Regulation Of Cell Polarity Research Articles

Abstract P6-14-09: Non-canonical Wnt/Ror2 signaling regulates tumor cell invasion and dissemination in breast cancer through cell-matrix crosstalk

Abstract BACKGROUND: Metastasis is the main cause of mortality for breast cancer patients. The early steps of cancer metastasis require that tumor cells actively invade and disseminate from the primary tumor to distant organs. Cell-extracellular matrix (ECM) interactions represent fundamental interactions during tumor invasion and metastasis, yet how particular signal-transduction factors prompt the conversion of tumor cells into migratory populations capable of systemic dissemination remains elusive. OBJECTIVES: Wnt signaling is a known regulator of cell fate, migration, and polarity during various key biological processes. We previously discovered an inverse correlation between the canonical Wnt signature and a non-canonical Wnt receptor, Ror2, across breast cancers in TCGA. The objective of this study is to investigate how canonical and non-canonical Wnt signaling orchestrate tumor cell behavior during cancer invasion and metastasis. METHODS: We used clinically relevant syngeneic TP53-null Genetically Engineered Mouse Model (GEMM) tumors that molecularly reflect the different breast cancer intrinsic subtypes. In vivo transplantation of GEMM models and in vitro 3D tumor organoids were employed to elucidate the molecular mechanism underlying the migration, invasion, and metastasis of tumor cells upon genetic perturbation of Wnt/Ror2 signaling. RESULTS/DISCUSSION: From 3-dimensional (3D) tumor organoid models, we identified novel transcriptional alterations encompassing cell-cell adhesion, cytoskeletal remodeling, and ECM organization upon Ror2 loss. At protein levels, we discovered a significant increase in collagen fibril organization and integrin-α5 and integrin-β3 expression following Ror2 depletion. In addition, the matrisomal protein fibronectin (FN) was concomitantly upregulated and assembled in Ror2-deficient tumor cells at sites of invasion. Consequently, we observed FAK activation and actin cytoskeleton alterations in Ror2-deficient tumor cells, leading to a promigratory tumor cell behavior. The altered cytoskeleton and cell-ECM interaction enhanced the rigidity of the Ror2-depleted cells. Furthermore, Inhibition of either integrin or FAK activation abrogated the increased invasion driven by Ror2-loss. Unexpectedly, these changes were distinct from processes regulated by Wnt/ß-catenin activation. From both spontaneous metastasis and experimental metastasis models, we discovered a shift from canonical to non-canonical Wnt signaling throughout the progression of lung metastasis, indicating that the balance of Wnt signaling may serve as a switch to dictate cell functions at different stages of metastatic development. Consistently, we found enhanced initial colonization in the lungs when Ror2 is depleted in these breast cancer cells. Together, these studies provide new insight into how canonical and alternative Wnt pathways coordinate cell-cell and cell-ECM exchanges during breast cancer progression and metastasis. Supported by grant NIH-CA016303 Citation Format: Hongjiang Si, Na Zhao, Andrea Pedroza, Chad Creighton, Jeffrey Rosen, Kevin Roarty. Non-canonical Wnt/Ror2 signaling regulates tumor cell invasion and dissemination in breast cancer through cell-matrix crosstalk [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-14-09.

Ror1 promotes PPARα-mediated fatty acid metabolism in astrocytes.

Ror1 signaling regulates cell polarity, migration, proliferation, and differentiation during developmental morphogenesis, and plays an important role in regulating neurogenesis in the embryonic neocortices. However, the role of Ror1 signaling in the brains after birth remains largely unknown. Here, we found that expression levels of Ror1 in the mouse neocortices increase during the postnatal period, when astrocytes mature and start expressing GFAP. Indeed, Ror1 is highly expressed in cultured postmitotic mature astrocytes. RNA-Seq analysis revealed that Ror1 expressed in cultured astrocytes mediates upregulated expression of genes related to fatty acid (FA) metabolism, including the gene encoding carnitine palmitoyl-transferase 1a (Cpt1a), the rate-limiting enzyme of mitochondrial fatty acid β-oxidation (FAO). We also found that Ror1 promotes the degradation of lipid droplets (LDs) accumulated in the cytoplasm of cultured astrocytes after oleic acid loading, and that suppressed expression of Ror1 decreases the amount of FAs localized at mitochondria, intracellular ATP levels, and expression levels of peroxisome proliferator-activated receptor α (PPARα) target genes, including Cpt1a. Collectively, these findings indicate that Ror1 signaling promotes PPARα-mediated transcription of FA metabolism-related genes, thereby facilitating the availability of FAs derived from LDs for mitochondrial FAO in the mature astrocytes.

