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Metastasis is the cause of over 90% of all deaths associated with breast cancer, yet the strategies to predict cancer spreading based on primary tumor profiles and therefore prevent metastasis are egregiously limited. As rare precursor cells to metastasis, circulating tumor cells (CTCs) in multicellular clusters in the blood are 20-50 times more likely to produce viable metastasis than single CTCs. However, the molecular mechanisms underlying various CTC clusters, such as homotypic tumor cell clusters and heterotypic tumor-immune cell clusters, are yet to be fully elucidated. Combining machine learning-assisted computational ranking with experimental demonstration to assess cell adhesion candidates, we identified a transmembrane protein Plexin- B2 (PB2) as a new therapeutic target that drives the formation of both homotypic and heterotypic CTC clusters. High PB2 expression in human primary tumors predicts an unfavorable distant metastasis-free survival and is enriched in CTC clusters compared to single CTCs in advanced breast cancers. Loss of PB2 reduces formation of homotypic tumor cell clusters as well as heterotypic tumor-myeloid cell clusters in triple-negative breast cancer. Interactions between PB2 and its ligand Sema4C on tumor cells promote homotypic cluster formation, and PB2 binding with Sema4A on myeloid cells (monocytes) drives heterotypic CTC cluster formation, suggesting that metastasizing tumor cells hijack the PB2/Sema family axis to promote lung metastasis in breast cancer. Additionally, using a global proteomic analysis, we identified novel downstream effectors of the PB2 pathway associated with cancer stemness, cell cycling, and tumor cell clustering in breast cancer. Thus, PB2 is a novel therapeutic target for preventing new metastasis.

Circulating tumor cell (CTC) clusters mediate metastasis at a higher efficiency and are associated with lower overall survival in breast cancer compared to single cells. Combining single-cell RNA sequencing and protein analyses, here we report the profiles of primary tumor cells and lung metastases of triple-negative breast cancer (TNBC). ICAM1 expression increases by 200-fold in the lung metastases of three TNBC patient-derived xenografts (PDXs). Depletion of ICAM1 abrogates lung colonization of TNBC cells by inhibiting homotypic tumor cell-tumor cell cluster formation. Machine learning-based algorithms and mutagenesis analyses identify ICAM1 regions responsible for homophilic ICAM1-ICAM1 interactions, thereby directing homotypic tumor cell clustering, as well as heterotypic tumor-endothelial adhesion for trans-endothelial migration. Moreover, ICAM1 promotes metastasis by activating cellular pathways related to cell cycle and stemness. Finally, blocking ICAM1 interactions significantly inhibits CTC cluster formation, tumor cell transendothelial migration, and lung metastasis. Therefore, ICAM1 can serve as a novel therapeutic target for metastasis initiation of TNBC., Circulating tumor cell (CTC) clusters are more efficient at mediating metastasis as compared to single cells and are associated with poor prognosis in breast cancer. Here, the authors show that ICAM1 is enriched in CTC clusters and its loss suppresses cell-cell interaction and CTC cluster formation, and propose ICAM1 as a therapeutic target for treating breast cancer metastasis.

Cancer metastasis is largely incurable and accounts for 90% of breast cancer deaths, especially for the aggressive basal-like or triple negative breast cancer (TNBC). Combining patient database analyses and functional studies, we examined the association of integrin family members with clinical outcomes as well as their connection with previously identified microRNA regulators of metastasis, such as miR-206 that inhibits stemness and metastasis of TNBC. Here we report that the integrin receptor CD49b-encoding ITGA2, a direct target of miR-206, promotes breast cancer stemness and metastasis. ITGA2 knockdown suppressed self-renewal related mammosphere formation and pluripotency marker expression, inhibited cell cycling, compromised migration and invasion, and therefore decreased lung metastasis of breast cancer. ITGA2 overexpression reversed miR-206-caused cell cycle arrest in G1. RNA sequencing analyses revealed that ITGA2 knockdown inhibits genes related to cell cycle regulation and lipid metabolism, including CCND1 and ACLY as representative targets, respectively. Knockdown of CCND1 or ACLY inhibits mammosphere formation of breast cancer cells. Overexpression of CCND1 rescues the phenotype of ITGA2 knockdown-induced cell cycle arrest. ACLY-encoded ATP citrate lyase is essential to maintain cellular acetyl-CoA levels. CCND1 knockdown further mimics ITGA2 knockdown in abolishing lung colonization of breast cancer cells. We identified that the low levels of miR-206 as well as high expression levels of ITGA2, ACLY and CCND1 are associated with an unfavorable relapse-free survival of the patients with estrogen receptor-negative or high grade breast cancer, especially basal-like or TNBC, possibly serving as potential biomarkers of cancer stemness and therapeutic targets of breast cancer metastasis.

