Microfibrillar-associated Protein 5 Research Articles

Multicompartment Polyion Complex Micelles Based on Triblock Polypept(o)ides Mediate Efficient siRNA Delivery to Cancer-Associated Fibroblasts for Antistromal Therapy of Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is the most frequent type of primary liver cancer and the third leading cause for cancer-related death worldwide. The tumor is difficult-to-treat due to its inherent resistance to chemotherapy. Antistromal therapy is a novel therapeutic approach, targeting cancer-associated fibroblasts (CAF) in the tumor microenvironment. CAF-derived microfibrillar-associated protein 5 (MFAP-5) is identified as a novel target for antistromal therapy of HCC with high translational relevance. Biocompatible polypept(o)ide-based polyion complex micelles (PICMs) constructed with a triblock copolymer composed of a cationic poly(l-lysine) complexing anti-MFAP-5 siRNA (siMFAP-5) via electrostatic interaction, a poly(γ-benzyl-l-glutamate) block loading cationic amphiphilic drug desloratatine (DES) via π-πinteraction as endosomal escape enhancer and polysarcosine poly(N-methylglycine) for introducing stealth properties, are generated for siRNA delivery. Intravenous injection of siMFAP-5/DES PICMs significantly reduces the hepatic tumor burden in a syngeneic implantation model of HCC, with a superior MFAP-5 knockdown effect over siMFAP-5 PICMs or lipid nanoparticles. Transcriptome and histological analysis reveal that MFAP-5 knockdown inhibited CAF-related tumor vascularization, suggesting the anti-angiogenic effect of RNA interference therapy. In conclusion, multicompartment PICMs combining siMFAP-5 and DES in a single polypept(o)ide micelle induce a specific knockdown of MFAP-5 and demonstrate a potent antitumor efficacy (80% reduced tumor burden vs untreated control) in a clinically relevant HCC model.

Extracellular matrix analysis of fibrosis: A step towards tissue engineering for urethral stricture disease.

The urogenital tract is a target for many congenital and acquired diseases, both benign and oncogenic. In males, the urethra that transports urine and semen can be obstructed by a fibrotic disease called urethral stricture disease (USD). In severe USD, the whole organ including the vascular embedding, the corpus spongiosum (CS), is affected. Recurrent or severe USD is treated by reconstructive surgery. Tissue engineering may improve the outcome of urethral reconstruction in patients with complicated USD. Currently in urethral reconstruction only the epithelial layer is replaced, no substitution for the CS is provided, while the CS is important for mechanical support and vascularization. To develop a tissue engineering strategy for the CS, it is necessary to know the protein composition of the CS. As the extracellular matrix (ECM) plays an important role in the formation of fibrosis, we analyzed the distribution and localization of ECM components in human healthy and fibrotic CS tissue using immunohistology. The morphology of components of the elastic network were affected in USD. After decellularization a clear enrichment of proteins belonging to the ECM was found. In the proteomic analysis collagens COL15A1 and COL4A2 as well as inter-alpha-trypsin inhibitor ITIH4 were upregulated in fibrotic samples. The glycoproteins Periostin (POSTN), Microfibrillar-associated protein 5 (MFAP5) and EMILIN2 are downregulated in fibrotic tissue. To our knowledge this is the first proteomic study of ECM proteins of the CS in healthy and in USD. With these results a regenerating approach for tissue engineered CS can be developed, including relevant ECM proteins that reduce fibrosis and promote healthy healing in urethral reconstructive surgery.

Tricellulin, α-Catenin and Microfibrillar-Associated Protein 5 Exhibit Concomitantly Altered Immunosignals along with Vascular, Extracellular and Cytoskeletal Elements after Experimental Focal Cerebral Ischemia.

Along with initiatives to understand the pathophysiology of stroke in detail and to identify neuroprotective targets, cell-stabilizing elements have gained increasing attention. Although cell culture experiments have indicated that tricellulin, α-catenin and microfibrillar-associated protein 5 (MFAP5) contribute to cellular integrity, these elements have not yet been investigated in the ischemic brain. Applying immunofluorescence labeling, this study explored tricellulin, MFAP5 and α-catenin in non-ischemic and ischemic brain areas of mice (24, 4 h of ischemia) and rats (4 h of ischemia), along with collagen IV and fibronectin as vascular and extracellular matrix constituents and microtubule-associated protein 2 (MAP2) and neurofilament light chain (NF-L) as cytoskeletal elements. Immunosignals of tricellulin and notably MFAP5 partially appeared in a fiber-like pattern, and α-catenin appeared more in a dotted pattern. Regional associations with vascular and extracellular constituents were found for tricellulin and α-catenin, particularly in ischemic areas. Due to ischemia, signals of tricellulin, MFAP5 and α-catenin decreased concomitantly with MAP2 and NF-L, whereby MFAP5 provided the most sensitive reaction. For the first time, this study demonstrated ischemia-related alterations in tricellulin, MFAP5 and α-catenin along with the vasculature, extracellular matrix and cytoskeleton. Confirmatory studies are needed, also exploring their role in cellular integrity and the potential for neuroprotective approaches in stroke.

