (mammalian SWI/SNF) complexes, leading to the degradation of canonical (cBAF) complex and amplified presence of an SS18::SSX-containing non-canonical BAF (ncBAF or GBAF) that drives an SS-specific transcription program and tumorigenesis. We demonstrate that SS18::SSX activates the SUMOylation program and SSs are sensitive to the small molecule SAE1/2 inhibitor, TAK-981. Mechanistically, TAK-981 de-SUMOylates the cBAF subunit SMARCE1, stabilizing and restoring cBAF on chromatin, shifting away from SS18::SSX-ncBAF-driven transcription, associated with DNA damage and cell death and resulting in tumor inhibition across both human and mouse SS tumor models. TAK-981 synergized with cytotoxic chemotherapy through increased DNA damage, leading to tumor regression. Targeting the SUMOylation pathway in SS restores cBAF complexes and blocks the SS18::SSX-ncBAF transcriptome, identifying a therapeutic vulnerability in SS, positioning the in-clinic TAK-981 to treat SS.

Antitumor active trans­platinum complexes through metabolic stability and enhanced cellular accumulation.

Utilizing isoquinoline as a carrier ligand, we have evaluated the reactivity of selected trans­platinum planar amine (TPA) carboxylate compounds by varying the leaving carboxylate group (acetate, hydroxyacetate, and lactate) in an effort to optimize the cytotoxic and metabolic efficiency. To measure the pharmacological properties of these compounds, a combination of systematic biophysical and biological studies were carried out mainly involving substitution reaction with NAM (N-acetyl-methionine), effects on DNA structural perturbation, cytotoxicity, cellular accumulation, metabolic stability, and cell cycle effects. TPA compounds showed minimal losses in cytotoxic efficacy and outperformed cisplatin after pre-incubation with serum, while displaying a distinct micromolar cytotoxic activity with minimal DNA binding and unaltered cell cycle. Monitoring the TPA compounds with NAM suggests the following trend for the reactivity: hydroxyacetate > lactate > acetate. The same trend was seen for the cytotoxicity in tumor cells and DNA binding, while the rate of drug inactivation/protein binding in cells was not significantly different among these leaving groups. Thus, our results show superior cellular efficacy of TPA compounds and distinct micromolar cytotoxic activities different than cisplatin. Moreover, we found the TPA compounds had prolonged survival and decreased tumor burden compared to the control mice in a relevant human ovarian cancer mouse model with A2780 cells expressing luciferase. Therefore, we propose that further optimization of the basic TPA structure can give further enhanced in vivo activity and may eventually be translated into the development of clinically relevant non-traditional platinum drugs.

Targeting Oncogenic Mutant p53 and BCL-2 for Small Cell Lung Cancer Treatment.

Through a unique genomics and drug screening platform with ~800 solid tumor cell lines, we have found a subset of SCLC cell lines are hypersensitive to venetoclax, an FDA-approved inhibitor of BCL-2. SCLC-A (ASCL1 positive) and SCLC-P (POU2F3 positive), which make up almost 80% of SCLC, frequently express high levels of BCL-2. We found that a subset of SCLC-A and SCLC-P showed high BCL-2 expression but were venetoclax-resistant. In addition, most of these SCLC cell lines have TP53 missense mutations, which make a single amino acid change. These mutants not only lose wild-type (WT) p53 tumor suppressor functions, but also acquire novel cancer-promoting activities (oncogenic, gain-of-function). A recent study with oncogenic mutant (Onc)-p53 knock-in mouse models of SCLC suggests gain-of-function activity can attenuate chemotherapeutic efficacy. Based on these observations, we hypothesize that Onc-p53 confers venetoclax resistance and that simultaneous inhibition of BCL-2 and Onc-p53 induces synergistic anticancer activity in a subset of SCLC-A and SCLC-P. We show here that (1) down-regulation of Onc-p53 increases the expression of a BH3-only pro-apoptotic BIM and sensitizes to venetoclax in SCLC-P cells; (2) targeting Onc-p53 by the HSP90 inhibitor, ganetespib, increases BIM expression and sensitizes to venetoclax in SCLC-P and SCLC-A cells. Although there are currently many combination studies for venetoclax proposed, the concept of simultaneous targeting of BCL-2 and Onc-p53 by the combination of venetoclax and HSP90 inhibitors would be a promising approach for SCLC treatment.