A mutational hotspot in AMOTL1 defines a new syndrome of orofacial clefting, cardiac anomalies, and tall stature.

AMOTL1 encodes angiomotin-like protein 1, an actin-binding protein that regulates cell polarity, adhesion, and migration. The role of AMOTL1 in human disease is equivocal. We report a large cohort of individuals harboring heterozygous AMOTL1 variants and define a core phenotype of orofacial clefting, congenital heart disease, tall stature, auricular anomalies, and gastrointestinal manifestations in individuals with variants in AMOTL1 affecting amino acids 157-161, a functionally undefined but highly conserved region. Three individuals with AMOTL1 variants outside this region are also described who had variable presentations with orofacial clefting and multi-organ disease. Our case cohort suggests that heterozygous missense variants in AMOTL1, most commonly affecting amino acid residues 157-161, define a new orofacial clefting syndrome, and indicates an important functional role for this undefined region.

Structural analysis of human papillomavirus E6 interactions with Scribble PDZ domains.

The cell polarity regulator Scribble has been shown to be a critical regulator of the establishment and development of tissue architecture, and its dysregulation promotes or suppresses tumour development in a context-dependent manner. Scribble activity is subverted by numerous viruses. This includes human papillomaviruses (HPVs), who target Scribble via the E6 protein. Binding of E6 from high-risk HPV strains to Scribble via a C-terminal PDZ-binding motif leads to Scribble degradation in vivo. However, the precise molecular basis for Scribble-E6 interactions remains to be defined. We now show that Scribble PDZ1 and PDZ3 are the major interactors of HPV E6 from multiple high-risk strains, with each E6 protein displaying a unique interaction profile. We then determined crystal structures of Scribble PDZ1 and PDZ3 domains in complex with the PDZ-binding motif (PBM) motifs of E6 from HPV strains 16, 18 and 66. Our findings reveal distinct interaction patterns for each E6 PBM motif from a given HPV strain, suggesting that a complex molecular interplay exists that underpins the overt Scribble-HPV E6 interaction and controls E6 carcinogenic potential.

The role of Rho GTPase family in cochlear hair cells and hearing.

Rho GTPases are essential regulators of the actin cytoskeleton. They are involved in various physiological and biochemical processes such as the regulation of cytoskeleton dynamics, development, proliferation, survival, and regeneration. During the development of cochlear hair cells, Rho GTPases are activated by various extracellular signals through membrane receptors to further stimulate multiple downstream effectors. Specifically, RhoA, Cdc42, and Rac1, members of the classical subfamily of the Rho GTPase family, regulate the development and maintenance of cilia by inducing the polymerization of actin monomers and stabilizing actin filaments. In addition, they also regulate the normal morphology orientation of ciliary bundles in auditory hair cells, which is an important element of cell polarity regulation. Moreover, the actin-related pathways mediated by RhoA, Cdc42, and Rac1 also play a role in the motility of outer hair cells, indicating that the function of Rho GTPases is crucial in the highly polar auditory sensory system. In this review, we focus on the expression of RhoA, Cdc42, and Rac1 in cochlear hair cells and how these small molecules participate in ciliary bundle morphogenesis and cochlear hair cell movement. We also discuss the progress of current research investigating the use of these small molecules as drug targets for deafness treatment.

An isoform-specific function of Cdc42 in regulating mammalian Exo70 during axon formation.

The highly conserved GTPase Cdc42 is an essential regulator of cell polarity and promotes exocytosis through the exocyst complex in budding yeast and Drosophila In mammals, this function is performed by the closely related GTPase TC10, whereas mammalian Cdc42 does not interact with the exocyst. Axon formation is facilitated by the exocyst complex that tethers vesicles before their fusion to expand the plasma membrane. This function depends on the recruitment of the Exo70 subunit to the plasma membrane. Alternative splicing generates two Cdc42 isoforms that differ in their C-terminal 10 amino acids. Our results identify an isoform-specific function of Cdc42 in neurons. We show that the brain-specific Cdc42b isoform, in contrast to the ubiquitous isoform Cdc42u, can interact with Exo70. Inactivation of Arhgef7 or Cdc42b interferes with the exocytosis of post-Golgi vesicles in the growth cone. Cdc42b regulates exocytosis and axon formation downstream of its activator Arhgef7. Thus, the function of Cdc42 in regulating exocytosis is conserved in mammals but specific to one isoform.