Triple-negative breast cancer (TNBC) is one of the most aggressive and metastatic breast cancer subtypes lacking targeted therapy. Our recent work demonstrated that circulating tumor cell (CTC) clusters and polyclonal metastasis of TNBC are driven by aggregation of CD44+ cancer stem cells (CSC) and associated with an unfavorable prognosis, such as low overall survival. However, there is no existing therapeutic that can specifically block CTC or CSC cluster formation. Methods: Using patient-derived xenograft (PDX) models, we established an ex vivo tumor cell clustering assay for a pilot screening of blockade antibodies. After identifying EGFR as a target candidate, we modulated the gene expression and inhibited its kinase activity to determine its functional importance in tumor cell clustering and therapeutic inhibition of lung metastasis. We also examined the molecular regulation network of EGFR and a potential connection to CSC marker CD44 and microRNAs, which regulate CTC clustering. Results: We report here that EGFR inhibition successfully blocks circulating CSC (cCSC) clustering and lung metastasis of TNBC. EGFR enhances CD44-mediated tumor cell aggregation and CD44 stabilizes EGFR. Importantly, blocking EGFR by a novel anti-EGFR monoclonal antibody (clone LA1) effectively blocked cell aggregation in vitro and reduced lung metastasis in vivo. Furthermore, our data demonstrated that the tumor suppressor microRNA-30c serves as another negative regulator of cCSC clustering and lung metastasis by targeting CD44 as well as its downstream effector EGFR. Conclusion: Our studies identify a novel anti-EGFR therapeutic strategy to inhibit cCSC aggregation and therefore abolish cCSC cluster-mediated metastasis of TNBC.

CD44 molecule (CD44) is a well-known surface glycoprotein on tumor-initiating cells or cancer stem cells. However, its utility as a therapeutic target for managing metastases remains to be fully evaluated. We previously demonstrated that CD44 mediates homophilic interactions for circulating tumor cell (CTC) cluster formation, which enhances cancer stemness and metastatic potential in association with an unfavorable prognosis. Furthermore, CD44 self-interactions activate the P21-activated kinase 2 (PAK2) signaling pathway. Here, we further examined the biochemical properties of CD44 in homotypic tumor cell aggregation. The standard CD44 form (CD44s) mainly assembled as intercellular homodimers (trans-dimers) in tumor clusters rather than intracellular dimers (cis-dimers) present in single cells. Machine learning–based computational modeling combined with experimental mutagenesis tests revealed that the extracellular Domains I and II of CD44 are essential for its trans-dimerization and predicted high-score residues to be required for dimerization. Substitutions of 10 these residues in Domain I (Ser-45, Glu-48, Phe-74, Cys-77, Arg-78, Tyr-79, Ile-88, Arg-90, Asn-94, and Cys-97) or 5 residues in Domain II (Ile-106, Tyr-155, Val-156, Gln-157, and Lys-158) abolished CD44 dimerization and reduced tumor cell aggregation in vitro. Importantly, the substitutions in Domain II dramatically inhibited lung colonization in mice. The CD44 dimer-disrupting substitutions decreased downstream PAK2 activation without affecting the interaction between CD44 and PAK2, suggesting that PAK2 activation in tumor cell clusters is CD44 trans-dimer–dependent. These results shed critical light on the biochemical mechanisms of CD44-mediated tumor cell cluster formation and may help inform the development of therapeutic strategies to prevent tumor cluster formation and block cluster-mediated metastases.