MFAP5 in Cancer-associated Fibroblasts Regulates the Progression of the PDAC

Introduction: Cancer-associated fibroblasts (CAFs) are one of the main components of the tumor microenvironment in pancreatic ductal adenocarcinoma (PDAC), and microfibrillar-associated protein 5 (MFAP5) is significantly highly expressed in CAFs. However, the exact role of MFAP5 in CAFs on progression of PDAC remains unclear.

Microfibrillar-associated protein 5 regulates osteogenic differentiation by modulating the Wnt/\u03b2-catenin and AMPK signaling pathways

BackgroundDysfunctional osteogenesis of bone marrow mesenchymal stem cells (BMSCs) plays an important role in osteoporosis occurrence and development. However, the molecular mechanisms of osteogenic differentiation remain unclear. This study explored whether microfibrillar-associated protein 5 (MFAP5) regulated BMSCs osteogenic differentiation.MethodsWe used shRNA or cDNA to knock down or overexpress MFAP5 in C3H10 and MC3T3-E1 cells. AR-S- and ALP-staining were performed to quantify cellular osteogenic differentiation. The mRNA levels of the classical osteogenic differentiation biomarkers Runx2, Col1α1, and OCN were quantified by qRT-PCR. Finally, we employed Western blotting to measure the levels of Wnt/β-catenin and AMPK signaling proteins.ResultsAt days 0, 3, 7, and 14 after osteogenic induction, AR-S- and ALP-staining was lighter in MFAP5 knockdown compared to control cells, as were the levels of Runx2, Col1α1 and OCN. During osteogenesis, the levels of β-catenin, p-GSK-3β, AMPK, and p-AMPK were upregulated, while that of GSK-3β was downregulated, indicating that Wnt/β-catenin and AMPK signaling were activated. The relevant molecules were expressed at lower levels in the knockdown than control group; the opposite was seen for overexpressing cell lines.ConclusionsMFAP5 regulates osteogenesis via Wnt/β‑catenin- and AMPK-signaling; MFAP5 may serve as a therapeutic target in patients with osteoporosis.

CAFs-derived MFAP5 promotes bladder cancer malignant behavior through NOTCH2/HEY1 signaling.

Cancer-associated fibroblasts (CAFs) are an important component of the tumor microenvironment and contribute to tumor cell proliferation and metastasis. Microfibrillar-associated protein 5 (MFAP5), a component of elastic microfibers and an oncogenic protein in several types of tumors, is secreted by CAFs. However, the role of MFAP5 in the bladder cancer remains unclear. Here, we report that MFAP5 is upregulated in bladder cancer and is associated with poor patient survival. Downregulation of MFAP5 in CAFs led to an impairment in proliferation and invasion of bladder cancer cells. Luciferase reporter assays and electrophoretic mobility shift assays (EMSA) showed QKI directly downregulates MFAP5 in CAFs. In addition, CAFs-derived MFAP5 led to an activation of the NOTCH2/HEY1 signaling pathway through direct interaction with the NOTCH2 receptor, thereby stimulating the N2ICD release. RNA-sequencing revealed that MFAP5-mediated PI3K-AKT signaling activated the DLL4/NOTCH2 pathway axis in bladder cancer. Moreover, downregulation of NOTCH2 by short hairpin RNA or the inactivating anti-body NRR2Mab was able to reverse the adverse effects of MFAP5 stimulation in vitro and in vivo. Together, these results demonstrate CAFs-derived MFAP5 promotes the bladder cancer proliferation and metastasis and provides new insight for targeting CAFs as novel diagnostic and therapeutic strategy.

Hypoxia-induced MFAP5 Promotes Tumor Migration and Invasion via AKT Pathway in Head and Neck Squamous Cell Carcinoma.

