Protein Profilin-1 Research Articles

40 Preclinical evaluation of a novel dendritic cell vaccine for kidney cancer

Abstract Background Dendritic cell (DC)-based vaccines have been previously shown to promote therapeutic T cell responses in both preclinical tumor models and in cancer patients, where extended patient overall survival has been noted in many cases. The goal of the present study is to develop a novel DC-based vaccine as an effective immuno-oncology (IO) agent for the prevention/treatment of kidney cancer. We previously demonstrated that actin-binding protein profilin1 (Pfn1) is overexpressed in tumor-associated vascular endothelial cells (ECs), where it may serve as a prognostic factor in human clear cell renal cancer (ccRCC). We further established a direct causal relationship between EC Pfn1 dysregulation, immune microenvironment alterations, and tumor progression in mouse models of RCC. The current work specifically explores whether Pfn1-targeted DC-based vaccines are effective in preventing orthotopic RCC in mice. Methods To generate Pfn1-targeted DC vaccines, we harvested and matured DCs from Balb/C mouse bone marrow-derived precursor cells in cultures containing rmIL4 and rmGM-CSF. DCs loaded with individual Pfn1 synthetic peptides (these sequences were identified using three MHC class I/II peptide-binding algorithms) were injected into syngeneic mice on days 0, 7 and 14. Spleens were harvested from immunized mice on day 21 to isolate CD4+ and CD8+ T cells, with T cells then stimulated with unmanipulated DCs or DCs pulsed with individual immunizing peptides to detect specific T cell activation, as measured by IFN-gamma release quantitated by ELISA. Tissue histology was performed to assess immune-related adverse events (iRAE) in vaccinated mice as a safety index. For cancer studies, Balb/c mice were immunized with DCs loaded with pooled Pfn1 peptides a few days prior to establishing either subcutaneous or orthotopic RCC tumors. Tumor-bearing mice were subjected to an identical booster vaccine prior one week later in advance of euthanasia and study end-point analyses. Results Our studies show that vaccination of Balb/c mice with DCs pulsed with Pfn1 peptides elicit specific CD4+ and/or CD8+ T cell responses without promoting irAEs. When applied in the prophylactic setting, DC-Pfn1 peptide vaccines substantially slow the growth of subcutaneous RCC tumors. In support of these findings, we have also developed preliminary data supporting the therapeutic effects of DC/Pfn1-peptide vaccines in a murine orthotopic model of RCC. Conclusions In summary, our studies provide first evidence for Pfn1-targeted IO agent in the cancer setting. Given that the survival benefit for dual immune-check point inhibitors (ICI)/anti-angiogenic therapy remains modest and is limited to a small minority of ccRCC patients, Pfn1-targeted vaccines could represent a novel therapeutic agent for improved patient outcomes when applied alone or in combination with in-clinic ICI agents. DOD CDMRP Funding: yes

The actin binding protein profilin 1 localizes inside mitochondria and is critical for their function

The monomer-binding protein profilin 1 (PFN1) plays a crucial role in actin polymerization. However, mutations in PFN1 are also linked to hereditary amyotrophic lateral sclerosis, resulting in a broad range of cellular pathologies which cannot be explained by its primary function as a cytosolic actin assembly factor. This implies that there are important, undiscovered roles for PFN1 in cellular physiology. Here we screened knockout cells for novel phenotypes associated with PFN1 loss of function and discovered that mitophagy was significantly upregulated. Indeed, despite successful autophagosome formation, fusion with the lysosome, and activation of additional mitochondrial quality control pathways, PFN1 knockout cells accumulate depolarized, dysmorphic mitochondria with altered metabolic properties. Surprisingly, we also discovered that PFN1 is present inside mitochondria and provide evidence that mitochondrial defects associated with PFN1 loss are not caused by reduced actin polymerization in the cytosol. These findings suggest a previously unrecognized role for PFN1 in maintaining mitochondrial integrity and highlight new pathogenic mechanisms that can result from PFN1 dysregulation.

Haplo-insufficiency of Profilin1 in vascular endothelial cells is beneficial but not sufficient to confer protection against experimentally induced atherosclerosis.

