Regulation Of Cell Polarity Research Articles

CDC42 enhances phagocytic activity and exhibits antimicrobial functions through the recruitment of IQGAP1 in Apostichopus japonicus

Cell Division Cycle 42 protein (CDC42) is a pivotal member of the Rho family of small GTPases, integral to cell signaling and the regulation of cell polarity. CDC42 is extensively documented to participate in host immunity, particularly in processes such as phagocytosis and cell migration. Nevertheless, the precise mechanisms by which CDC42 affects host-bacteria interactions remain unclear in Echinoderms. In this study, we have isolated and characterized the CDC42 gene from Apostichopus japonicus, referred to as AjCDC42. The complete cDNA sequence of AjCDC42 extends 1282 base pairs, including a typical classical RHO domain, spanning amino acids from 6 to 179. Spatial expression analysis demonstrated that AjCDC42 is ubiquitously expressed across all examined tissues. Following infection with Vibrio splendidus and stimulation with LPS, the protein and mRNA levels of AjCDC42 were significantly upregulated in coelomocytes. Moreover, the knockdown of AjCDC42 significantly decreased V. splendidus-induced phagocytic activity in coelomocytes, leading to a reduced intracellular load of V. splendidus, as evidenced by flow cytometry and bacterial plate counting assays. Mechanistically, AjCDC42 was identified as an interacting partner of IQ Motif Containing GTPase Activating Protein 1 (AjIQGAP1) through the use of co-immunoprecipitation, immunofluorescence, and GST pull-down assays combined with mass spectrometry techniques. Additional functional analyses revealed that the knockdown of AjIQGAP1 similarly decreased the phagocytic activity and the intracellular load of V. splendidus in coelomocytes. Furthermore, we detected that decreased levels of AjCDC42 and AjIQGAP1 impaired cytoskeletal rearrangement and delayed lysosomal clearance of V. splendidus. In summary, our findings underscore the pivotal role of the AjCDC42-AjIQGAP1 axis in regulating coelomocytes phagocytosis in A. japonicus during V. splendidus infection.

Therapeutic targeting of the protein tyrosine kinase-7 in cancer: an overview.

The protein tyrosine kinase-7 (PTK7) is an evolutionarily conserved transmembrane receptor that has emerged as a potential therapeutic target for human tumors. PTK7 is a pseudokinase that is involved in the modulation of the Wnt signaling pathway through interactions with other receptors. These interactions result in targeted gene activation that regulates cell polarity, migration, and proliferation during embryogenesis. Aside of this role during development, PTK7 has been shown as overexpressed in numerous cancers including colon carcinoma, leukemia, neuroblastoma, hepatoma, and ovarian cancer. The activity of PTK7 and the direct correlation with poor prognosis have fostered preclinical investigations and phase I clinical trials, aiming at inhibiting PTK7 and inducing antitumoral effects. In this review, we provide an exhaustive overview of the diverse approaches that use PTK7 as a new molecular target for cancer therapy in different tumor types. We discuss current therapies and future strategies including chimeric antigen receptor-T cells, antibody-drug conjugates, aptamers, based on up-to-date literature and ongoing research progress.

SARS-CoV-2-derived protein Orf9b enhances MARK2 activity via interaction with the autoinhibitory KA1 domain.

Microtubule affinity-regulating kinase 2 (MARK2) is a Ser/Thr protein kinase that regulates cell polarity and immune responses. Here, we report that Orf9b, one of the accessory proteins encoded in the SARS-CoV-2 genome, increases MARK2 activity via interaction with the autoinhibitory KAI domain. We found that co-expression of Orf9b enhances the kinase activity of MARK2 in HEK293 cells. Orf9b does not bind to or enhance the activity of the mutant form of MARK2 lacking the KA1 domain. Orf9b lowers inhibitory phosphorylation of MARK2 at T595 while mutation experiments indicate that this site is dispensable for Orf9b-mediated enhancement of MARK2 activity. Our results suggest that Orf9b enhances MARK2 activity by binding the autoinhibitory KA1 domain, which closely interacts with the kinase domain.

Early Cell Lineage Formation in Mammals: Complexity, Species Diversity, and Susceptibility to Disruptions Impacting Embryo Viability.