Objective: Microfibrillar-associated protein 5 (MFAP5) is highly expressed in many types of cancers. Our previous study has observed that overexpression of MFAP5 was correlated with lymph nodes metastasis and poor prognosis in head and neck squamous cell carcinoma (HNSCC), but the underlying mechanism is poorly understood.Materials and methods: The MFAP5 expression is detected under hypoxia condition. HNSCC cell lines are transfected with MFAP5-expressing lentivirus vector to establish stable overexpression model. Wound-healing, migration and invasion assay are used to determine the effect of MFAP5 on HNSCC and metastasis-related proteins are examined by Western blot. In vivo lung metastasis assays are conducted by the tail vein injection. In addition, immunohistochemistry is applied to analyze the correlation of MFAP5, hypoxia-induced factor-1 α (HIF-1α), and vimentin in 84 HNSCC patients' tissue samples.Results: Firstly, MFAP5 expression can be markedly induced under hypoxia condition in HNSCC cell lines. Cell lines with MFAP5 overexpression has a significant higher ability of migration and invasion. In addition, in vivo assay observes that overexpression of MFAP5 can promote tumor lung metastasis. Furthermore, MFAP5 facilitates this process by activating epithelial-mesenchymal transition (EMT) program via AKT pathway in HNSCC cell lines. The pro-metastatic effect of MFAP5 can be reversed by MK2206, an AKT phosphorylation inhibitor. Lastly, the positive correlation among HIF-1α, MFAP5 and vimentin from tissue samples and TCGA dataset are also observed in HNSCC.Conclusion: Our study demonstrates MFAP5 plays a critical role in hypoxia-induced EMT program via AKT pathway in HNSCC, which would be a very promising therapeutic target.

Prognostic and clinicopathological values of tissue expression of MFAP5 and ITM2A in triple-negative breast cancer: an immunohistochemical study.

IntroductionTriple-negative breast cancer (TNBC) is a markedly aggressive molecular subtype of breast cancer; there is an urgent need to clarify the molecular mechanisms underlying the progression and metastases of BLBC, in order to find a novel targeted therapy. Microfibrillar-associated protein 5 (MFAP5) plays an essential role in the regulation of cell behaviour and survival. Integral membrane protein 2A (ITM2A) is a type II transmembrane protein, which is a member of a family of autophagy related proteins.The aim of this study was to assess the expression of MFAP5 and ITM2A proteins in tissues of BLBC using immunohistochemistry, in order to correlate the expression with clinicopathological and prognostic parameters of such aggressive cancer.Material and methodsThe present study included sections from archived paraffin blocks retrieved from 120 patients with TNBC. We collected cases from three years, i.e. from 2016 to 2019. We assessed expression of MFAP5 and ITM2A using immunohistochemistry.ResultsHigh expression of MFAP5 and low expression of ITM2A was associated with advanced stage (p = 0.007), higher grade of tumour (p = 0.005 and p = 0.004, respectively), the presence of lymph nodes metastases (p < 0.001 and p = 0.002, respectively), lower three-year RFS rate (p < 0.001 and p = 0.016, respectively), and lower three-year OS rate (p < 0.001).ConclusionsMFAP5 and ITM2A are novel prognostic biomarkers for breast cancer and might be considered as promising therapeutic targets for patients with breast cancer, particularly TNBC molecular subtype, in the future.

MFAP5 facilitates the aggressiveness of intrahepatic Cholangiocarcinoma by activating the Notch1 signaling pathway

BackgroundIntrahepatic cholangiocarcinoma (ICC) is the second most common primary liver cancer. The dismal outcome of ICC patients is due to lack of early diagnosis, the aggressive biological behavior of ICC and the lack of effective therapeutic options. Early diagnosis and prognosis of ICC by non-invasive methods would be helpful in providing valuable information and developing effective treatment strategies.MethodsExpression of microfibrillar-associated protein 5 (MFAP5) in the serum of ICC patients was detected by ELISA. Human ICC specimens were immunostained by MFAP5 antibodies. The growth rate of human ICC cell lines treated with MFAP5 or MFAP5 shRNAs was examined by CCK8 and colony formation assays. Cell cycle analysis was performed with PI staining. The effect of MFAP5 inhibition was assessed by xenograft models in nude mice. RNA-seq and ATAC-seq analyses were used to dissect the molecular mechanism by which MFAP5 promoted ICC aggressiveness.ResultsWe identified MFAP5 as a biomarker for the diagnosis and prognosis of ICC. Upregulated MFAP5 is a common feature in aggressive ICC patients’ tissues. Importantly, MFAP5 level in the serum of ICC patients and healthy individuals showed significant differential expression profiles. Furthermore, we showed that MFAP5 promoted ICC cell growth and G1 to S-phase transition. Using RNA-seq expression and ATAC-seq chromatin accessibility profiling of ICC cells with suppressed MFAP5 secretion, we showed that MFAP5 regulated the expression of genes involved in the Notch1 signaling pathway. Furthermore, FLI-06, a Notch signaling inhibitor, completely abolished the MFAP5-dependent transcriptional programs.ConclusionsRaised MFAP5 serum level is useful for differentiating ICC patients from healthy individuals, and could be helpful in ICC diagnosis, prognosis and therapies.