Actin cytoskeleton plays an important role in various aspects of atherosclerosis, a key driver of ischemic heart disease. Actin-binding protein Profilin1 (Pfn1) is overexpressed in atherosclerotic plaques in human disease, and Pfn1, when partially depleted globally in all cell types, confers atheroprotection in vivo. This study investigates the impact of endothelial cell (EC)-specific partial loss of Pfn1 expression in atherosclerosis development. We utilized mice engineered for conditional heterozygous knockout of the Pfn1 gene in ECs, with atherosclerosis induced by depletion of hepatic LDL receptor by gene delivery of PCSK9 combined with high-cholesterol diet. Our studies show that partial depletion of EC Pfn1 has certain beneficial effects marked by dampening of select pro-atherogenic cytokines (CXCL10 and IL7) with concomitant reduction in cytotoxic T cell abundance but is not sufficient to reduce hyperlipidemia and confer atheroprotection in vivo. In light of these findings, we conclude that atheroprotective phenotype conferred by global Pfn1 haplo-insufficiency requires contributions of additional cell types that are relevant for atherosclerosis progression.

Actin-binding protein profilin1 is an important determinant of cellular phosphoinositide control

Membrane polyphosphoinositides (PPIs) are lipid-signaling molecules that undergo metabolic turnover and influence a diverse range of cellular functions. PPIs regulate the activity and/or spatial localization of a number of actin-binding proteins (ABPs) through direct interactions; however, it is much less clear whether ABPs could also be an integral part in regulating PPI signaling. In this study, we show that ABP profilin1 (Pfn1) is an important molecular determinant of the cellular content of PI(4,5)P2 (the most abundant PPI in cells). In growth factor (EGF) stimulation setting, Pfn1 depletion does not impact PI(4,5)P2 hydrolysis but enhances plasma membrane (PM) enrichment of PPIs that are produced downstream of activated PI3-kinase, including PI(3,4,5)P3 and PI(3,4)P2, the latter consistent with increased PM recruitment of SH2-containing inositol 5' phosphatase (SHIP2) (a key enzyme for PI(3,4)P2 biosynthesis). Although Pfn1 binds to PPIs invitro, our data suggest that Pfn1's affinity to PPIs and PM presence in actual cells, if at all, is negligible, suggesting that Pfn1 is unlikely to directly compete with SHIP2 for binding to PM PPIs. Additionally, we provide evidence for Pfn1's interaction with SHIP2 in cells and modulation of this interaction upon EGF stimulation, raising an alternative possibility of Pfn1 binding as a potential restrictive mechanism for PM recruitment of SHIP2. In conclusion, our findings challenge the dogma of Pfn1's binding to PM by PPI interaction, uncover a previously unrecognized role of Pfn1 in PI(4,5)P2 homeostasis and provide a new mechanistic avenue of how an ABP could potentially impact PI3K signaling byproducts in cells through lipid phosphatase control.

Vascular endothelial cell-specific disruption of the profilin1 gene leads to severe multiorgan pathology and inflammation causing mortality.

Actin-binding protein Profilin1 is an important regulator of actin cytoskeletal dynamics in cells and critical for embryonic development in higher eukaryotes. The objective of the present study was to examine the consequence of loss-of-function of Pfn1 in vascular endothelial cells (ECs) in vivo. We utilized a mouse model engineered for tamoxifen-inducible biallelic inactivation of the Pfn1 gene selectively in EC (Pfn1EC-KO). Widespread deletion of EC Pfn1 in adult mice leads to severe health complications presenting overt pathologies (endothelial cell death, infarct, and fibrosis) in major organ systems and evidence for inflammatory infiltrates, ultimately compromising the survival of animals within 3 weeks of gene ablation. Mice deficient in endothelial Pfn1 exhibit selective bias toward the proinflammatory myeloid-derived population of immune cells, a finding further supported by systemic elevation of proinflammatory cytokines. We further show that triggering Pfn1 depletion not only directly upregulates proinflammatory cytokine/chemokine gene expression in EC but also potentiates the paracrine effect of EC on proinflammatory gene expression in macrophages. Consistent with these findings, we provide further evidence for increased activation of Interferon Regulatory Factor 7 (IRF7) and STAT1 in EC when depleted of Pfn1. Collectively, these findings for the first time demonstrate a prominent immunological consequence of loss of endothelial Pfn1 and an indispensable role of endothelial Pfn1 in mammalian survival unlike tolerable phenotypes of Pfn1 loss in other differentiated cell types.