The emergence of the earliest cell lineages in mammalian embryos is a complex process that utilizes an extensive network of chromatin regulators, transcription factors, cell polarity regulators, and cellular signaling pathways. These factors and pathways operate over a protracted period of time as embryos cleave, undergo compaction, and form blastocysts. The first cell fate specification event separates the pluripotent inner cell mass from the trophectoderm lineage. The second event separates pluripotent epiblast from hypoblast. This review summarizes over 50 years of study of these early lineage forming events, addressing the complexity of the network of interacting molecules, cellular functions and pathways that drive them, interspecies differences, and aspects of these mechanisms that likely underlie their high susceptibility to disruption by numerous environmental factors that can compromise embryo viability, such as maternal health and diet, environmental toxins, and other stressors.

Four-dimensional trapped ion mobility spectrometry proteomics reveals circulating extracellular vesicles encapsulated drivers of nasopharyngeal carcinoma distant dissemination

Nasopharyngeal carcinoma (NPC) is a head and neck cancer with a high propensity for early metastatic spread. Emerging evidence shows that extracellular vesicles (EVs) are key players in cancer metastasis, but their role in NPC metastasis remains poorly understood. We here present the first description of the proteomic and functional profiles of serum-derived circulating small EVs in metastatic NPC patients. To enhance the capture of low-abundance signaling proteins in EVs, timsTOF-based four-dimensional label-free quantitative proteomics was employed. We found that metastatic NPC patients (M-NPC-EVs) exhibited the highest serum EV levels compared to locoregional patients (L-NPC-EVs) and healthy subjects (Normal-EVs). The proteome of M-NPC-EVs differed substantially from L-NPC-EVs and was functionally enriched in pathways regulating cell polarity and motility, glucose metabolism, and angiogenesis. Functional assays testing individual EV samples demonstrated that M-NPC-EVs pronouncedly enhanced NPC cell migration, invasion, and the formation of lamellipodia and filopodia in vitro, and promoted angiogenesis in subcutaneous Matrigel plugs in vivo. In silico analyses suggested that PTPRA, TPI1 and GPI highly enriched in M-NPC-EVs were putative drivers underlying the motogenic and angiogenic activities of M-NPC-EVs, and their high expression levels were associated with a poor prognosis of NPC patients. The increased expression of PTPRA, TPI1 and GPI in M-NPC-EVs was then validated in an independent cohort consisting of 175 NPC patients (locoregional n = 114; metastatic n = 61). Together, utilizing patient-derived EVs, we mimicked the potential pro-metastatic functions of EVs in NPC patients in vitro and in vivo and provided novel insights into their bioactive cargoes.

JNK Kinase regulates cachexia like syndrome in scribble knockdown tumor model of Drosophila melanogaster

Cachexia and systemic organ wasting are metabolic syndrome often associated with cancer. However, the exact mechanism of cancer associated cachexia like syndrome still remain elusive. In this study, we utilized a scribble (scrib) knockdown induced hindgut tumor to investigate the role of JNK kinase in cachexia like syndrome. Scrib, a cell polarity regulator, also acts as a tumor suppressor gene. Its loss and mis-localization are reported in various type of malignant cancer-like breast, colon and prostate cancer. The scrib knockdown flies exhibited male lethality, reduced life span, systemic organ wasting and increased pJNK level in hindgut of female flies. Interestingly, knocking down of human JNK Kinase analogue, hep, in scrib knockdown background in hindgut leads to restoration of loss of scrib mediated lethality and systemic organ wasting. Our data showed that scrib loss in hindgut is capable of inducing cancer associated cachexia like syndrome. Here, we firstly report that blocking the JNK signaling pathway effectively rescued the cancer cachexia induced by scrib knockdown, along with its associated gut barrier disruption. These findings have significantly advanced our understanding of cancer cachexia and have potential implications for the development of therapeutic strategies. However, more research is needed to fully understand the complex mechanisms underlying this condition.