Immunohistochemical Expression of Microfibrillar-associated Protein 5 (MFAP5) in Invasive Breast Carcinoma of No Special Type.

Breast cancer (BC) remains the most prevalent female cancer in Egypt and worldwide. Microfibrillar-associated protein 5 (MFAP5) is a multifunctional glycoprotein. Although MFAP5 gene was among the genes that found globally expressed in human cancers, it had been only recently reported in few cancer research studies. This is a retrospective study that has been conducted on 66 Egyptian patients who had invasive carcinoma of no special type. Immunohistochemical staining for MFAP5 was applied on the archival formalin-fixed paraffin-embedded blocks. Staining was assessed semiquantitatively and correlated with the available clinicopathologic parameters and immunohistochemical subtypes of BC. MFAP5 epithelial cytoplasmic expression was observed in 89.4% (59/66) of cases. In contrast, nuclear expression was seen in non-neoplastic breast lobules and premalignant lesions adjacent to tumors that also exhibited constant staining in myoepithelial layer. Statistical analysis of epithelial cytoplasmic expression revealed association of MFAP5 expression with tumor size (P=0.046), high histologic grade (P=0.007), presence of lymph node metastasis (P=0.014), poor Nottingham Prognostic Index (NPI) (P=0.001), late stage (P=0.008), immunohistochemical subtypes of BC (P=0.018), and increased microvessel density using CD34 immunostianing (P=0.04). MFAP5 cytoplasmic expression was also observed in an adjacent duct carcinoma in situ component in 37/45 cases (82.2%). This study showed that MFAP5 is a novel myoepithelial cell marker that appears to be upregulated in duct epithelium in duct carcinoma in situ and invasive carcinoma of no special type during tumorogenesis and that its cytoplasmic expression in invasive tumors seems to have a poor prognostic role manifested by its association with poor prognostic parameters such as high grade, late stage, lymph node invasion, and increased microvessel density.

Stromal fibroblast-derived MFAP5 promotes the invasion and migration of breast cancer cells via Notch1/slug signaling.

The tumor microenvironment (TME) regulates tumor progression, and cancer-associated fibroblasts (CAFs) are the primary stromal components of the TME, with the potential to drive tumor metastasis via the secretion of paracrine factors, but the specific mechanisms driving this process have not been defined. Proteins secreted from CAFs and normal fibroblasts (NFs) were analyzed via proteomic analysis (fold change > 2, p < 0.05) to identify tumor-promoting proteins secreted by CAFs. Proteomic analysis revealed that microfibrillar-associated protein 5 (MFAP5) is preferentially expressed and secreted by CAFs relative to NFs, which was confirmed by Western blotting and RT-qPCR. Transwell and wound healing assays confirmed that MFAP5 is secreted by CAFs, and drives the invasion and migration of MCF7 breast cancer cells. We further found that in MCF7 cells MFAP5 promoted epithelial-mesenchymal transition, activating Notch1 signaling and consequently upregulating NICD1 and slug. When Notch1 was knocked down in MCF7 cells, the ability of MFAP5 to promote invasion and migration decreased. CAFs promote cancer cells invasion and migration via MFAP5 secretion and activation of the Notch1/slug signaling. These data highlight this pathway as a therapeutic target to disrupt tumor progression through the interference of CAF-tumor crosstalk.

MFAP5 promotes basal-like breast cancer progression by activating the EMT program

PurposeHuman basal-like breast cancer (BLBC) is an aggressive malignancy with poor prognosis. Since most current treatments are ineffective, there is an urgent need to identify therapeutic targets for BLBC. Microfibrillar-associated protein 5 (MFAP5) plays an important role in the integration of elastic microfibers and the regulation of endothelial cell behaviors. We previously demonstrated that MFAP5 was significantly overexpressed in BLBC tissues and associated with poor metastasis-free survival of patients with BLBC. However, the detailed role of MFAP5 in BLBC is unclear. Thereby, the current study aimed to investigate the underlying function of MFAP5 in BLBC.MethodFunctional analyses were conducted for the role of MFAP5 in BLBC in vitro and in vivo.ResultsOverexpression of MFAP5 resulted in a significant increase in the proliferation, migration, invasion and epithelial–mesenchymal transition (EMT) markers in BLBC in vitro and in vivo. In addition, other metastasis animal models by tail intravenous injection of BT20 cells further confirmed that MFAP5 overexpression promoted BLBC proliferation and BT20 cells metastasis. We found that the TGF-β or Notch inhibitor significantly reversed the tumorigenicity and metastasis of MFAP5-induced BLBC cells.ConclusionOur findings suggest that MFAP5 may promote EMT in BLBC metastasis via the TGF-β/Notch pathway.