A Comparison of Di- and Trinuclear Platinum Complexes Interacting with Glycosaminoglycans for Targeted Chemotherapy.

Heparan sulfate proteoglycans (HSPGs) and their associated proteins aid in tumor progression through modulation of biological events such as cell invasion, angiogenesis, metastasis, and immunological responses. Metalloshielding of the anionic heparan sulfate (HS) chains by cationic polynuclear platinum complexes (PPCs) prevents the HS from interacting with HS-associated proteins and thus diminishes the critical functions of HSPG. Studies herein exploring the PPC-HS interactions demonstrated that a series of PPCs varying in charge, nuclearity, distance between Pt centers, and hydrogen-bonding ability influence HS affinity. We report that the polyamine-linked complexes have high HS affinity and display excellent in vivo activity against breast cancer metastases and those arising in the bone and liver compared to carboplatin. Overall, the PPC-HS niche offers an attractive approach for targeting HSPG-expressing tumor cells.

Melanoma differentiation associated gene-9/syndecan binding protein promotes hepatocellular carcinoma.

BACKGROUND AND AIMS: The oncogene Melanoma differentiation associated gene-9/syndecan binding protein (MDA-9/SDCBP) is overexpressed in many cancers, promoting aggressive, metastatic disease. However, the role of MDA-9 in regulating hepatocellular carcinoma (HCC) has not been well studied. APPROACH AND RESULTS: To unravel the function of MDA-9 in HCC, we generated and characterized a transgenic mouse with hepatocyte-specific overexpression of MDA-9 (Alb/MDA-9). Compared with wild-type (WT) littermates, Alb/MDA-9 mice demonstrated significantly higher incidence of N-nitrosodiethylamine/phenobarbital-induced HCC, with marked activation and infiltration of macrophages. RNA sequencing (RNA-seq) in naive WT and Alb/MDA-9 hepatocytes identified activation of signaling pathways associated with invasion, angiogenesis, and inflammation, especially NF-κB and integrin-linked kinase signaling pathways. In nonparenchymal cells purified from naive livers, single-cell RNA-seq showed activation of Kupffer cells and macrophages in Alb/MDA-9 mice versus WT mice. A robust increase in the expression of Secreted phosphoprotein 1 (Spp1/osteopontin) was observed upon overexpression of MDA-9. Inhibition of NF-κB pathway blocked MDA-9-induced Spp1 induction, and knock down of Spp1 resulted in inhibition of MDA-9-induced macrophage migration, as well as angiogenesis. CONCLUSIONS: Alb/MDA-9 is a mouse model with MDA-9 overexpression in any tissue type. Our findings unravel an HCC-promoting role of MDA-9 mediated by NF-κB and Spp1 and support the rationale of using MDA-9 inhibitors as a potential treatment for aggressive HCC.

Pharmacologic Inhibition of SHP2 Blocks Both PI3K and MEK Signaling in Low-epiregulin HNSCC via GAB1.

Preclinical and clinical studies have evidenced that effective targeted therapy treatment against receptor tyrosine kinases (RTKs) in different solid tumor paradigms is predicated on simultaneous inhibition of both the PI3K and MEK intracellular signaling pathways. Indeed, re-activation of either pathway results in resistance to these therapies. Recently, oncogenic phosphatase SHP2 inhibitors have been developed with some now reaching clinical trials. To expand on possible indications for SHP099, we screened over 800 cancer cell lines covering over 25 subsets of cancer. We found HNSCC was the most sensitive adult subtype of cancer to SHP099. We found that, in addition to the MEK pathway, SHP2 inhibition blocks the PI3K pathway in sensitive HNSCC, resulting in downregulation of mTORC signaling and anti-tumor effects across several HNSCC mouse models, including an HPV+ patient-derived xenograft (PDX). Importantly, we found low levels of the RTK ligand epiregulin identified HNSCCs that were sensitive to SHP2 inhibitor, and, adding exogenous epiregulin mitigated SHP099 efficacy. Mechanistically, epiregulin maintained SHP2-GAB1 complexes in the presence of SHP2 inhibition, preventing downregulation of the MEK and PI3K pathways. We demonstrate HNSCCs were highly dependent on GAB1 for their survival and knockdown of GAB1 is sufficient to block the ability of epiregulin to rescue MEK and PI3K signaling. These data connect the sensitivity of HNSCC to SHP2 inhibitors and to a broad reliance on GAB1-SHP2, revealing an important and druggable signaling axis. Overall, SHP2 inhibitors are being heavily developed and may have activity in HNSCCs, and in particular those with low levels of epiregulin.