PDLIM2 is highly expressed in Breast Cancer tumour-associated macrophages and is required for M2 macrophage polarization.

The PDZ-LIM domain-containing protein 2 (PDLIM2) regulates cell polarity and the protein stability of key transcription factors in epithelial and hemopoietic cells. We previously reported that PDLIM2 is more highly expressed in Triple Negative Breast Cancer (TNBC) than in other breast cancer types or normal breast tissue. In the course of the TNBC study, it was noted that PDLIM2 was highly expressed in the stroma of PDLIM2-expressing tumours. Here, we investigated the phenotype of these stromal cells and whether any infiltrating immune population was linked to PDLIM2 expression. We found that high PDLIM2 expression in breast tumours was associated with higher levels of infiltrating M2 macrophages, but was not associated with infiltrating T cell sub-populations. We then tested whether PDLIM2 contributes to macrophage differentiation or function by using cultures of bone marrow-derived macrophages from wildtype and Pdlim2 knockout mice. This demonstrated that PDLIM2 is required for naïve macrophage migration and for the full adoption of IL-4-induced M2 polarization, including expression of M2 phenotypic markers, cell adhesion and cell migration. TLR4-, TLR3- or IFNγ-induced M1 macrophage activity was less dependent on PDLIM2. Finally, analysis of publicly available breast cancer datasets showed that high PDLIM2 expression is associated with increased M2 macrophage infiltration. We conclude that PDLIM2 expression influences the tumour associated stroma and, in particular, M2 macrophage infiltration that may contribute to the progression of TNBC or other subsets of breast cancer.

Cell polarity signalling at the birth of multicellularity: What can we learn from the first animals.

The innovation of multicellularity has driven the unparalleled evolution of animals (Metazoa). But how is a multicellular organism formed and how is its architecture maintained faithfully? The defining properties and rules required for the establishment of the architecture of multicellular organisms include the development of adhesive cell interactions, orientation of division axis, and the ability to reposition daughter cells over long distances. Central to all these properties is the ability to generate asymmetry (polarity), coordinated by a highly conserved set of proteins known as cell polarity regulators. The cell polarity complexes, Scribble, Par and Crumbs, are considered to be a metazoan innovation with apicobasal polarity and adherens junctions both believed to be present in all animals. A better understanding of the fundamental mechanisms regulating cell polarity and tissue architecture should provide key insights into the development and regeneration of all animals including humans. Here we review what is currently known about cell polarity and its control in the most basal metazoans, and how these first examples of multicellular life can inform us about the core mechanisms of tissue organisation and repair, and ultimately diseases of tissue organisation, such as cancer.

Neonatal lethality of mouse A/J-7SM consomic strain is caused by an insertion mutation in the Dchs1 gene.

Homosomic mice of the A/J-7SM consomic mouse strain that introduced the entire chromosome 7 (Chr 7) of SM/J into the A/J strain exhibited neonatal lethality. We tentatively maintained segregating inbred strains (A/J-7ASM and A/J-7DSM) in which the central portion of Chr 7 was heterozygous for the A/J and SM/J strains, and the centromeric and telomeric sides of Chr 7 were homozygous for the SM/J strain, instead of the A/J-7SM strain. Based on the chromosomal constitution of Chr 7 in A/J-7ASM and A/J-7DSM mice, the causative gene for neonatal lethality in homosomic mice was suggested to be located within an approximately 1.620Mb region between D7Mit125 (104.879Mb) and D7Mit355 (106.499Mb) on Chr 7. RT-PCR analysis revealed that homosomic mice lacked dachsous cadherin-related 1 (Dchs1), which is located within the D7Mit125 to D7Mit355 region and functions in the regulation of planar cell polarity. Screening for mutations in Dchs1 indicated that homosomic mice possessed an early transposable (ETn)-like sequence in intron 1 of Dchs1. Moreover, an allelism test between Dchs1 ETn-like-insertion alleles detected in homosomic mice and CRISPR/Cas9-induced Dchs1 deletion alleles revealed that Dchs1 is a causative gene for neonatal lethality in homosomic mice. Based on these results, we concluded that in the A/J-7SM strain, ETn-like elements were inserted into intron 1 of SM/J-derived Dchs1 during strain development, which dramatically reduced Dchs1 expression, thus resulting in neonatal lethality in homosomic mice. Additionally, it was suggested that the timing of lethality in Dchs1 mutant mice is influenced by the genetic background.