Claudin is a promising biomarker of intestinal wall permeability

To date, sufficient information is available on changes in the levels of tight contact proteins (TCPs) in various diseases. Of greatest interest is the study of claudin due to numerous studies confirming the important role of this protein in the development of pathologies. Deafness, behavioural changes, and nerve conduction disorders occur due to insufficient claudin production. Claudin is involved in the maintenance of cell polarity due to its apical location in the cell. When pathological processes occur, claudin may undergo phosphorylation, leading to changes in cell polarity and impaired regulation of APC levels. Irritable bowel syndrome is one of the possible causes of impaired regulation of claudin levels. In this pathology there is adecrease in claudin-1 expression. In addition, claudins play different roles in different tumour tissues, and the same protein may be differentially expressed in different forms of cancer. Claudin-4 expression is upregulated as disease progresses in colorectal cancer (CRC), oesophageal squamous cell carcinoma, and its upregulation is associated with decreased invasiveness and metastatic potential, which is positively correlated with improved prognosis. In contrast, overexpression of CLDN 4 in breast cancer (BC) and pancreatic cancer (PC) is positively correlated with invasion, metastasis, angiogenesis and poor prognosis. Aim: systematisation of available data on the biological role and clinical significance of claudin. Materials: review of Russian and foreign literature from 2013-2023.

PIP5K-Ras bistability initiates plasma membrane symmetry breaking to regulate cell polarity and migration.

Symmetry breaking, polarity establishment, and spontaneous cell protrusion formation are fundamental but poorly explained cell behaviors. Here, we demonstrate that a biochemical network, where the mutually inhibitory localization of PIP5K and Ras activities plays a central role, governs these processes. First, in resting cells devoid of cytoskeletal activity, PIP5K is uniformly elevated on the plasma membrane, while Ras activity remains minimal. Symmetry is broken by spontaneous local displacements of PIP5K, coupled with simultaneous activations of Ras and downstream signaling events, including PI3K activation. Second, knockout of PIP5K dramatically increases both the incidence and size of Ras-PI3K activation patches, accompanied by branched F-actin assembly. This leads to enhanced cortical wave formation, increased protrusive activity, and a shift in migration mode. Third, high inducible overexpression of PIP5K virtually eliminates Ras-PI3K signaling, cytoskeletal activity, and cell migration, while acute recruitment of cytosolic PIP5K to the membrane induces contraction and blebs in cancer cells. These arrested phenotypes are reversed by reducing myosin II activity, indicating myosin's involvement in the PIP5K-Ras-centered regulatory network. Remarkably, low inducible overexpression of PIP5K unexpectedly facilitates polarity establishment, highlighting PIP5K as a highly sensitive master regulator of these processes. Simulations of a computational model combining an excitable system, cytoskeletal loops, and dynamic partitioning of PIP5K recreates the experimental observations. Taken together, our results reveal that a bistable, mutually exclusive localization of PIP5K and active Ras on the plasma membrane triggers the initial symmetry breaking. Coupled actomyosin reduction and increased actin polymerization lead to intermittently extended protrusions and, with feedback from the cytoskeleton, self-organizing, complementary gradients of PIP5K versus Ras steepen, raising the threshold of the networks at the rear and lowering it at the front to generate polarity for cell migration.

Abstract PR-19: Cell polarity proteins as novel regulators of macropinocytosis

Abstract Macropinocytosis has emerged as a nutrient-scavenging pathway that cancer cells exploit to survive the nutrient-deprived conditions of the tumor microenvironment. Cancer cells are especially reliant on glutamine for their survival, and in pancreatic ductal adenocarcinoma (PDAC) cells, glutamine deficiency can enhance the stimulation of macropinocytosis, allowing the cells to escape metabolic stress through the production of extracellular-protein-derived amino acids. Here, we identify the atypical protein kinase C (aPKC) enzymes, PKCz and PKCi, as novel regulators of macropinocytosis. In normal epithelial cells, aPKCs are known to regulate cell polarity in association with the scaffold proteins Par3 and Par6, controlling the function of several targets, including the microtubule-associated Par1 kinases. In PDAC cells, we identify that each of these cell polarity proteins are required for glutamine stress-induced macropinocytosis. Mechanistically, we find that the aPKCs are regulated by EGFR signaling or by the transcription factor CREM to promote the relocation of Par3 to microtubules, facilitating macropinocytosis in a dynein-dependent manner. Importantly, we determine that cell fitness impairment caused by aPKC depletion in glutamine stress is rescued by the restoration of macropinocytosis and that aPKCs support PDAC growth in vivo. These results identify a previously unappreciated role for the cell polarity protein network in the regulation of macropinocytosis and provide a better understanding of the mechanistic underpinnings that control macropinocytic uptake in the context of metabolic stress. Citation Format: Guillem Lambies Barjau, Szu-Wei Lee, Karen Duong-Polk Duong-Polk, David Dawson, Cheska Marie Galapate, Swetha Maganti, Cosimo Commisso. Cell polarity proteins as novel regulators of macropinocytosis [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr PR-19.