Radiation induces ESCRT pathway dependent CD44v3+ extracellular vesicle production stimulating pro-tumor fibroblast activity in breast cancer.

Despite recent advances in radiotherapeutic strategies, acquired resistance remains a major obstacle, leading to tumor recurrence for many patients. Once thought to be a strictly cancer cell intrinsic property, it is becoming increasingly clear that treatment-resistance is driven in part by complex interactions between cancer cells and non-transformed cells of the tumor microenvironment. Herein, we report that radiotherapy induces the production of extracellular vesicles by breast cancer cells capable of stimulating tumor-supporting fibroblast activity, facilitating tumor survival and promoting cancer stem-like cell expansion. This pro-tumor activity was associated with fibroblast production of the paracrine signaling factor IL-6 and was dependent on the expression of the heparan sulfate proteoglycan CD44v3 on the vesicle surface. Enzymatic removal or pharmaceutical inhibition of its heparan sulfate side chains disrupted this tumor-fibroblast crosstalk. Additionally, we show that the radiation-induced production of CD44v3+ vesicles is effectively silenced by blocking the ESCRT pathway using a soluble pharmacological inhibitor of MDA-9/Syntenin/SDCBP PDZ1 domain activity, PDZ1i. This population of vesicles was also detected in the sera of human patients undergoing radiotherapy, therefore representing a potential biomarker for radiation therapy and providing an opportunity for clinical intervention to improve treatment outcomes.

Exploitation of Sulfated Glycosaminoglycan Status for Precision Medicine of Triplatin in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is a subtype of breast cancer lacking targetable biomarkers. TNBC is known to be most aggressive and when metastatic is often drug-resistant and uncurable. Biomarkers predicting response to therapy improve treatment decisions and allow personalized approaches for patients with TNBC. This study explores sulfated glycosaminoglycan (sGAG) levels as a predictor of TNBC response to platinum therapy. sGAG levels were quantified in three distinct TNBC tumor models, including cell line-derived, patient-derived xenograft (PDX) tumors, and isogenic models deficient in sGAG biosynthesis. The in vivo antitumor efficacy of Triplatin, a sGAG-directed platinum agent, was compared in these models with the clinical platinum agent, carboplatin. We determined that >40% of TNBC PDX tissue microarray samples have high levels of sGAGs. The in vivo accumulation of Triplatin in tumors as well as antitumor efficacy of Triplatin positively correlated with sGAG levels on tumor cells, whereas carboplatin followed the opposite trend. In carboplatin-resistant tumor models expressing high levels of sGAGs, Triplatin decreased primary tumor growth, reduced lung metastases, and inhibited metastatic growth in lungs, liver, and ovaries. sGAG levels served as a predictor of Triplatin sensitivity in TNBC. Triplatin may be particularly beneficial in treating patients with chemotherapy-resistant tumors who have evidence of residual disease after standard neoadjuvant chemotherapy. More effective neoadjuvant and adjuvant treatment will likely improve clinical outcome of TNBC.

Genomic screening reveals ubiquitin-like modifier activating enzyme 1 as a potent and druggable target in c-MYC-high triple negative breast cancer models.

Triple negative breast cancer (TNBC) accounts for over 30% of all breast cancer (BC)-related deaths, despite accounting for only 10% to 15% of total BC cases. Targeted therapy development has largely stalled in TNBC, underlined by a lack of traditionally druggable addictions like receptor tyrosine kinases (RTKs). Here, through full genome CRISPR/Cas9 screening of TNBC models, we have uncovered the sensitivity of TNBCs to the depletion of the ubiquitin-like modifier activating enzyme 1 (UBA1). Targeting UBA1 with the first-in-class UBA1 inhibitor TAK-243 induced unresolvable endoplasmic reticulum (ER)-stress and activating transcription factor 4 (ATF4)-mediated upregulation of proapoptotic NOXA, leading to cell death. c-MYC expression correlates with TAK-243 sensitivity and cooperates with TAK-243 to induce a stress response and cell death. Importantly, there was an order of magnitude greater sensitivity of TNBC lines to TAK-243 compared to normal tissue-derived cells. In five patient derived xenograft models (PDXs) of TNBC, TAK-243 therapy led to tumor inhibition or frank tumor regression. Moreover, in an intracardiac metastatic model of TNBC, TAK-243 markedly reduced metastatic burden, indicating UBA1 is a potential new target in TNBC expressing high levels of c-MYC.