Loss of Cell Fate Determinant LLGL1 Impairs Leukemia Development in Acute Myeloid Leukemia

Cell fate determinants and polarity regulators influence the fine balance between self-renewal and differentiation in HSC and leukemic stem cells (LSC). Previously our group provided first evidence that inactivation of the cell fate regulator Llgl1 in murine HSCs leads to alterations in self-renewal, proliferative capacity and differentiation (Heidel et al., 2013). The impact of Llgl1 on malignant transformation and especially acute myeloid leukemia (AML) development had not been investigated in detail. Inactivation of LLGL1 in several human AML cell lines (MOLM13, HEL, MV4;11, HL-60) by CRISPR/Cas9 resulted in decreased proliferation of leukemic cells. Consistently, xenograft studies using LLGL1-depleted human AML cells resulted in delayed disease development compared to non-targeting control (median survival NT-control: 37.5 days vs. LLGL1-KD: 92 days; p=0.0002). Genetic inactivation of LLGL1 in primary AML patient cells resulted in reduced colony formation in vitro and improved survival of recipient mice in a patient-derived xenograft model of AML. To recapitulate these findings in defined model systems of murine AML, we used a conditional Llgl1 knockout mouse model (Klezovitch et al, 2004) and intercrossed it with AML-models harboring different driver mutations: MllAF9 (Cerral et al., 1995) or Flt3ITD/ITD (Lee et al., 2006). Conditional deletion of Llgl1 in MllAF9 knockin mice using an Mx1-Cre-recombinase (Cre+) resulted in significant delay of leukemia development (median survival +/+: 174 days; -/-: 203 days; p=0.0361). To confirm these findings, we investigated a second oncogenic model and induced deletion of Llgl1 in Flt3ITD/ITD knockin mice. Leukemic Flt3ITD/ITD; Llgl1+/+; Cre+ animals (n=12) showed rapid onset of leukemia development, whereas the Flt3ITD/ITD; Llgl1-/-; Cre+ animals (n=25) showed significant delay in leukemia progression (median survival +/+: 38 days; -/-: 51 days; p=0.0005). Llgl1-/- animals show reduced peripheral blast counts (WBC +/+: 332.558 Gpt/l; -/-: 141.083 Gpt/l; p=0.0012) and decreased organ infiltration. Importantly, immunophenotypic analysis of bone marrow (BM) compartments indicated a shift of immature cells towards a more differentiated state. The overall abundance of leukemic Lin-Kit+Sca-1+ (LSK) cells was reduced in Flt3ITD/ITD; Llgl1-/- animals compared to Flt3ITD/ITD; Llgl1+/+ controls (LSK +/+: 13099±3144/106 BMC; -/-: 8117±2810 /106 BMC; p=0.0227). In contrast, we found increased numbers of Lin-Kit+Sca-1+FcgR+CD34+ of leukemic granulocyte-macrophage progenitors (LGMPs: +/+: 8306±1963/106 BMC; -/-: 14755±4783/106 BMC; p=0.0141) upon deletion of Llgl1 in Flt3ITD/ITD animals. These findings indicate that genetic inactivation of Llgl1 results in loss of leukemia stemness and shifts the leukemia stem cell phenotype from HSC-like to GMP-like LSCs. To test for this hypothesis, we performed global transcriptome analysis of sorted LSK cells of Flt3ITD/ITD mice early after genetic deletion of Llgl1. Among the significantly downregulated genes, we found several members of the HoxA-cluster, consistent with the immunophenotypic loss of stemness. Gene-set-enrichment analysis revealed downregulation of leukemia stem cell signatures (Gal et al., 2006) and enrichment of a gene set associated with LGMPs (compared to leukemic-HSC) signatures (Krivtsov et al., 2006). To validate whether loss of Hox-gene expression is relevant for impaired leukemia development, we performed rescue experiments in vivo. LSK cells derived from Flt3ITD/ITD; Llgl1+/+ or Flt3ITD/ITD; Llgl1-/- animals were retrovirally transduced with HoxA9 or empty vector control (eV). When transplanted into sublethally irradiated recipient mice, overexpression of HoxA9 (OE) in Llgl1-/-; Flt3ITD/ITD cells rescued the disease phenotype comparable to Flt3ITD/ITD; Llgl1+/+ cells (median survival +/+;eV: 62.50%; -/-; OE: 55.56%). In contrast, empty vector control Flt3ITD/ITD; Llgl1-/- recipients showed no disease development (median survival -/-; eV: 100%). Taken together our results may provide first evidence for a functional role of Llgl1 in models of AML. In contrast to its function in normal HSCs, deletion of Llgl1 appears to reduce stemness of leukemic stem cells and impair leukemia development in vitro and in vivo. Experiments to assess for the effects of Llgl1 on LSC polarity and cell fate decisions are currently under way.