Vascular endothelial profilin-1 drives a protumorigenic tumor microenvironment and tumor progression in renal cancer

Overexpression of actin-binding protein profilin-1 (Pfn1) correlates with advanced disease features and adverse clinical outcome of patients with clear cell renal carcinoma, the most prevalent form of renal cancer. We previously reported that Pfn1 is predominantly overexpressed in tumor-associated vascular endothelial cells in human clear cell renal carcinoma. In this study, we combined invivo strategies involving endothelial cell-specific depletion and overexpression of Pfn1 to demonstrate a role of vascular endothelial Pfn1 in promoting tumorigenicity and enabling progressive growth and metastasis of renal carcinoma cells in a syngeneic orthotopic mouse model of kidney cancer. We established an important role of endothelial Pfn1 in tumor angiogenesis and further identified endothelial Pfn1-dependent regulation of several pro- (VEGF, SERPINE1, CCL2) and anti-angiogenic factors (platelet factor 4) invivo. Endothelial Pfn1 overexpression increases tumor infiltration by macrophages and concomitantly diminishes tumor infiltration by T cells including CD8+ T cells invivo, correlating with the pattern of endothelial Pfn1-dependent changes in tumor abundance of several prominent immunomodulatory cytokines. These data were also corroborated by multiplexed quantitative immunohistochemistry and immune deconvolution analyses of RNA-seq data of clinical samples. Guided by Upstream Regulator Analysis of tumor transcriptome data, we further established endothelial Pfn1-induced Hif1α elevation and suppression of STAT1 activation. In conclusion, this study demonstrates for the first time a direct causal relationship between vascular endothelial Pfn1 dysregulation, immunosuppressive tumor microenvironment, and disease progression with mechanistic insights in kidney cancer. Our study also provides a conceptual basis for targeting Pfn1 for therapeutic benefit in kidney cancer.

The Impact of Profilin‐1 Mutations on Protein Homeostasis in Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is a progressive, fatal disease characterized by the dysfunction and degeneration of motor neurons in the central nervous system. ALS has been linked to a number of genetic mutations, including point mutations in the actin‐binding protein profilin‐1 (Pfn1). While the prevalence of Pfn1 mutations is low, around 1% of inherited forms of the disease, an understanding of their impact on motor neuron cell death can provide insight into the mechanisms underlying ALS onset and progression. As Pfn1 is an important regulator of the actin cytoskeleton, we hypothesize that ALS‐linked mutations of Pfn1 promote defects in protein homeostasis and synaptic communication. To test this hypothesis, neuronal cells were transfected with Pfn1 constructs (including wild‐type, C71G, and M118V) and subjected to cellular stressors (including heat shock and sodium arsenite treatments). Immunostaining was then used to visualize and quantify differences in protein aggregation and stress granule formation. Our results have established the baseline response with the wild‐type construct and indicate that there is an increase in protein aggregation as well as in the number and size of stress granules with the mutant constructs. Our ongoing studies are aimed at validating these preliminary results, and our goal is to provide mechanistic insight pertaining to Pfn1’s role in ALS disease pathogenesis.