Cell Polarity-related Gene PTK7, a Potential Diagnostic Biomarker in Pan-cancer.

PTK7 (Protein Tyrosine Kinase 7), a member of the receptor protein tyrosine kinase family, was originally discovered in colon cancer cells. It plays a pivotal role in numerous developmental and physiological processes, particularly in the regulation of cell polarity. Despite accumulating evidence of PTK7's significant influence on tumor development, a comprehensive pan-cancer analysis of PTK7 has yet to be conducted. We conducted a comprehensive analysis of PTK7's expression, prognostic value, and mutational patterns across various tumor types. We further explored the correlations between PTK7 expression and tumor stemness, immune-related genes, immune scores, and immune cell infiltration. Enrichment analysis revealed PTK7's critical involvement in pan-cancer functions and processes, including the WNT pathway, Epithelial-Mesenchymal Transition (EMT), and cell polarity regulation. Additionally, we validated PTK7's expression in gastric cancer via immunohistochemistry. Our study indicates that PTK7 holds promise as an ideal pan-cancer biomarker due to its involvement in tumor progression and tumor immunity.

Identification of diagnostic biomarkers and potential therapeutic targets for biliary atresia via WGCNA and machine learning methods.

Biliary atresia (BA) is a severe and progressive biliary obstructive disease in infants that requires early diagnosis and new therapeutic targets. This study employed bioinformatics methods to identify diagnostic biomarkers and potential therapeutic targets for BA. Our analysis of mRNA expression from Gene Expression Omnibus datasets revealed 3,273 differentially expressed genes between patients with BA and those without BA (nBA). Weighted gene coexpression network analysis determined that the turquoise gene coexpression module, consisting of 298 genes, is predominantly associated with BA. The machine learning method then filtered out the top 2 important genes, CXCL8 and TMSB10, from the turquoise module. The area under receiver operating characteristic curves for TMSB10 and CXCL8 were 0.961 and 0.927 in the training group and 0.819 and 0.791 in the testing group, which indicated a high diagnostic value. Besides, combining TMSB10 and CXCL8, a nomogram with better diagnostic performance was built for clinical translation. Several studies have highlighted the potential of CXCL8 as a therapeutic target for BA, while TMSB10 has been shown to regulate cell polarity, which was related to BA progression. Our analysis with qRT PCR and immunohistochemistry also confirmed the upregulation of TMSB10 at mRNA and protein levels in BA liver samples. These findings highlight the sensitivity of CXCL8 and TMSB10 as diagnostic biomarkers and their potential as therapeutic targets for BA.

The dishevelled associated activator of morphogenesis protein 2 (Daam2) regulates neural tube closure.

The Wnt signaling pathway is highly conserved in metazoans and regulates a large array of cellular processes including motility, polarity and fate determination, and stem cell homeostasis. Modulation of the actin cytoskeleton via the non-canonical Wnt pathway regulate cell polarity and cell migration that are required for proper vertebrate gastrulation and subsequent neurulation. However, the mechanism(s) of how the non-canonical pathway mediates actin cytoskeleton modulation is not fully understood. Herein, we characterize the role of the Formin-homology protein; dishevelled associated activator of morphogenesis 2 (Daam2) protein in the Wnt signaling pathway. Co-immunoprecipitation assays confirm the binding of Daam2 to dishevelled2 (Dvl2) as well as the domains within these proteins required for interaction; additionally, the interaction between Daam2 and Dvl2 was Wnt-regulated. Sub-cellular localization studies reveal Daam2 is cytoplasmic and regulates the cellular actin cytoskeleton by modulating actin filament formation. During Xenopus development, a knockdown or loss of Daam2 specifically produces neural tube closure defects indicative of a role in non-canonical signaling. Additionally, our studies did not identify any role for Daam2 in canonical Wnt signaling in mammalian culture cells or the Xenopus embryo. Our studies together identify Daam2 as a component of the non-canonical Wnt pathway and Daam2 is a regulator of neural tube morphogenesis during vertebrate development.