MFAP5 suppression inhibits migration/invasion, regulates cell cycle and induces apoptosis via promoting ROS production in cervical cancer

Cervical cancer is one of the most lethal types of cancer among female. Microfibrillar-associated protein 5 (MFAP5) is an extracellular matrix (ECM) glycoprotein, and is confirmed to be involved in cell signaling during microfibril assembly, elastinogenesis and cell survival. However, the role of MFAP5 in cervical cancer development and progression remains poorly understood. In the study, MFAP5 was over-expressed in human cervical cancers, and in different cervical cancer cell lines. Patients suffering from cervical cancer with low MFAP5 expression exhibited better survival rate. Suppressing MFAP5 in cervical cancer cells markedly reduced the cell proliferation, migration and invasion by modulating epithelial-mesenchymal transition (EMT)-related signaling pathway. In addition, MFAP5 knockdown induced large number of cells distributed in G2/M phase, along with reduced Cyclin B1, Cyclin D1 and cyclin-dependent kinase 4 (CDK4) expressions, and enhanced p21 and p53 levels. Moreover, apoptosis was highly induced by MFAP5 silence through reducing Bcl-xl and Bcl-2 expressions, and promoting Bax, cleaved Caspase-3 and poly (ADP-Ribose) polymerase (PARP) expressions in cervical cancer cells. Reactive oxygen species (ROS) production levels were also higher in MFAP5-knockdown cells, along with Jun-N-terminal kinase (JNK) activation. Importantly, we found that MFAP5 knockdown-inhibited cervical cancer cell growth was dependent on ROS production. Finally, the depletion of MFAP5 prevented cervical cancer progression in vivo. In summary, our study identified a critical role played by MFAP5 in the progression of cervical cancer and the potential mechanisms by which exerted its effects, indicating that targeting MFAP5-related pathways could be conducive to the therapies for cervical cancer.

PO-174 Evaluation of immunohistochemical expression of microfibrillar-associated protein 5 (MFAP5) in invasive breast carcinoma of no special type

IntroductionBreast cancer is one of the most malignant cancer types which is the second leading cause of cancer-associated deaths in women. Breast cancer begins as a local disease which can then metastasize to lymph nodes and distant sites. The metastasis of breast cancer is mainly to bone, lung and liver. Being the leading cause of cancer related deaths, it is essential to understand the complex metastasis phenomenon to develop new diagnostic and therapeutic strategies. In this study, we aim to develop a lab-on-a-chip (LOC) system to detect the metastatic capacities of breast cancer cells to different target tissues.Material and methodsThe triple negative breast cancer cell line MDA-MB-231 was selected based on its high metastatic capacity, while normal lung, liver and bone fibroblasts WI-38, BRL3A and HS-5 and human umbilical vein endothelial cell line HUVEC-C were used to generate target tissue microenvironments and endothelial monolayer, respectively. A three-channel LOC system, one for endothelial monolayer formation, one for mimicking target tissue microenvironment and one for media supply, was used. The seeding cell densities, common medium composition and ECM components for microenvironment were optimised for the LOC system. Following addition of cancer cell lines to the endothelial monolayer channel, invasion and extravasation capacities to different target sites were determined by tracking 3D distribution of fluorescently labelled cancer cells up to four days.Results and discussionsMDA-MB-231 cells were shown to invade and extravasate preferentially to lung and liver microenvironments, while their movement to bone or cell-free target sites was limited. The observed results were in agreement with the previously reported phenotype of MDA-MB-231 cell lines.ConclusionOur results show that tissue- specific cell lines in a LOC system can be used to mimic target tissue microenvironments and evaluate different invasion and extravasation capacities of breast cancer cell lines to different target sites.