ROP signaling regulates spatial pattern of cell division and specification of meristem notch

The formation of cell polarity is essential for many developmental processes such as polar cell growth and spatial patterning of cell division. A plant-specific ROP (Rho-like GTPases from Plants) subfamily of conserved Rho GTPase plays a crucial role in the regulation of cell polarity. However, the functional study of ROPs in angiosperm is challenging because of their functional redundancy. The Marchantia polymorpha genome encodes a single ROP gene, MpROP, providing an excellent genetic system to study ROP-dependent signaling pathways. Mprop knockout mutants exhibited rhizoid growth defects, and MpROP was localized at the tip of elongating rhizoids, establishing a role for MpROP in the control of polar cell growth and its functional conservation in plants. Furthermore, the Mprop knockout mutant showed defects in the formation of meristem notches associated with disorganized cell division patterns. These results reveal a critical function of MpROP in the regulation of plant development. Interestingly, these phenotypes were complemented not only by MpROP but also Arabidopsis AtROP2, supporting the conservation of ROP's function among land plants. Our results demonstrate a great potential for M. polymorpha as a powerful genetic system for functional and mechanistic elucidation of ROP signaling pathways during plant development.

Regulation of Cdc42 protein turnover modulates the filamentous growth MAPK pathway.

Rho GTPases are central regulators of cell polarity and signaling. How Rho GTPases are directed to function in certain settings remains unclear. Here, we show the protein levels of the yeast Rho GTPase Cdc42p are regulated, which impacts a subset of its biological functions. Specifically, the active conformation of Cdc42p was ubiquitinated by the NEDD4 ubiquitin ligase Rsp5p and HSP40/HSP70 chaperones and turned over in the proteasome. A GTP-locked (Q61L) turnover-defective (TD) version, Cdc42pQ61L+TD, hyperactivated the MAPK pathway that regulates filamentous growth (fMAPK). Cdc42pQ61L+TD did not influence the activity of the mating pathway, which shares components with the fMAPK pathway. The fMAPK pathway adaptor, Bem4p, stabilized Cdc42p levels, which resulted in elevated fMAPK pathway signaling. Our results identify Cdc42p turnover regulation as being critical for the regulation of a MAPK pathway. The control of Rho GTPase levels by stabilization and turnover may be a general feature of signaling pathway regulation, which can result in the execution of a specific developmental program.

HPV-18E6 Inhibits Interactions between TANC2 and SNX27 in a PBM-Dependent Manner and Promotes Increased Cell Proliferation.

Cancer-causing HPV E6 oncoproteins contain a PDZ-binding motif at the extreme carboxy terminus, which plays an important role in the viral life cycle and in the development of malignancy. Through this motif, HPV E6 targets a large number of cellular substrates, many of which are involved in processes related to the regulation of cell polarity. Recent studies also demonstrated E6's PDZ binding motif (PBM)-dependent association with SNX27, with a potential role in the perturbation of endocytic transport. Here, we have performed a proteomic analysis to identify SNX27-interacting partners whose binding to SNX27 is specifically perturbed in an E6-dependent manner. Extracts of HeLa cells that express GFP-tagged SNX27, transfected with control siRNA or siRNA targeting E6AP, were subject to GFP immunoprecipitation followed by mass spectroscopy, which identified TANC2 as an interacting partner of SNX27. Furthermore, we demonstrate that HPV E6 inhibits association between SNX27 and TANC2 in a PBM-dependent manner, resulting in an increase in TANC2 protein levels. In the absence of E6, SNX27 directs TANC2 toward lysosomal degradation. TANC2, in the presence of HPV-18E6, enhances cell proliferation in a PBM-dependent manner, indicating that HPV E6 targets the SNX27-mediated transport of TANC2 to promote cellular proliferation. IMPORTANCE While a great deal is known about the role of the E6 PDZ binding motif (PBM) in modulating the cellular proteins involved in regulating cell polarity, much less is known about the consequences of E6's interactions with SNX27 and the endocytic sorting machinery. We reasoned that a potential consequence of such interactions could be to affect the fate of multiple SNX27 endosomal partners, such as transmembrane proteins or soluble accessory proteins. Using a proteomic approach in HPV-18-positive cervical tumor-derived cells, we demonstrate that TANC2 is an interacting partner of SNX27, whose interaction is blocked by E6 in a PBM-dependent manner. This study therefore begins to shed new light on how E6 can regulate the endocytic transport of multiple SNX27-binding proteins, thereby expanding our understanding of the functions of the E6 PBM.