Interfering effects on the bioactivities of several key proteins of COVID-19/variants in diabetes by compounds from Lianqiao leaves: In silico and in vitro analyses

Diabetes is considered to be one of the diseases most associated with COVID-19. In this study, interfering effects and potential mechanisms of several compounds from Lianqiao (Forsythia suspensa (Thunb.) Vahl) leaves on the bioactivities of some key proteins of COVID-19 and its variants, as well as diabetic endothelial dysfunctions were illuminated through in vitro and in silico analyses. Results showed that, among the main ingredients in the leaves, forsythoside A showed the strongest docking affinities with the proteins SARS-CoV-2-RBD-hACE2 of COVID-19 and its variants (Alpha (B.1.1.7), Beta (B.1.351), and Delta (B.1.617)), as well as neuropilin-1 (NRP1), and SARS-CoV-2 main protease (MPro) to interfere coronavirus entering into the human body. Moreover, forsythoside A was the most stable in binding to receptors in Delta (B.1.617) system. It also has good antiviral activities and drug properties and has the strongest binding force to the RBD domain of COVID-19. In addition, forsythoside A reduced ROS production in AGEs-induced EA.hy926 cells, maintained endothelial integrity, and bound closely to protein profilin-1 (PFN1) receptor. This work may provide useful knowledge for further understanding the interfering effects and potential mechanisms of compounds, especially forsythoside A, from Lianqiao leaves on the bioactivities of key proteins of COVID-19/variants in diabetes.

Biochemical characterization of actin assembly mechanisms with ALS-associated profilin variants

Eight separate mutations in the actin-binding protein profilin-1 have been identified as a rare cause of amyotrophic lateral sclerosis (ALS). Profilin is essential for many neuronal cell processes through its regulation of lipids, nuclear signals, and cytoskeletal dynamics, including actin filament assembly. Direct interactions between profilin and actin monomers inhibit actin filament polymerization. In contrast, profilin can also stimulate polymerization by simultaneously binding actin monomers and proline-rich tracts found in other proteins. Whether the ALS-associated mutations in profilin compromise these actin assembly functions is unclear. We performed a quantitative biochemical comparison of the direct and formin mediated impact for the eight ALS-associated profilin variants on actin assembly using classic protein-binding and single-filament microscopy assays. We determined that the binding constant of each profilin for actin monomers generally correlates with the actin nucleation strength associated with each ALS-related profilin. In the presence of formin, the A20T, R136W, Q139L, and C71G variants failed to activate the elongation phase of actin assembly. This diverse range of formin-activities is not fully explained through profilin-poly-L-proline (PLP) interactions, as all ALS-associated variants bind a formin-derived PLP peptide with similar affinities. However, chemical denaturation experiments suggest that the folding stability of these profilins impact some of these effects on actin assembly. Thus, changes in profilin protein stability and alterations in actin filament polymerization may both contribute to the profilin-mediated actin disruptions in ALS.

Protein Network Analysis Reveals a Functional Connectivity of Dysregulated Processes in ALS and SMA.

Spinal Muscular Atrophy (SMA) and Amyotrophic Lateral Sclerosis (ALS) are neurodegenerative diseases which are characterized by the loss of motoneurons within the central nervous system. SMA is a monogenic disease caused by reduced levels of the Survival of motoneuron protein, whereas ALS is a multi-genic disease with over 50 identified disease-causing genes and involvement of environmental risk factors. Although these diseases have different causes, they partially share identical phenotypes and pathomechanisms. To analyze and identify functional connections and to get a global overview of altered pathways in both diseases, protein network analyses are commonly used. Here, we used an in silico tool to test for functional associations between proteins that are involved in actin cytoskeleton dynamics, fatty acid metabolism, skeletal muscle metabolism, stress granule dynamics as well as SMA or ALS risk factors, respectively. In network biology, interactions are represented by edges which connect proteins (nodes). Our approach showed that only a few edges are necessary to present a complex protein network of different biological processes. Moreover, Superoxide dismutase 1, which is mutated in ALS, and the actin-binding protein profilin1 play a central role in the connectivity of the aforementioned pathways. Our network indicates functional links between altered processes that are described in either ALS or SMA. These links may not have been considered in the past but represent putative targets to restore altered processes and reveal overlapping pathomechanisms in both diseases.

Adaptor SH3BGRL promotes breast cancer metastasis through PFN1 degradation by translational STUB1 upregulation.