MFAP5 promotes tumor progression and bone metastasis by regulating ERK/MMP signaling pathways in breast cancer

Breast cancer accounts for about 30% of all cancers in women, while approximately 70% breast cancer patients developed bone metastases throughout the course of their disease, highlighting the importance of exploring new therapeutic targets. Microfibrillar-associated protein 5 (MFAP5) is a component of extracellular elastic microfibril which has been confirmed to function in tissue development and cancer progression. But the role of MFAP5 in breast cancer remains unclear. The present study demonstrated that MFAP5 was up-regulated in breast cancers compared with that in normal breast tissues, and further increased in breast cancer bone metastasis. Functionally, MFAP5 overexpression accelerated breast cancer cell proliferation and migration, while an opposite effect was observed when MFAP5 was knocked down. In addition, up-regulation of MFAP5 increased the expression of MMP2 and MMP9 and activated the ERK signaling pathway. Conversely, inhibition of MFAP5 suppressed the expression of MMP2, MMP9, p-FAK, p-Erk1/2 and p-cJun. These findings may provide a better understanding about the mechanism of breast cancer and suggest that MFAP5 may be a potential prognostic biomarker and therapeutic target for breast cancer, especially for bone metastasis of breast cancer.

Cancer-associated fibroblasts regulate endothelial adhesion protein LPP to promote ovarian cancer chemoresistance.

The molecular mechanism by which cancer-associated fibroblasts (CAFs) confer chemoresistance in ovarian cancer is poorly understood. The purpose of the present study was to evaluate the roles of CAFs in modulating tumor vasculature, chemoresistance, and disease progression. Here, we found that CAFs upregulated the lipoma-preferred partner (LPP) gene in microvascular endothelial cells (MECs) and that LPP expression levels in intratumoral MECs correlated with survival and chemoresistance in patients with ovarian cancer. Mechanistically, LPP increased focal adhesion and stress fiber formation to promote endothelial cell motility and permeability. siRNA-mediated LPP silencing in ovarian tumor-bearing mice improved paclitaxel delivery to cancer cells by decreasing intratumoral microvessel leakiness. Further studies showed that CAFs regulate endothelial LPP via a calcium-dependent signaling pathway involving microfibrillar-associated protein 5 (MFAP5), focal adhesion kinase (FAK), ERK, and LPP. Thus, our findings suggest that targeting endothelial LPP enhances the efficacy of chemotherapy in ovarian cancer. Our data highlight the importance of CAF-endothelial cell crosstalk signaling in cancer chemoresistance and demonstrate the improved efficacy of using LPP-targeting siRNA in combination with cytotoxic drugs.

YAP promotes proliferation, chemoresistance, and angiogenesis in human cholangiocarcinoma through TEAD transcription factors.

The Yes-associated protein (YAP)/Hippo pathway has been implicated in tissue development, regeneration, and tumorigenesis. However, its role in cholangiocarcinoma (CC) is not established. We show that YAP activation is a common feature in CC patient biopsies and human CC cell lines. Using microarray expression profiling of CC cells with overexpressed or down-regulated YAP, we show that YAP regulates genes involved in proliferation, apoptosis, and angiogenesis. YAP activity promotes CC growth in vitro and in vivo by functionally interacting with TEAD transcription factors (TEADs). YAP activity together with TEADs prevents apoptosis induced by cytotoxic drugs, whereas YAP knockdown sensitizes CC cells to drug-induced apoptosis. We further show that the proangiogenic microfibrillar-associated protein 5 (MFAP5) is a direct transcriptional target of YAP/TEAD in CC cells and that secreted MFAP5 promotes tube formation of human microvascular endothelial cells. High YAP activity in human CC xenografts and clinical samples correlates with increased MFAP5 expression and CD31(+) vasculature. These findings establish YAP as a key regulator of proliferation and antiapoptotic mechanisms in CC and provide first evidence that YAP promotes angiogenesis by regulating the expression of secreted proangiogenic proteins.

Abstract 4184: CAF-derived MFAP5 promotes tumor angiogenesis and confers paclitaxel resistance in high-grade serous ovarian cancer