Atypical Protein Kinase C Promotes its own Asymmetric Localisation by Phosphorylating Cdc42 in the C. elegans zygote.

Atypical protein kinase C (aPKC) is a major regulator of cell polarity. Acting in conjunction with Par6, Par3 and the small GTPase Cdc42, aPKC becomes asymmetrically localised and drives the polarisation of cells. aPKC activity is crucial for its own asymmetric localisation, suggesting a hitherto unknown feedback mechanism contributing to polarisation. Here we show in the C. elegans zygote that the feedback relies on aPKC phosphorylation of Cdc42 at serine 71. The turnover of CDC-42 phosphorylation ensures optimal aPKC asymmetry and activity throughout polarisation by tuning Par6/aPKC association with Par3 and Cdc42. Moreover, turnover of Cdc42 phosphorylation regulates actomyosin cortex dynamics that are known to drive aPKC asymmetry. Given the widespread role of aPKC and Cdc42 in cell polarity, this form of self-regulation of aPKC may be vital for the robust control of polarisation in many cell types.

Focal adhesions are controlled by microtubules through local contractility regulation

Microtubules regulate cell polarity and migration via local activation of focal adhesion turnover, but the mechanism of this process is insufficiently understood. Molecular complexes containing KANK family proteins connect microtubules with talin, the major component of focal adhesions. Here, local optogenetic activation of KANK1-mediated microtubule/talin linkage promoted microtubule targeting to an individual focal adhesion and subsequent withdrawal, resulting in focal adhesion centripetal sliding and rapid disassembly. This sliding is preceded by a local increase of traction force due to accumulation of myosin-II and actin in the proximity of the focal adhesion. Knockdown of the Rho activator GEF-H1 prevented development of traction force and abolished sliding and disassembly of focal adhesions upon KANK1 activation. Other players participating in microtubule-driven, KANK-dependent focal adhesion disassembly include kinases ROCK, PAK, and FAK, as well as microtubules/focal adhesion-associated proteins kinesin-1, APC, and αTAT. Based on these data, we develop a mathematical model for a microtubule-driven focal adhesion disruption involving local GEF-H1/RhoA/ROCK-dependent activation of contractility, which is consistent with experimental data.

The cellular localization and oncogenic or tumor suppressive effects of angiomiotin-like protein 2 in tumor and normal cells.

Angiomiotin (AMOT) family comprises three members: AMOT, AMOT-like protein 1 (AMOTL1), and AMOT-like protein 2 (AMOTL2). AMOTL2 is widely expressed in endothelial cells, epithelial cells, and various cancer cells. Specifically, AMOTL2 predominantly localizes in the cytoplasm and nucleus in human normal cells, whereas associates with cell-cell junctions and actin cytoskeleton in non-human cells, and locates at cell junctions or within the recycling endosomes in cancer cells. AMOTL2 is implicated in regulation of tube formation, cell polarity, and shape, although the specific impact on tumorigenesis remains to be conclusively determined. It has been shown that AMOTL2 enhances tumor growth and metastasis in pancreatic, breast, and colon cancer, however inhibits cell proliferation and migration in lung, hepatocellular cancer, and glioblastoma. In addition to its role in cell shape and cytoskeletal dynamics through co-localization with F-actin, AMOTL2 modulates the transcription of Yes-associated protein (YAP) by binding to it, thereby affecting its phosphorylation and cellular sequestration. Furthermore, the stability and cellular localization of AMOTL2, influenced by its phosphorylation and ubiquitination mediated by specific proteins, affects its cellular function. Additionally, we observe that AMOTL2 is predominantly downregulated in some tumors, but significantly elevated in colorectal adenocarcinoma (COAD). Moreover, overall analysis, GSEA and ROC curve analysis indicate that AMOTL2 exerts as an oncogenic protein in COAD by modulating Wnt pathway, participating in synthesis of collagen formation, and interacting with extracellular matrix receptor. In addition, AMOTL2 potentially regulates the distribution of immune cells infiltration in COAD. In summary, AMOTL2 probably functions as an oncogene in COAD. Consequently, further in-depth mechanistic research is required to elucidate the precise roles of AMOTL2 in various cancers.