Metastatic recurrence is still a major challenge in breast cancer treatment, but the underlying mechanisms remain unclear. Here, we report that a small adaptor protein, SH3BGRL, is upregulated in the majority of breast cancer patients, especially elevated in those with metastatic relapse, indicating it as a marker for the poor prognosis of breast cancer. Physiologically, SH3BGRL can multifunctionally promote breast cancer cell tumorigenicity, migration, invasiveness, and efficient lung colonization in nude mice. Mechanistically, SH3BGRL downregulates the acting-binding protein profilin 1 (PFN1) by accelerating the translation of the PFN1 E3 ligase, STUB1 via SH3BGRL interaction with ribosomal proteins, or/and enhancing the interaction of PFN1 with STUB1 to accelerate PFN1 degradation. Loss of PFN1 consequently contributes to downstream multiple activations of AKT, NF-kB, and WNT signaling pathways. In contrast, the forced expression of compensatory PFN1 in SH3BGRL-high cells efficiently neutralizes SH3BGRL-induced metastasis and tumorigenesis with PTEN upregulation and PI3K-AKT signaling inactivation. Clinical analysis validates that SH3BGRL expression is negatively correlated with PFN1 and PTEN levels, but positively to the activations of AKT, NF-kB, and WNT signaling pathways in breast patient tissues. Our results thus suggest that SH3BGRL is a valuable prognostic factor and a potential therapeutic target for preventing breast cancer progression and metastasis.

Application of proteomic to investigate the different degrees of meat tenderness in Nellore breed

This study describes the association between meat tenderness and abundance of soluble muscle proteins in Nellore bulls (Bos indicus) using a proteomic approach. We evaluated shear force (SF) of Longissimus thoracis muscle 24 h after slaughter and selected three experimental groups of animals with moderately tender (TE; SF = 3.9 ± 0.7 kg), moderately tough (TO; SF = 5.6 ± 0.7 kg) and very tough meat (TO+; SF = 7.9 ± 1.4 kg). Proteome was investigated by two-dimensional electrophoresis (2D-PAGE) in combination with electrospray ionization-tandem mass spectrometry (ESI–MS/MS). The metabolic proteins triosephosphate isomerase (TPI1) and phosphoglucomutase 1 (PGM1), the structural protein profilin 1 (PFN1), and cytosol aminopeptidase (LAP3) were up-regulated (P < 0.05) in the TE meat group when compared to the TO and TO+ groups. Actin structural proteins (ACTA1, ACTB, and ACTG1), the oxidative stress protein peroxiredoxin (PRDX6, PRDX2, PRDX1, and PARK7), heat shock protein isoforms, and co-chaperones (CDC37 and STIP1) were up-regulated (P < 0.05) in the TO and TO+ meat groups. In addition, we also identified proteins PFN1, LAP3, PRDX1, PRDX2, HSPD1, and ARHGDIA to be associated with beef tenderness. The results reported herein demonstrated that meat tenderness in Nellore cattle depends on the modulation and expression of a set of proteins involved in different biological pathways. SignificanceThe manuscript entitled “Application of proteomic to investigate the different degrees of meat tenderness in Nellore breed” describes a classical proteomics work using two-dimensional gel electrophoresis (2D-PAGE), followed by mass spectrometry coupled to electrospray ionization ion trap (ESI-MS/MS) in order to understand the biochemical engineering involved in the process of meat tenderness. We evaluated shear force (SF) of Longissimus thoracis muscle samples of Nellore cattle (n = 90) and select three experimental groups of animals with moderately tender (TE; SF = 3.9 ± 0.7), moderately tough (TO; SF = 5.6 ± 0.7) and very tough meat (TO+; SF = 7.9 ± 1.4). The proteomic approach allowed observing that meat tenderness is influenced by structural proteins (ACTA1, ACTG1, ACTB, MYL1 and PFN1), co-chaperones (CDC37 and STIP1), heat shock proteins (HSP90AA1, HSP90AB1, HSPD1, HSPA1L, HSPA1A and HSPB1), regulatory protein (ARHGDIA), metabolic proteins (TPI1 and PGM1) and oxidative stress proteins (PRDX1, PRDX2, PRDX6, PARK7). Our results suggest that meat tenderness in Nellore depends on the modulation and expression of a set of proteins involved in different biological pathways.