Abstract High-grade serous ovarian cancer (HGSOC) is the most lethal gynecologic malignancy. Although most patients respond to chemotherapy, relapse often occurs as chemoresistance disease. We previously reported the tumor-promoting role of Microfibrillar-Associated Protein 5 (MFAP5) in ovarian cancer. Follow up studies suggested a pro-angiogenic and chemo-protective role of MFAP5. Based on these findings, the present study aims at delineating the mechanism by which stromal MFAP5 modulates tumor angiogenesis and chemoresistance, and evaluating the potential of targeting MFAP5 as therapy for ovarian cancer. Immunostaining of HGSOC tissue sections revealed a significant correlation between stromal MFAP5 expression and intratumoral microvessel density. Further endothelial cell motility and tube formation assays on human endothelial cells demonstrated the pro-angiogenic roles of exogenous MFAP5. Furthermore, anti-αVβ3 integrin antibody and cell permeant calcium chelator, BAPTA, attenuated MFAP5's effects, suggesting that MFAP5 exerts its biological activity on endothelial cells through the αVβ3 integrin receptor and calcium signaling. In vivo studies showed that extensive tubular network was formed in the matrigel mixed with MFAP5 injected into mice (p&amp;lt;0.01). Co-injection of MFAP5 overexpressing fibroblasts and cancer cells subcutaneously into mice led to increased tumor size and number of CD34 positive intratumor microvessels. Microarray analysis on endothelial cells invaded into Matrigel plug identified a set of motility promoting genes that were associated with calcium signaling. Among them, upregulation of Lipoma-Peferred Partner (LPP), an actin cytoskeleton protein, was validated by qRT-PCR and western blot analyses. Silencing LPP by siRNA abolished MFAP5 stimulated motility in both HMEC-1 and TIME. Further western blot analyses showed that MFAP5 modulated endothelial cell motility and invasion potential through calcium dependent ERK/CREB/LPP signaling pathways. In an orthotopic mouse model, silencing of stromal MFAP5 by siRNA delivered by nanoparticles demonstrated a significant reduction in tumor weight (p&amp;lt;0.001) and intratumor microvessel densities. In addition, analysis on stromal gene expression profiles revealed that stromal MFAP5 expression is significantly increased in the group of patients with shorter progression-free survival when compared to those with longer progression-free survival. Further cell survival assays showed that pretreating to HGSOC cell lines with recombinant MFAP5 confers resistance to ovarian cancer cells towards paclitaxel treatment. RNA-Seq analysis was performed to delineate the underlying mechanism of such induced chemoresistance. To conclude, stromal MFAP5 contributes to ovarian tumor angiogenesis and chemoresistance. Hence, targeting stromal MFAP5 could be a potential therapy for ovarian cancer treatment. Citation Format: Cecilia S. Leung, Tsz-Lun Yeung, Kay-Pong Yip, Kwong-Kwok Wong, Anil K. Sood, Michael J. Birrer, Samuel C. Mok. CAF-derived MFAP5 promotes tumor angiogenesis and confers paclitaxel resistance in high-grade serous ovarian cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4184. doi:10.1158/1538-7445.AM2015-4184

MFAP5is related to obesity-associated adipose tissue and extracellular matrix remodeling and inflammation

Low-grade inflammation is involved in adipose tissue (AT) and extracellular matrix (ECM) remodeling and induces deposition of ECM proteins in AT. We have previously shown that MFAP5 (microfibrillar-associated protein 5) expression decreases in AT after weight loss. The aim of this study was to investigate MFAP5 localization in human AT and gene expression in adipocytes and the role of MFAP5 in adipocyte metabolism and inflammation. MFAP5 protein localization and gene expression were studied with immunohistochemistry and quantitative reverse transcriptase PCR (RT-qPCR) in human subcutaneous AT and cultured Simpson-Golabi-Behmel syndrome (SGBS) adipocytes, respectively. The effect of MFAP5 knock-down by siRNA on gene expression and insulin action was examined with RT-qPCR, western blot, and insulin-stimulated glucose uptake. The effect of different cytokines on MFAP5 gene and protein expression was investigated in cultured human SGBS preadipocytes. MFAP5 protein was highly expressed in AT, and gene expression decreased during adipocyte differentiation in SGBS cells. Treatment of preadipocytes with TNFα and TGFβ1 increased MFAP5 gene and protein expression. Furthermore, MFAP5 knock-down decreased the expression of genes involved in inflammation. Our results demonstrate that factors involving low-grade inflammation modulate MFAP5 expression and that the modified expression of MFAP5 may further regulate AT inflammation.