Abstract PO4-24-03: Adiponectin modulates cell polarity according to ERα expression in breast cancer cells

Abstract INTRODUCTION: Tumor microenvironment (TME) is a complex and heterogeneous network, consisting of stromal and immune cells, embedded with cytokines, growth factors, soluble receptors, and exosomes. Mutual and dynamic crosstalk among cancer, stromal and immune cells predispose cancer cells to metastasis. Particularly, the main component of breast TME is represented by adipocytes, which secretory activity is compromised in obesity. Indeed, hypertrophic, and hyperplastic adipocytes undergo to a modified cytokines secretory pattern, characterized by a reduced secretion of Adiponectin, now recognized as a crucial factor in the pathogenesis of breast cancer. LKB1 is one the most important effector of Adiponectin transduction pathways, and recently it has been demonstrated its role as ERα coactivator in promoting breast cancer cell growth. Moreover, LKB1 is a key regulator in controlling cell polarity along with E-cadherin and Cdc42. Cell polarity has been considered essential for mammary epithelial integrity, and disruption of individual polarity proteins is sufficient to induce tumor progression. The aim of the present study was to investigate the effects of adiponectin on the regulation and alteration of cell polarity, essential for breast cancer cell growth and invasiveness. METHODS: Immunofluorescence analyses were done to detect protein localization in breast cancer cells. Protein expression was performed by immunoblotting in the presence or absence of ERa and LKB1 siRNA. LKB1/Cdc42 and LKB1/E-cadherin interaction was detected by Proximity ligation and immunoprecipitation assay respectively. Wound healing assay was carried out to evaluate GM130 orientation. Growth and metastatic potential of breast cancer cells were assessed in vivo by orthotopic model and metastatic study respectively. RESULTS: In Adiponectin-treated MCF-7 cells LKB1 is mostly present in the nucleus, recruited as ERa-coactivator, while in BT20 cells LKB1 is localized with E-cadherin in the cytosolic compartment, suggesting its role in polarity. Our results showed that in MCF-7 cells Adiponectin increases Cdc42 level depending on LKB1 expression. Moreover, Cdc42 and LKB1 interact and colocalized in the nucleus of Adiponectin-treated MCF-7 cells. On the contrary, LKB1 and Cdc42 are located with E-cadherin in the cytosol of BT20 cells, addressing their cytosolic cooperation in maintaining cell polarity, as confirmed by an increased Cdc42 activity. Immunofluorescence analyses demonstrated a lack of Golgi realignment toward the front of the leading edge in MCF-7 cells treated with Adiponectin, not observed in BT20 cells. All this suggest a loss of cell polarity organization that predispose to cancer growth and progression, as confirmed by an increased tumor volume mass and a major number of lung metastatic events upon tail vein injection of adiponectin-treated ERα-positive cells with respect to untreated cells. CONCLUSION: In ERα-positive breast cancer cells Adiponectin modulates the expression of the main proteins involved in cell polarity, leading to increased growth, progression, and distant metastasis. Citation Format: Giuseppina Daniela Naimo, Martina Forestiero, Alessandro Paolì, Luca Gelsomino, Rocco Malivindi, Adele Elisabetta Leonetti, Francesca Giordano, Maria Luisa Panno, Loredana Mauro, Sebastiano Andò. Adiponectin modulates cell polarity according to ERα expression in breast cancer cells [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-24-03.

Abstract 261: Akt1 and Cyclin D1 govern polarity of self-renewing divisions in breast cancer stem cells