Pharmaceutical Interference of the EWS-FLI1-driven Transcriptome By Cotargeting H3K27ac and RNA Polymerase Activity in Ewing Sarcoma.

The EWSR1-FLI1 t(11;22)(q24;q12) translocation is the hallmark genomic alteration of Ewing sarcoma, a malignancy of the bone and surrounding tissue, predominantly affecting children and adolescents. Although significant progress has been made for the treatment of localized disease, patients with metastasis or who relapse after chemotherapy have less than a 30% five-year survival rate. EWS-FLI1 is currently not clinically druggable, driving the need for more effective targeted therapies. Treatment with the H3K27 demethylase inhibitor, GSK-J4, leads to an increase in H3K27me and a decrease in H3K27ac, a significant event in Ewing sarcoma because H3K27ac associates strongly with EWS-FLI1 binding at enhancers and promoters and subsequent activity of EWS-FLI1 target genes. We were able to identify targets of EWS-FLI1 tumorigenesis directly inhibited by GSK-J4. GSK-J4 disruption of EWS-FLI1-driven transcription was toxic to Ewing sarcoma cells and slowed tumor growth in patient-derived xenografts (PDX) of Ewing sarcoma. Responses were markedly exacerbated by cotreatment with a disruptor of RNA polymerase II activity, the CDK7 inhibitor THZ1. This combination together suppressed EWS-FLI1 target genes and viability of ex vivo PDX Ewing sarcoma cells in a synergistic manner. In PDX models of Ewing Sarcoma, the combination shrank tumors. We present a new therapeutic strategy to treat Ewing sarcoma by decreasing H3K27ac at EWS-FLI1-driven transcripts, exacerbated by blocking phosphorylation of the C-terminal domain of RNA polymerase II to further hinder the EWS-FLI1-driven transcriptome.

Unmasking BCL-2 Addiction in Synovial Sarcoma by Overcoming Low NOXA.

Synovial sarcoma (SS) is frequently diagnosed in teenagers and young adults and continues to be treated with polychemotherapy with variable success. The SS18-SSX gene fusion is pathognomonic for the disease, and high expression of the anti-apoptotic BCL-2 pathologically supports the diagnosis. As the oncogenic SS18-SSX fusion gene itself is not druggable, BCL-2 inhibitor-based therapies are an appealing therapeutic opportunity. Venetoclax, an FDA-approved BCL-2 inhibitor that is revolutionizing care in some BCL-2-expressing hematological cancers, affords an intriguing therapeutic possibility to treat SS. In addition, there are now dozens of venetoclax-based combination therapies in clinical trials in hematological cancers, attributing to the limited toxicity of venetoclax. However, preclinical studies of venetoclax in SS have demonstrated an unexpected ineffectiveness. In this study, we analyzed the response of SS to venetoclax and the underlying BCL-2 family biology in an effort to understand venetoclax treatment failure and find a therapeutic strategy to sensitize SS to venetoclax. We found remarkably depressed levels of the endogenous MCL-1 inhibitor, NOXA, in SS compared to other sarcomas. Expressing NOXA led to sensitization to venetoclax, as did the addition of the MCL-1 BH3 mimetic, S63845. Importantly, the venetoclax/S63845 combination induced tumor regressions in SS patient-derived xenograft (PDX) models. As a very close analog of S63845 (S64315) is now in clinical trials with venetoclax in AML (NCT03672695), the combination of MCL-1 BH3 mimetics and venetoclax should be considered for SS patients as a new therapy.

MYCN-Amplified Neuroblastoma Is Addicted to Iron and Vulnerable to Inhibition of the System Xc-/Glutathione Axis.