Global Analysis of Protein Expression in A549 Cells After Prolonged Nicotine Exposure by Using Label-free Quantification

Lung cancer is the leading cause of cancer death worldwide. Cigarette smoke is the most important risk factor for cancer development. Growing evidence indicates that prolonged nicotine exposure is a potential factor associated with tumorigenesis. Here, the effect of prolonged nicotine exposure on A549 cells was investigated, using label-free quantitative proteomics. Selection of an invasive subpopulation from the A549 cell line was performed to reveal the differential expression of proteins in relation to prolonged nicotine exposure, using Boyden chamber assays in combination with a proteomics approach. One hundred proteins from the NicoA549-L5 subline showed significant change in expression compared to those from the A549-L5 subline and their A549 parental cell line. Heat shock protein, protein disulfide isomerase A3, profilin-1 and legumain were expressed at higher levels in A549 cells after prolonged nicotine exposure. These aberrant proteins might serve as novel cancer biomarkers for cigarette smokers.

The role of profilin-1 in cardiovascular diseases.

Dynamic remodeling of the actin cytoskeleton is an essential feature for virtually all actin-dependent cellular processes, including cell migration, cell cycle progression, chromatin remodeling and gene expression, and even the DNA damage response. An altered actin cytoskeleton is a structural hallmark associated with numerous pathologies ranging from cardiovascular diseases to immune disorders, neurological diseases and cancer. The actin cytoskeleton in cells is regulated through the orchestrated actions of a myriad of actin-binding proteins. In this Review, we provide a brief overview of the structure and functions of the actin-monomer-binding protein profilin-1 (Pfn1) and then discuss how dysregulated expression of Pfn1 contributes to diseases associated with the cardiovascular system.

Actin-binding protein profilin1 promotes aggressiveness of clear-cell renal cell carcinoma cells

Clear-cell renal cell carcinoma (ccRCC), the most common subtype of renal cancer, has a poor clinical outcome. A hallmark of ccRCC is genetic loss-of-function of VHL (von Hippel-Lindau) that leads to a highly vascularized tumor microenvironment. Although many ccRCC patients initially respond to antiangiogenic therapies, virtually all develop progressive, drug-refractory disease. Given the role of dysregulated expressions of cytoskeletal and cytoskeleton-regulatory proteins in tumor progression, we performed analyses of The Cancer Genome Atlas (TCGA) transcriptome data for different classes of actin-binding proteins to demonstrate that increased mRNA expression of profilin1 (Pfn1), Arp3, cofilin1, Ena/VASP, and CapZ, is an indicator of poor prognosis in ccRCC. Focusing further on Pfn1, we performed immunohistochemistry-based classification of Pfn1 staining in tissue microarrays, which indicated Pfn1 positivity in both tumor and stromal cells; however, the vast majority of ccRCC tumors tend to be Pfn1-positive selectively in stromal cells only. This finding is further supported by evidence for dramatic transcriptional up-regulation of Pfn1 in tumor-associated vascular endothelial cells in the clinical specimens of ccRCC. In vitro studies support the importance of Pfn1 in proliferation and migration of RCC cells and in soluble Pfn1's involvement in vascular endothelial cell tumor cell cross-talk. Furthermore, proof-of-concept studies demonstrate that treatment with a novel computationally designed Pfn1-actin interaction inhibitor identified herein reduces proliferation and migration of RCC cells in vitro and RCC tumor growth in vivo Based on these findings, we propose a potentiating role for Pfn1 in promoting tumor cell aggressiveness in the setting of ccRCC.

Arp2/3 and Mena/VASP Require Profilin 1 for Actin Network Assembly at the Leading Edge