Abstract Background. Stem-like cells contribute to the development and progression of human cancers. Genes that control symmetric cell divisions have an evolutionarily conserved role in the regulation of cell polarity and in tumorigenesis. Two cells generated by asymmetric divisions have reduced proliferative potential compared with symmetric divisions. The cell survival oncoprotein Akt (Protein Kinase B) is encoded by three isoforms that are altered in human cancers. The Akt isoform-specific impact on breast cancer stem cell function and polarity is controversial, with Akt1 either reducing (1) or promoting (2) cell polarity and migration in tissue culture. Deletion of Akt1 reduced mammary tumor progression and metastasis in transgenic mice (2,3). Methods. Analysis of Akt1, cyclin D1 and Notch signaling was conducted from public databases, and human as well as murine breast cancer samples. Cells derived from Akt1 gene deletion MMTV-ErbB2 breast oncomice (MMTV-ErbB2/Akt1+/+ and MMTV-ErbB2/Akt1−/−) and from the human MCF-7 breast cancer cell line with regulated expression of cyclin D1 and p53 were analyzed for the symmetry of cell division, stem cell function, and signaling pathways. Results. In breast tumors derived from MMTV-ErbB2 transgenics, associated with reduced metastasis, Akt1 genetic deletion reduced the frequency of symmetric cell divisions and the production of mammospheres. Akt1 deletion increased p53 and reduced cyclin D1. Pharmacological reactivation of p53 with Nutlin correlated with the reinduction of asymmetric cellular divisions and reduction in mammospheres in murine and human BCa cells. Akt1 deletion abrogated Nutlin-mediated induction of asymmetric cell divisions. Cyclin D1 induced symmetrical cell divisions, mammosphere formation and reduced p53 and Numb. Conclusions. Akt1 promoted symmetrical cell division at least in part via cyclin D1 suppression of Numb, and induction of γ secretase activity and thereby Notch signaling. Reference. (1) Irie HY, Pearline RV, Grueneberg D, Hsia M, Ravichandran P, Kothari N, et al. Distinct roles of Akt1 and Akt2 in regulating cell migration and epithelial-mesenchymal transition. J Cell Biol 2005;171:1023-34. (2) Ju X, Katiyar S, Wang C, Liu M, Jiao X, Li S, et al. Akt1 governs breast cancer progression in vivo. Proc Natl Acad Sci U S A 2007;104:7438-43. (3) Chen X, Ariss MM, Ramakrishnan G, Nogueira V, Blaha C, Putzbach W, et al. Cell-Autonomous versus Systemic Akt Isoform Deletions Uncovered New Roles for Akt1 and Akt2 in Breast Cancer. Mol Cell 2020;80:87-101 e5 Citation Format: Xuanmao Jiao, Xin Sun, Zhiping Li, Xiaoming Ju, Danni Li, Ritika Harish, Gordon Robertson, Anthony W. Ashton, Richard G. Pestell. Akt1 and Cyclin D1 govern polarity of self-renewing divisions in breast cancer stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 261.

MulStack: An ensemble learning prediction model of multilabel mRNA subcellular localization

Subcellular localization of mRNA is related to protein synthesis, cell polarity, cell movement and other biological regulation mechanisms. The distribution of mRNAs in subcellulars is similar to that of proteins, and most mRNAs are distributed in multiple subcellulars. Recently, some computational methods have been designed to predict the subcellular localization of mRNA. However, these methods only employed a sin-gle level of mRNA features and did not employ the position encoding of nucleotides in mRNA. In this paper, an ensemble learning prediction model is proposed, named MulStack, which is based on random forest and deep learning for multilabel mRNA subcellular localization. The proposed method employs two levels of mRNA features, including sequence-level and residue-level features, and position encoding is employed for the first time in the field of subcellular localization of mRNA. Random forest is employed to learn mRNA sequence-level feature, deep learning is employed to learn mRNA sequence-level feature and mRNA residue-level combined with position encoding. And the outputs of random forest and deep learning model will be weighted sum as the prediction probability. Compared with existing methods, the results show that MulStack is the best in the localization of the nucleus, cytosol and exosome. In addition, position weight matrices (PWMs) are extracted by convolutional neural networks (CNNs) that can be matched with known RNA binding protein motifs. Gene ontology (GO) enrichment analysis shows biological processes, molecular functions and cellular components of mRNA genes. The prediction web server of MulStack is freely accessible at http://bliulab.net/MulStack.

Comprehensive analysis of basement membrane-related gene based on single-cell and bulk RNA sequencing data to predict prognosis and evaluate immune characteristics in colorectal cancer.