MYCN is amplified in 20% to 25% of neuroblastoma, and MYCN-amplified neuroblastoma contributes to a large percent of pediatric cancer-related deaths. Therapy improvements for this subtype of cancer are a high priority. Here we uncover a MYCN-dependent therapeutic vulnerability in neuroblastoma. Namely, amplified MYCN rewires the cell through expression of key receptors, ultimately enhancing iron influx through increased expression of the iron import transferrin receptor 1. Accumulating iron causes reactive oxygen species (ROS) production, and MYCN-amplified neuroblastomas show enhanced reliance on the system Xc- cystine/glutamate antiporter for ROS detoxification through increased transcription of this receptor. This dependence creates a marked vulnerability to targeting the system Xc-/glutathione (GSH) pathway with ferroptosis inducers. This reliance can be exploited through therapy with FDA-approved rheumatoid arthritis drugs sulfasalazine (SAS) and auranofin: in MYCN-amplified, patient-derived xenograft models, both therapies blocked growth and induced ferroptosis. SAS and auranofin activity was largely mitigated by the ferroptosis inhibitor ferrostatin-1, antioxidants like N-acetyl-L-cysteine, or by the iron scavenger deferoxamine (DFO). DFO reduced auranofin-induced ROS, further linking increased iron capture in MYCN-amplified neuroblastoma to a therapeutic vulnerability to ROS-inducing drugs. These data uncover an oncogene vulnerability to ferroptosis caused by increased iron accumulation and subsequent reliance on the system Xc-/GSH pathway. SIGNIFICANCE: This study shows how MYCN increases intracellular iron levels and subsequent GSH pathway activity and demonstrates the antitumor activity of FDA-approved SAS and auranofin in patient-derived xenograft models of MYCN-amplified neuroblastoma.

Anillin is an emerging regulator of tumorigenesis, acting as a cortical cytoskeletal scaffold and a nuclear modulator of cancer cell differentiation.

Remodeling of the intracellular cytoskeleton plays a key role in accelerating tumor growth and metastasis. Targeting different cytoskeletal elements is important for existing and future anticancer therapies. Anillin is a unique scaffolding protein that interacts with major cytoskeletal structures, e.g., actin filaments, microtubules and septin polymers. A well-studied function of this scaffolding protein is the regulation of cytokinesis at the completion of cell division. Emerging evidence suggest that anillin has other important activities in non-dividing cells, including control of intercellular adhesions and cell motility. Anillin is markedly overexpressed in different solid cancers and its high expression is commonly associated with poor prognosis of patient survival. This review article summarizes rapidly accumulating evidence that implicates anillin in the regulation of tumor growth and metastasis. We focus on molecular and cellular mechanisms of anillin-dependent tumorigenesis that include both canonical control of cytokinesis and novel poorly understood functions as a nuclear regulator of the transcriptional reprogramming and phenotypic plasticity of cancer cells.

Conformational Modulation of Iduronic Acid-Containing Sulfated Glycosaminoglycans by a Polynuclear Platinum Compound and Implications for Development of Antimetastatic Platinum Drugs.

1 H NMR spectroscopic studies on the 1:1 adduct of the pentasaccharide Fondaparinux (FPX) and the substitution-inert polynuclear platinum complex TriplatinNC show significant modulation of geometry around the glycosidic linkages of the FPX constituent monosaccharides. FPX is a valid model for the highly sulfated cell signalling molecule heparan sulfate (HS). The conformational ratio of the 1 C4 :2 S0 forms of the FPX residue IdoA(2S) is altered from ca. 35:65 (free FPX) to ca. 75:25 in the adduct; the first demonstration of a small molecule affecting conformational changes on a HS oligosaccharide. Functional consequences of such binding are suggested to be inhibition of HS cleavage in MDA-MB-231 triple-negative breast cancer (TNBC) cells. We further describe inhibition of metastasis by TriplatinNC in the TNBC 4T1 syngeneic tumour model. Our work provides insight into a novel approach for design of platinum drugs (and coordination compounds in general) with intrinsic anti-metastatic potential.

Inhibition of the Activity of Cyclophilin A Impedes Prolactin Receptor-Mediated Signaling, Mammary Tumorigenesis, and Metastases.