Cells have many types of actin structures, which must assemble from a common monomer pool. Yet, it remains poorly understood how monomers are distributed to and shared between different filament networks. Simplified model systems suggest that monomers are limited and heterogeneous, which alters actin network assembly through biased polymerization and internetwork competition. However, less is known about how monomers influence complex actin structures, where different networks competing for monomers overlap and are functionally interdependent. One example is the leading edge of migrating cells, which contains filament networks generated by multiple assembly factors. The leading edge dynamically switches between the formation of different actin structures, such as lamellipodia or filopodia, by altering the balance of these assembly factors' activities. Here, we sought to determine how the monomer-binding protein profilin 1 (PFN1) controls the assembly and organization of actin in mammalian cells. Actin polymerization in PFN1 knockout cells was severely disrupted, particularly at the leading edge, where both Arp2/3 and Mena/VASP-based filament assembly was inhibited. Further studies showed that in the absence of PFN1, Arp2/3 no longer localizes to the leading edge and Mena/VASP is non-functional. Additionally, we discovered that discrete stages of internetwork competition and collaboration between Arp2/3 and Mena/VASP networks exist at different PFN1 concentrations. Low levels of PFN1 caused filopodia to form exclusively at the leading edge, while higher concentrations inhibited filopodia and favored lamellipodia and pre-filopodia bundles. These results demonstrate that dramatic changes to actin architecture can be made simply by modifying PFN1 availability.

Abstract LB-043: MRTF|Profilin is an important signaling axis for metastatic outgrowth of triple negative breast cancer cells

Abstract Treatment of triple negative breast cancer (TNBC) is particularly difficult due to lack of molecular targets. The vast majority of breast cancer patient mortality is a result of emergence of disseminated cancer cells from a so-called dormancy-like state allowing subsequent aggressive growth at the metastatic sites. Actin assembly and important regulators of actin assembly play a key role in promoting the dormancy to proliferation switch of extravasated breast cancer cells. MRTF (myocardin-related transcription factor - an important family of transcription cofactors for SRF)/SRF transcriptional axis plays a major role in transcriptional regulation of many major molecular components of actin cytoskeletal system. We hypothesize that MRTF plays a critical role in post-extravasation survival and outgrowth of TNBC cells. To test this hypothesis, we first performed matrigel-on-top assay (3D culture experiments which successfully predicts the post-extravasation pulmonary metastatic outgrowth competency of breast cancer cells) and found that loss-of-function (LOF) of MRTF, either by knockdown or small molecule inhibitor CCG-1423 (and its analog CCG-203971), significantly retards the outgrowth of isolated TNBC cells. In this model, LOF of MRTF also dramatically suppresses the progression of established outgrowth (an in vitro mimic of micro-to-macrometastasis progression) of TNBC cells. Translating these findings in vivo, in intracardiac-injection model of experimental metastasis, administration of MRTF inhibitor reduces the metastatic burden of TNBC cells in immunocompetent mice. These experimental data are consistent with clinical findings demonstrating significantly shorter progression-free survival with MRTF upregulation in breast cancer patients. To obtain further insight into the mechanism behind MRTF’s regulation of metastatic outgrowth, we performed RNAseq from TNBC grown in 3D culture treated with CCG and identified a number of differentially expressed genes related to cell proliferation/survival and invasion. In addition to these findings, we also found that LOF of MRTF causes a dramatic reduction of intracellular content of actin-binding protein Profilin-1 (Pfn1), an important regulator of actin cytoskeletal dynamics. Using a tetracycline-inducible Pfn1 knockdown system, we further demonstrated that acute Pfn1 depletion alone is sufficient to cause a major reduction of metastatic colonization of TNBC cells in vivo. Collectively, these findings suggest that MRTF-Pfn1 is an important signaling axis for metastatic outgrowth of TNBC cells. Citation Format: David M. Gau, Souvik Chakraborty, David Boone, Alan Wells, Partha Roy. MRTF|Profilin is an important signaling axis for metastatic outgrowth of triple negative breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-043.