Basement membrane-related genes (BMs) participate in regulating cell polarity, invasion, metastasis, and survival across different tumor types. Nevertheless, the specific functions of BMs in colorectal cancer (CRC) remain uncertain. To investigate the clinical relevance of BMs in CRC, we retrieved both gene expression and clinical data from The Cancer Genome Atlas (TCGA) datasets for subsequent analysis. The Kaplan-Meier (K-M) survival curve was employed to evaluate prognosis in high- and low-risk groups. Furthermore, additional analyses, including nomogram construction, functional enrichment, examination of the tumor immune microenvironment, prediction of small-molecule drugs, and more, were conducted to delve into the significance of BM-related signatures in CRC. Single-cell data from seven CRC patients were obtained from the TISCH2 database, and expression validation and cell source exploration of BM-related signatures were performed. Lastly, the expression and function of TIMP1, a key gene in BMs that may play a role in the progression of CRC, was validated in vitro through a series of basic experiments. We constructed a seven BMs-based model to categorize CRC patients into high-risk and low-risk groups. K-M survival analysis indicated a poorer prognosis for high-risk CRC patients. Cox regression analysis further identified the risk score as an independent prognostic factor for CRC patients. The nomogram model exhibited superior discrimination and calibration abilities of CRC patients. Based on the results from GO/KEGG and GSEA, genes in the high-risk subgroup were implicated in immune-related pathways and exhibited a positive correlation with immune checkpoints. In single-cell data, we found that TIMP1 is highly expressed in many cells, especially in malignant tumor cells. We also observed up-regulation of TIMP1 in CRC cell lines, promoting cancer invasion and migration in vitro. Our study has discovered a novel prognostic index derived from BM-related genes in CRC patients. Specifically, the new model enables patient stratification, improving the selection of individuals likely to benefit from immunotherapy.

PARD3 drives tumorigenesis through activating Sonic Hedgehog signalling in tumour-initiating cells in liver cancer

BackgroundPar-3 Family Cell Polarity Regulator (PARD3) is a cellular protein essential for asymmetric cell division and polarized growth. This study aimed to study the role of PARD3 in hepatic tumorigenesis.MethodsThe essential role of PARD3 in mediating hepatic tumorigenesis was assessed in diet-induced spontaneous liver tumour and syngeneic tumour models. The mechanism of PARD3 was delineated by bulk and single-cell RNA sequencing. The clinical significance of PARD3 was identified by tissue array analysis.ResultsPARD3 was overexpressed in tumour tissues and PARD3 overexpression was positively correlated with high tumour stage as well as the poor prognosis in patients. In models of spontaneous liver cancer induced by choline-deficient, amino acid-defined (CDAA) and methionine-choline-deficient (MCD) diets, upregulation of PARD3 was induced specifically at the tumorigenesis stage rather than other early stages of liver disease progression. Site-directed knockout of PARD3 using an adeno-associated virus 8 (AAV8)-delivered CRISPR/Cas9 single-guide RNA (sgRNA) plasmid blocked hepatic tumorigenesis, while PARD3 overexpression accelerated liver tumour progression. In particular, single-cell sequencing analysis suggested that PARD3 was enriched in primitive tumour cells and its overexpression enhanced tumour-initiating cell (TICs). Overexpression of PARD3 maintained the self-renewal ability of the CD133+ TIC population within hepatocellular carcinoma (HCC) cells and promoted the in vitro and in vivo tumorigenicity of CD133+ TICs. Transcriptome analysis revealed that Sonic Hedgehog (SHH) signalling was activated in PARD3-overexpressing CD133+ TICs. Mechanistically, PARD3 interacted with aPKC to further activate SHH signalling and downstream stemness-related genes. Suppression of SHH signalling and aPKC expression attenuated the in vitro and in vivo tumorigenicity of PARD3-overexpressing CD133+ TICs. Tissue array analysis revealed that PARD3 expression was positively associated with the phosphorylation of aPKC, SOX2 and Gli1 and that the combination of these markers could be used to stratify HCC patients into two clusters with different clinicopathological characteristics and overall survival prognoses. The natural compound berberine was selected as a potent suppressor of PARD3 expression and could be used as a preventive agent for liver cancer that completely blocks diet-induced hepatic tumorigenesis in a PARD3-dependent manner.ConclusionThis study revealed PARD3 as a potential preventive target of liver tumorigenesis via TIC regulation.