CAF reprogramming inhibits ovarian cancer progression

Development and progression of cancer are often facilitated by deregulation of gene expression and accumulation of somatic mutations. Therefore, major research efforts have focused on cancer cells to identify these disease-related genes and mutations, and investigators have developed therapies that target such deregulated genes and mutations and used them for a variety of cancers. In addition to cancer cells, the complex and relatively unexplored tumor microenvironment plays an important role in cancer progression. However, therapeutic regimens targeting tumor stromal cells, particularly cancer-associated fibroblasts (CAFs), have yet to be developed. Because the tumor-supportive roles of CAFs are increasingly being recognized, researchers have begun to evaluate the potential of inhibiting tumor progression via stromal ablation. However, multiple studies demonstrated that ablation of activated myofibroblasts in tumors actually promotes tumor progression and reduces survival.1,2 Although inhibiting tumor progression by targeting stroma via global depletion of CAFs may not be viable, reprogramming CAFs by targeting deregulated genes presents the opportunity for developing novel cancer therapy. CAF reprogramming allows silencing of CAF-derived tumor-promoting factors while preserving the overall structural integrity of the extracellular matrix. We believe that reprogramming CAFs in the tumor microenvironment can inhibit tumor progression, thereby presenting an alternative approach to the treatment of cancer. Transcriptome profiling of laser-microdissected ovarian tumor samples enabled us to identify stroma-exclusive genes that are deregulated in tumors. This cannot be achieved using bulk tumor samples containing various amounts of epithelial and stromal components. Using laser-microdissected tissue samples, we identified a CAF gene expression signature for high-grade serous ovarian cancer (HGSOC).3 Immunolocalization of microfibrillar-associated protein 5 (MFAP5) in tissues samples obtained from HGSOC patients and cell culture studies demonstrated that whereas MFAP5 was expressed by cancer cells in a small group of patients, CAFs had markedly higher MFAP5 expression than that in cancer cells, and MFAP5 was exclusively expressed by CAFs in the majority of HGSOC patients.4,5 We further evaluated the functional roles of MFAP5 in ovarian cancer progression. We observed that MFAP5 was actively secreted by CAFs into the extracellular matrix, where MFAP5 binds to the αvβ3 integrin receptors on the surface of ovarian cancer cells. Engagement of αvβ3 integrin with MFAP5 on cancer cells subsequently activates the calcium-dependent focal adhesion kinase/extracellular signal-regulated kinase/cAMP response element-binding protein signaling pathway, which leads to upregulation of troponin C type 1 (TNNC1) expression. TNNC1 facilitates the formation and rearrangement of the F-actin cytoskeleton and enhances the motility of ovarian cancer cells via induction of traction force.5 TNNC1 is commonly expressed by muscle cells, including those in skeletal and cardiac muscles, and is involved in the contraction of these muscles. Further survival analysis demonstrated that TNNC1 is an independent biomarker for poor prognosis for HGSOC. Besides stromal-epithelial cross-talk mediated by CAF-derived MFAP5, we found a positive correlation between MFAP5 expression and microvessel density,4,5 suggesting that CAF-derived MFAP5 modulates tumor progression via increased angiogenesis. However, the underlying molecular mechanism of how MFAP5 stimulates angiogenesis remains to be elucidated. Taken together with the fact that high stromal MFAP5 expression is closely associated with poor outcome of HGSOC and functional studies demonstrated the stimulatory effects of MFAP5 on the motility and invasive potential of ovarian cancer cells, reprogramming of CAFs by targeting MFAP5 may be a novel therapeutic strategy for ovarian cancer that may improve survival in HGSOC patients. To determine the effect of reprogramming of CAFs by targeting MFAP5 on ovarian cancer progression, we injected murine MFAP5-targeting small interfering RNA (siRNA) incorporated into chitosan nanoparticles into ovarian cancer-bearing mice. We observed that this treatment successfully silenced MFAP5 expression in CAFs, decreased the expression of MFAP5's downstream target gene TNNC1 in tumor cells, and inhibited ovarian tumor growth, invasion, and metastasis.5 These findings suggested that delivery of siRNAs specific to CAF-expressing genes could reprogram CAFs and lead to inhibition of tumor progression (Fig. 1). CAF reprogramming can be further improved with targeted delivery of siRNA sequences using vehicles that specifically target CAFs. This improved specificity can certainly increase treatment efficacy, reduce treatment dosages required for gene silencing, and possibly reduce side effects. A number of such targeted delivery platforms are being developed and evaluated.6 Figure 1. CAF reprogramming using MFAP5-tareting siRNA delivered by chitosan nanoparticles significantly reduces the level of CAF-derived MFAP5 protein in the tumor microenvironment. Shutting down MFAP5-modulated activation of αvβ3 integrin and ... In summary, CAF-derived MFAP5 plays important roles in modulating cancer progression and has proven to be a potential therapeutic target for ovarian cancer. These findings suggest that CAF reprogramming via targeted gene silencing in these cells is a new paradigm for ovarian cancer treatment. Also, it may synergize with other treatment regimens that target cancer cells to increase treatment efficacy.