Parafibromin governs cell polarity and centrosome assembly in Drosophila neural stem cells.

Neural stem cells (NSCs) divide asymmetrically to balance their self-renewal and differentiation, an imbalance in which can lead to NSC overgrowth and tumor formation. The functions of Parafibromin, a conserved tumor suppressor, in the nervous system are not established. Here, we demonstrate that Drosophila Parafibromin/Hyrax (Hyx) inhibits ectopic NSC formation by governing cell polarity. Hyx is essential for the asymmetric distribution and/or maintenance of polarity proteins. hyx depletion results in the symmetric division of NSCs, leading to the formation of supernumerary NSCs in the larval brain. Importantly, we show that human Parafibromin rescues the ectopic NSC phenotype in Drosophila hyx mutant brains. We have also discovered that Hyx is required for the proper formation of interphase microtubule-organizing center and mitotic spindles in NSCs. Moreover, Hyx is required for the proper localization of 2 key centrosomal proteins, Polo and AurA, and the microtubule-binding proteins Msps and D-TACC in dividing NSCs. Furthermore, Hyx directly regulates the polo and aurA expression in vitro. Finally, overexpression of polo and aurA could significantly suppress ectopic NSC formation and NSC polarity defects caused by hyx depletion. Our data support a model in which Hyx promotes the expression of polo and aurA in NSCs and, in turn, regulates cell polarity and centrosome/microtubule assembly. This new paradigm may be relevant to future studies on Parafibromin/HRPT2-associated cancers.

I.12 Modulating muscle stem cells to enhance regeneration to ameliorate DMD disease progression

Duchenne muscular dystrophy (DMD) is a lethal X-linked muscle-wasting disease. Dystrophin functions as an essential component of the large oligomeric dystrophin-glycoprotein complex (DGC). The DGC acts to connect the actin cytoskeleton of the myofiber to the surrounding extracellular matrix through the sarcolemma. In the absence of dystrophin, DGC assembly is impaired which weakens the muscle fibers rendering them highly susceptible to injury, causing accumulation of inflammation and fibrosis, and progressive loss of muscle mass and function. We discovered that dystrophin is highly expressed in activated muscle stem cells and associates with the serine-threonine kinase Mark2/Par1b, a regulator of cell polarity. In the absence of dystrophin, Mark2 is downregulated, resulting in the inability to localize the cell polarity regulator Pard3 to the opposite side of the cell. Consequently, the number of asymmetric divisions and hence the generation of myogenic progenitors is significantly reduced. Our findings indicate that muscle wasting in DMD not only is caused by myofiber fragility, but also is exacerbated by impaired regeneration owing to intrinsic satellite cell dysfunction. Through a small-molecule screen, we made the discovery of a novel signal transduction pathway involving epidermal growth factor receptor (EGFR) and Aurora kinase A (Aurka) as key regulators of asymmetric satellite cell divisions. Inhibiting EGFR causes a substantial shift from asymmetric to symmetric division modes, whereas EGF treatment increases asymmetric divisions. EGFR activation acts through Aurka to orient mitotic centrosomes, and inhibiting Aurka blocks EGF stimulation-induced asymmetric division. In vivo EGF treatment markedly activates asymmetric divisions of dystrophin-deficient satellite cells in mdx mice, increasing progenitor numbers, enhancing regeneration, and restoring muscle strength. Therefore, activating an EGFR-dependent polarity pathway promotes functional rescue of dystrophin-deficient satellite cells and enhances muscle force generation. In collaboration with Satellos Bioscience, we have been developing small molecule drugs that stimulate asymmetric division by modulating the EGFR/Aurka and other pathways. Our goal is to develop small molecule modulators of muscle stem cell asymmetric division as a treatment for DMD.