Breast cancer cell invasiveness is stimulated by loss of membrane interaction of actinbinding protein profilin1 via altered phosphoinositide metabolism

Profilin1 (Pfn1) belongs to a class of actin‐binding protein cytoskeletal function of which is negatively regulated by its interaction with membrane phosphoinositides (PPI). In stark contrast to most physiological scenarios where loss of Pfn1 significantly impairs normal cell migration and invasion through inhibition of actin polymerization, breast cancer cells exhibit hyper‐migratory phenotype in vitro and enhanced hematogenous dissemination from primary tumor in vivo upon depletion of Pfn1, the underlying mechanism of which is not easily explained by Pfn1's cytoskeletal function. Through knockdown and overexpression studies in multiple cell lines, we show that Pfn1 plays a key role in determining the cellular profile of various PPIs including PI(4,5)P2, PIP3 and PI(3,4)P2 Specifically, Pfn1 promotes PI(4,5)P2 and inhibits membrane accumulation of PI3K‐generated PPIs including PIP3 and PI(3,4)P2, a feature related to Pfn1's PPI interaction. Kinetics of PI3K signaling output suggest that loss of Pfn1 enhances sustenance of PI3K signaling by inhibiting an antagonistic pathway, a finding that correlates to Pfn1‐dependent regulation of PTEN. We establish Pfn1‐dependent alteration of multiple pro‐metastatic cytokines and membrane recruitment of selective PI(3,4)P2‐dependent pro‐migratory protein complexes, the latter in particular linking to hyper‐migratory and hyper‐invasive phenotypes of Pfn1‐deficient breast cancer cells. In summary, these findings establish novel regulation of cell migration by lipid signaling via membrane interaction of actin‐binding protein.Support or Funding InformationNIH 2R01CA 108607This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The VASP–profilin1 (Pfn1) interaction is critical for efficient cell migration and is regulated by cell–substrate adhesion in a PKA-dependent manner

Dynamic regulation of the actin cytoskeleton is an essential feature of cell motility. Action of Enabled (Ena)/vasodilator-stimulated phosphoprotein (VASP), a family of conserved actin-elongating proteins, is an important aspect of regulation of the actin cytoskeletal architecture at the leading edge that controls membrane protrusion and cell motility. In this study, we performed mutagenesis experiments in overexpression and knockdown-rescue settings to provide, for the first time, direct evidence of the role of the actin-binding protein profilin1 (Pfn1) in VASP-mediated regulation of cell motility. We found that VASP's interaction with Pfn1 is promoted by cell-substrate adhesion and requires down-regulation of PKA activity. Our experimental data further suggest that PKA-mediated Ser137 phosphorylation of Pfn1 potentially negatively regulates the Pfn1-VASP interaction. Finally, Pfn1's ability to be phosphorylated on Ser137 was partly responsible for the anti-migratory action elicited by exposing cells to a cAMP/PKA agonist. On the basis of these findings, we propose a mechanism of adhesion-protrusion coupling in cell motility that involves dynamic regulation of Pfn1 by PKA activity.

Profilin binding couples chloride intracellular channel protein CLIC4 to RhoA–mDia2 signaling and filopodium formation

Chloride intracellular channel 4 (CLIC4) is a cytosolic protein implicated in diverse actin-based processes, including integrin trafficking, cell adhesion, and tubulogenesis. CLIC4 is rapidly recruited to the plasma membrane by RhoA-activating agonists and then partly colocalizes with β1 integrins. Agonist-induced CLIC4 translocation depends on actin polymerization and requires conserved residues that make up a putative binding groove. However, the mechanism and significance of CLIC4 trafficking have been elusive. Here, we show that RhoA activation by either lysophosphatidic acid (LPA) or epidermal growth factor is necessary and sufficient for CLIC4 translocation to the plasma membrane and involves regulation by the RhoA effector mDia2, a driver of actin polymerization and filopodium formation. We found that CLIC4 binds the G-actin–binding protein profilin-1 via the same residues that are required for CLIC4 trafficking. Consistently, shRNA-induced profilin-1 silencing impaired agonist-induced CLIC4 trafficking and the formation of mDia2-dependent filopodia. Conversely, CLIC4 knockdown increased filopodium formation in an integrin-dependent manner, a phenotype rescued by wild-type CLIC4 but not by the trafficking-incompetent mutant CLIC4(C35A). Furthermore, CLIC4 accelerated LPA-induced filopodium retraction. We conclude that through profilin-1 binding, CLIC4 functions in a RhoA–mDia2–regulated signaling network to integrate cortical actin assembly and membrane protrusion. We propose that agonist-induced CLIC4 translocation provides a feedback mechanism that counteracts formin-driven filopodium formation.