Prolactin (PRL) and its receptor (PRLr) play important roles in the pathogenesis of breast cancer. Cyclophilin A (CypA) is a cis-trans peptidyl-prolyl isomerase (PPI) that is constitutively associated with the PRLr and facilitates the activation of the tyrosine kinase Jak2. Treatment with the non-immunosuppressive prolyl isomerase inhibitor NIM811 or CypA short hairpin RNA inhibited PRL-stimulated signaling, breast cancer cell growth, and migration. Transcriptomic analysis revealed that NIM811 inhibited two-thirds of the top 50 PRL-induced genes and a reduction in gene pathways associated with cancer cell signaling. In vivo treatment of NIM811 in a TNBC xenograft lessened primary tumor growth and induced central tumor necrosis. Deletion of CypA in the MMTV-PyMT mouse model demonstrated inhibition of tumorigenesis with significant reduction in lung and lymph node metastasis. The regulation of PRLr/Jak2-mediated biology by NIM811 demonstrates that a non-immunosuppressive prolyl isomerase inhibitor can function as a potential breast cancer therapeutic.

Anillin regulates breast cancer cell migration, growth, and metastasis by non-canonical mechanisms involving control of cell stemness and differentiation.

BACKGROUND: Breast cancer metastasis is driven by a profound remodeling of the cytoskeleton that enables efficient cell migration and invasion. Anillin is a unique scaffolding protein regulating major cytoskeletal structures, such as actin filaments, microtubules, and septin polymers. It is markedly overexpressed in breast cancer, and high anillin expression is associated with poor prognosis. The aim of this study was to investigate the role of anillin in breast cancer cell migration, growth, and metastasis. METHODS: CRISPR/Cas9 technology was used to deplete anillin in highly metastatic MDA-MB-231 and BT549 cells and to overexpress it in poorly invasive MCF10AneoT cells. The effects of anillin depletion and overexpression on breast cancer cell motility in vitro were examined by wound healing and Matrigel invasion assays. Assembly of the actin cytoskeleton and matrix adhesion were evaluated by immunofluorescence labeling and confocal microscopy. In vitro tumor development was monitored by soft agar growth assays, whereas cancer stem cells were examined using a mammosphere formation assay and flow cytometry. The effects of anillin knockout on tumor growth and metastasis in vivo were determined by injecting control and anillin-depleted breast cancer cells into NSG mice. RESULTS: Loss-of-function and gain-of-function studies demonstrated that anillin is necessary and sufficient to accelerate migration, invasion, and anchorage-independent growth of breast cancer cells in vitro. Furthermore, loss of anillin markedly attenuated primary tumor growth and metastasis of breast cancer in vivo. In breast cancer cells, anillin was localized in the nucleus; however, knockout of this protein affected the cytoplasmic/cortical events, e.g., the organization of actin cytoskeleton and cell-matrix adhesions. Furthermore, we observed a global transcriptional reprogramming of anillin-depleted breast cancer cells that resulted in suppression of their stemness and induction of the mesenchymal to epithelial trans-differentiation. Such trans-differentiation was manifested by the upregulation of basal keratins along with the increased expression of E-cadherin and P-cadherin. Knockdown of E-cadherin restored the impaired migration and invasion of anillin-deficient breast cancer cells. CONCLUSION: Our study demonstrates that anillin plays essential roles in promoting breast cancer growth and metastatic dissemination in vitro and in vivo and unravels novel functions of anillin in regulating breast cancer stemness and differentiation.

Syndecan-1 facilitates breast cancer metastasis to the brain.

PURPOSE: Although survival rates for patients with localized breast cancer have increased, patients with metastatic breast cancer still have poor prognosis. Understanding key factors involved in promoting breast cancer metastasis is imperative for better treatments. In this study, we investigated the role of syndecan-1 (Sdc1) in breast cancer metastasis. METHODS: To assess the role of Sdc1 in breast cancer metastasis, we silenced Sdc1 expression in the triple-negative breast cancer human MDA-MB-231 cell line and overexpressed it in the mouse mammary carcinoma 4T1 cell line. Intracardiac injections were performed in an experimental mouse metastasis model using both cell lines. In vitro transwell blood-brain barrier (BBB) and brain section adhesion assays were utilized to specifically investigate how Sdc1 facilitates brain metastasis. A cytokine array was performed to evaluate differences in the breast cancer cell secretome when Sdc1 is silenced. RESULTS: Silencing expression of Sdc1 in breast cancer cells significantly reduced metastasis to the brain. Conversely, overexpression of Sdc1 increased metastasis to the brain. We found that silencing of Sdc1 expression had no effect on attachment of breast cancer cells to brain endothelial cells or astrocytes, but migration across the BBB was reduced as well as adhesion to the perivascular regions of the brain. Loss of Sdc1 also led to changes in breast cancer cell-secreted cytokines/chemokines, which may influence the BBB. CONCLUSIONS: Taken together, our study demonstrates a role for Sdc1 in promoting breast cancer metastasis to the brain. These findings suggest that Sdc1 supports breast cancer cell migration across the BBB through regulation of cytokines, which may modulate the BBB. Further elucidating this mechanism will allow for the development of therapeutic strategies to combat brain metastasis.