116 (PB106) - Preclinical activity for TPX-4589 (LM-302), an antibody-drug conjugate targeting tight junction protein CLDN18.2 in solid tumors

Background: Claudin 18 isoform 2 (CLDN18.2) is a tight junction protein involved in the regulation of epithelial cell permeability, barrier function, and polarity. In healthy tissue, CLDN18.2 is selectively expressed in tight junctions of gastric epithelial tissue and hence largely inaccessible to intravenous antibodies. On malignant transformation, CLDN18.2 is exposed on the cancer cell surface and expressed in many solid tumors, including gastric cancer (GC) and pancreatic cancer (PC). CLDN18.2 expression is associated with tumor pathogenesis, proliferation, and metastasis, making it a promising target for cancer therapies. TPX-4589, a novel antibody-drug conjugate (ADC) coupled with cytotoxic payload monomethyl auristatin E (MMAE), was developed to target CLDN18.2. Materials and methods: Preclinical studies were conducted to characterize TPX-4589 in CLDN18.2-positive cells, including binding affinity, antibody-dependent cell-mediated cytotoxicity (ADCC), cell proliferation inhibition, and in vivo efficacy in gastric and pancreatic tumor models. Results: TPX-4589 bound to endogenous CLDN18.2 GC GAXC031 cells in a dose-dependent manner, with an EC50 of 47.25 nM. Internalization was shown in both high- and low-expressing CLDN18.2-positive cells, with superior internalization when compared to zolbetuximab, a monoclonal antibody (mAb) that specifically binds to CLDN18.2 (table). TPX-4589 showed strong concentration-dependent ADCC activity, with EC50 of 0.11 nM, and inhibited tumor cell proliferation in vitro with nanomolar potency in GAXC031 and PC MIA PaCa2-high CLDN18.2 cell lines. In a cell-line xenograft GC tumor model with high CLDN18.2 expression, TPX-4589 significantly reduced tumor volume in a dose-dependent manner and demonstrated superior efficacy to zolbetuximab. In a CLDN18.2-high patient-.derived xenograft (PDX) PC tumor model, increasing doses of TPX-4589 demonstrated superior tumor growth inhibition compared to gemcitabine and LM-102, a compound with identical anti-CLDN18.2 mAb component as LM-302, and similar efficacy to LM-102 + gemcitabine in combination. In a PDX PC tumor model with low CLDN18.2 expression, TPX-4589 showed potent inhibition of tumor growth.Table(abstract: 116 (PB106)). TPX-4589 internalization in CLDN18.2-positive cellsCell lineTPX-4589 (LM-102 conjugated with MMAE)LM-102 (CLDN18.2 mAb)ZolbetuximabControl hIgG1EC50 (nM)CLDN18.2/MKN45.#1(low CLDN18.2 expression)22.44.8NANAEC50 (nM)CLDN18.2/MKN45.#14(high CLDN18.2 expression)3.53.583.93NA Open table in a new tab Conclusions: TPX-4589, a novel CLDN18.2-targeting ADC, showed potent inhibitory effects on tumor cell proliferation in vitro and reduced tumor volume in both high- and low-expressing CDLN18.2 tumor models, with superior internalization and efficacy to zolbetuximab in a GC tumor model. These data suggest that TPX-4589 is a promising therapeutic candidate that warrants further investigation in clinical studies. Conflict of interest: Ownership: Stephanie Correia, Laura Rodon Turning Point Therapeutics Employee and Stock or Stock Options Other Substantive Relationships: Laura Rodon Turning Point Therapeutics Patents planned, issued or pending Yifan Li: Nothing to disclose Zhifang Liu: Nothing to disclose Runsheng Li: Nothing to disclose Yuan Li: Nothing to disclose Wentao Huang: Nothing to disclose

Spatiotemporal dynamics of membrane surface charge regulates cell polarity and migration.

During cell migration and polarization, numerous signal transduction and cytoskeletal components self-organize to generate localized protrusions. Although biochemical and genetic analyses have delineated many specific interactions, how the activation and localization of so many different molecules are spatiotemporally orchestrated at the subcellular level has remained unclear. Here we show that the regulation of negative surface charge on the inner leaflet of the plasma membrane plays an integrative role in the molecular interactions. Surface charge, or zeta potential, is transiently lowered at new protrusions and within cortical waves of Ras/PI3K/TORC2/F-actin network activation. Rapid alterations of inner leaflet anionic phospholipids-such as PI(4,5)P2, PI(3,4)P2, phosphatidylserine and phosphatidic acid-collectively contribute to the surface charge changes. Abruptly reducing the surface charge by recruiting positively charged optogenetic actuators was sufficient to trigger the entire biochemical network, initiate de novo protrusions and abrogate pre-existing polarity. These effects were blocked by genetic or pharmacological inhibition of key signalling components such as AKT and PI3K/TORC2. Conversely, increasing the negative surface charge deactivated the network and locally suppressed chemoattractant-induced protrusions or subverted EGF-induced ERK activation. Computational simulations involving excitable biochemical networks demonstrated that slight changes in feedback loops, induced by recruitment of the charged actuators, could lead to outsized effects on system activation. We propose that key signalling network components act on, and are in turn acted upon, by surface charge, closing feedback loops, which bring about the global-scale molecular self-organization required for spontaneous protrusion formation, cell migration and polarity establishment.

Implication of non-coding RNA-mediated ROCK1 regulation in various diseases.

Rho Associated Coiled-Coil Containing Protein Kinase 1 (ROCK1) is a protein serine/threonine kinase which is activated upon binding with the GTP-bound form of Rho. This protein can modulate actin-myosin contraction and stability. Moreover, it has a crucial role in the regulation of cell polarity. Therefore, it participates in modulation of cell morphology, regulation of expression of genes, cell proliferation and differentiation, apoptotic processes as well as oncogenic processes. Recent studies have highlighted interactions between ROCK1 and several non-coding RNAs, namely microRNAs, circular RNAs and long non-coding RNAs. Such interactions can be a target of medications. In fact, it seems that the interactions are implicated in therapeutic response to several medications. In the current review, we aimed to explain the impact of these interactions in the pathoetiology of cancers as well as non-malignant disorders.

Structural insight into the Scribble PDZ domains interaction with the oncogenic Human T-cell lymphotrophic virus-1 (HTLV-1) Tax1 PBM.

Scribble (Scrib) is a highly conserved cell polarity regulator that harbours potent tumour suppressor activity and plays an important role in cell migration. Dysregulation of polarity is associated with poor prognosis during viral infections. Human T-cell lymphotrophic virus-1 (HTLV-1) encodes for the oncogenic Tax1 protein, a modulator of the transcription of viral and human proteins that can cause cell cycle dysregulation as well as a loss of genomic integrity. Previous studies established that Scribble interacts with Tax1 via its C-terminal PDZ-binding motif (PBM), leading to aggregation of polarity regulators and subsequent perturbation of host cell adhesion, proliferation, and signalling. Using isothermal titration calorimetry, we now show that all four PDZ domains of Scribble bind to Tax1 PBM. We then determined crystal structures of Scribble PDZ1, PDZ2 and PDZ3 domains bound to Tax1 PBM. Our findings establish a structural basis for Tax1-mediated subversion of Scribble-mediated cell polarity signalling and provide the platform for mechanistic studies to examine Tax1 induced mislocalization of Scribble and the associated changes in cellular architecture and subsequent tumorigenesis.

Whole-Genome Sequencing Reveals the Genomic Characteristics and Selection Signatures of Hainan Black Goat.

Goats have become one of the most adaptive and important livestock species distributed in developing countries in recent years. The Hainan Black goat is a native goat breed of the Hainan region that is generally well-liked by the local population and is thus raised in large numbers. However, the genomic diversity and selective signals of the Hainan Black goat have not been clearly elucidated yet. Therefore, in this study, we performed whole-genome resequencing of 16 Hainan Black goats and compared the results with those of 71 goats of 6 other breeds from different geographic regions. Principal component analysis (PCA) and phylogenetic analysis identified seven lineages for all goats. Hainan Black goats showed the most similarity with Leizhou goats and the least similarity with Boer goats. Selective sweep analysis identified candidate genes associated with various functions, including immune resistance to disease (TNFAIP2 (TNF alpha induced protein 2) and EXOC3L4 (exocyst complex component 3 like 4)), melanin biosynthetic process (CDH15 (cadherin 15), ASIP (agouti signaling protein), and PARD3 (par-3 family cell polarity regulator)), and light sensitivity (CNGB3 (cyclic nucleotide gated channel subunit beta 3) and CNBD1 (cyclic nucleotide binding domain containing 1)), underlying strong selection signatures in Hainan Black goats. The melanin biosynthetic process, circadian entrainment, regulation of cyclic adenosine 3,5-monophosphate (cAMP)-mediated signaling, and the Rap-1 signaling pathway were significantly enriched in Hainan Black and Alashan Cashmere goats. This result may be important for understanding each trait. Selection signature analysis revealed candidate single nucleotide polymorphisms (SNPs) and genes correlated with the traits of Hainan Black goats. Collectively, our results provide valuable insights into the genetic basis of specific traits correlated with the Hainan island climate, artificial selection in certain local goat breeds, and the importance of protecting breed resources.