The Laminin-α1 Chain-Derived Peptide, AG73, Binds to Syndecans on MDA-231 Breast Cancer Cells and Alters Filopodium Formation.

Breast cancer is one of the most common forms of cancer affecting women in the United States, second only to skin cancers. Although treatments have been developed to combat primary breast cancer, metastasis remains a leading cause of death. An early step of metastasis is cancer cell invasion through the basement membrane. However, this process is not yet well understood. AG73, a synthetic laminin-α1 chain peptide, plays an important role in cell adhesion and has previously been linked to migration, invasion, and metastasis. Thus, we aimed to identify the binding partner of AG73 on breast cancer cells that could mediate cancer progression. We performed adhesion assays using MCF10A, T47D, SUM1315, and MDA-231 breast cell lines and found that AG73 binds to syndecans (Sdcs) 1, 2, and 4. This interaction was inhibited when we silenced Sdcs 1 and/or 4 in MDA-231 cells, indicating the importance of these receptors in this relationship. Through actin staining, we found that silencing of Sdc 1, 2, and 4 expression in MDA-231 cells exhibits a decrease in the length and number of filopodia bound to AG73. Expression of mouse Sdcs 1, 2, and 4 in MDA-231 cells provides rescue in filopodia, and overexpression of Sdcs 1 and 2 leads to increased filopodium length and number. Our findings demonstrate an intrinsic interaction between AG73 in the tumor environment and the Sdcs on breast cancer cells in supporting tumor cell adhesion and invasion through filopodia, an important step in cancer metastasis.

24R,25-Dihydroxyvitamin D3 regulates breast cancer cells in vitro and in vivo.

BACKGROUND: Epidemiological studies indicate high serum 25(OH)D3 is associated with increased survival in breast cancer patients. Pre-clinical studies attributed this to anti-tumorigenic properties of its metabolite 1α,25(OH)2D3. However, 1α,25(OH)2D3 is highly calcemic and thus has a narrow therapeutic window. Here we propose another metabolite, 24R,25(OH)2D3, as an alternative non-calcemic vitamin D3 supplement. METHODS: NOD-SCID-IL2γR null female mice with MCF7 breast cancer xenografts in the mammary fat pad were treated with 24R,25(OH)2D3 and changes in tumor burden and metastases were assessed. ERα66+ MCF7 and T47D cells, and ERα66- HCC38 cells were treated with 24R,25(OH)2D3in vitro to assess effects on proliferation and apoptosis. Effects on migration and metastatic markers were assessed in MCF7. RESULTS: 24R,25(OH)2D3 reduced MCF7 tumor growth and metastasis in vivo. In vitro results indicate that this was not due to an anti-proliferative effect; 24R,25(OH)2D3 stimulated DNA synthesis in MCF7 and T47D. In contrast, markers of invasion and metastasis were decreased. 24R,25(OH)2D3 caused dose-dependent increases in apoptosis in MCF7 and T47D, but not HCC38 cells. Inhibitors to palmitoylation, caveolae integrity, phospholipase-D, and estrogen receptors (ER) demonstrate that 24R,25(OH)2D3 acts on MCF7 cells through caveolae-associated, phospholipase D-dependent mechanisms via cross-talk with ERs. CONCLUSION: These results indicate that 24R,25(OH)2D3 shows promise in treatment of breast cancer by stimulating tumor apoptosis and reducing metastasis. GENERAL SIGNIFICANCE: 24R,25(OH)2D3 regulates breast cancer cell survival through ER-associated mechanisms similar to 24R,25(OH)2D3 effects on chondrocytes. Thus, 24R,25(OH)2D3 may modulate cell survival in other estrogen-responsive cell types, and its therapeutic potential should be investigated in ER-associated pathologies.