Tumor-initiating cells with reprogramming plasticity and/or de-differentiation attributes have been thought to initiate primary tumor development as well as to regenerate secondary tumors in metastatic organs; however, the molecular mechanisms are not fully understood. We previously found that breast tumor-initiating cell marker, CD44, directs multicellular aggregation and cluster formation of circulating tumor cells (CTCs), which further enhance stemness and survival of such cells, enabling metastatic colonization to the lungs. To further elucidate the molecular network underlying CTC cluster formation, we performed global proteomic profiling and discovered that the tetraspanin protein CD81, which is normally enriched in exosomes (small extracellular vesicles), is a new driver of cancer initiation and metastasis as a facilitator and target of CD44. Loss of CD81 compromises tumorigenicity and mammosphere formation of triple negative breast cancer (TNBC) cells. Assisted by machine learning-based algorithms and mutagenesis approach, we found that CD81 interacts with CD44 on the cellular membrane through their extracellular regions. Notably, genetic knockout of CD44 or CD81 results in loss of both CD81 and CD44 in secreted exosomes, a state which abolishes exosome-induced self-renewal of recipient cells, such as mammosphere formation. In addition, RNA sequencing analysis showed that CD81 knockdown up-regulates expression of a cell differentiation marker, SEMA7a, whose down-regulation partially rescues mammosphere formation inhibition by CD81 depletion. Clinically, CD81 expression was observed in >80% of CTCs and specifically enriched and co-expressed along with CD44 in clustered CTCs of breast cancer patients. Mimicing the phenotypes of CD44 deficiency, loss of CD81 also inhibited tumor cell aggregation and lung metastasis of TNBC in both human and mouse tumor models, supporting the clinical significance of CD81 in association with patient outcomes. Our study highlights a new driving role of CD81 in cancer exosome-induced stemness, clustered CTCs, and metastasis initiation of TNBC, reported for the first time to our knowledge.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes the coronavirus disease 2019 (COVID-19) with innate and adaptive immune response triggered in such patients by viral antigens. Both convalescent plasma and engineered high affinity human monoclonal antibodies have shown therapeutic potential to treat COVID-19. Whether additional antiviral soluble factors exist in peripheral blood remain understudied. Herein, we detected circulating exosomes that express the SARS-CoV-2 viral entry receptor angiotensin-converting enzyme 2 (ACE2) in plasma of both healthy donors and convalescent COVID-19 patients. We demonstrated that exosomal ACE2 competes with cellular ACE2 for neutralization of SARS-CoV-2 infection. ACE2-expressing (ACE2+) exosomes, but not the ACE2-negative controls, blocked the binding of the viral spike (S) protein RBD to ACE2+ cells in a dose dependent manner, which was 400- to 700-fold more potent than that of vesicle-free recombinant human ACE2 extracellular domain protein (rhACE2). As a consequence, exosomal ACE2 prevented SARS-CoV-2 pseudotype virus tethering and infection of human host cells at a 50–150 fold higher efficacy than rhACE2. A similar antiviral activity of exosomal ACE2 was further demonstrated to block wild-type live SARS-CoV-2 infection. Of note, depletion of ACE2+ exosomes from COVID-19 patient plasma impaired the ability to block SARS-CoV-2 RBD binding to host cells. Furthermore, a dramatic increase in plasma ACE2+ exosome levels were detected in patients with severe COVID-19 pathogenesis. Our data demonstrate that ACE2+ exosomes can serve as a decoy therapeutic and a possible innate antiviral mechanism to block SARS-CoV-2 infection.

CD44 molecule (CD44) is a well-known surface glycoprotein on tumor-initiating cells or cancer stem cells. However, its utility as a therapeutic target for managing metastases remains to be fully evaluated. We previously demonstrated that CD44 mediates homophilic interactions for circulating tumor cell (CTC) cluster formation, which enhances cancer stemness and metastatic potential in association with an unfavorable prognosis. Furthermore, CD44 self-interactions activate the P21-activated kinase 2 (PAK2) signaling pathway. Here, we further examined the biochemical properties of CD44 in homotypic tumor cell aggregation. The standard CD44 form (CD44s) mainly assembled as intercellular homodimers (trans-dimers) in tumor clusters rather than intracellular dimers (cis-dimers) present in single cells. Machine learning–based computational modeling combined with experimental mutagenesis tests revealed that the extracellular Domains I and II of CD44 are essential for its trans-dimerization and predicted high-score residues to be required for dimerization. Substitutions of 10 these residues in Domain I (Ser-45, Glu-48, Phe-74, Cys-77, Arg-78, Tyr-79, Ile-88, Arg-90, Asn-94, and Cys-97) or 5 residues in Domain II (Ile-106, Tyr-155, Val-156, Gln-157, and Lys-158) abolished CD44 dimerization and reduced tumor cell aggregation in vitro. Importantly, the substitutions in Domain II dramatically inhibited lung colonization in mice. The CD44 dimer-disrupting substitutions decreased downstream PAK2 activation without affecting the interaction between CD44 and PAK2, suggesting that PAK2 activation in tumor cell clusters is CD44 trans-dimer–dependent. These results shed critical light on the biochemical mechanisms of CD44-mediated tumor cell cluster formation and may help inform the development of therapeutic strategies to prevent tumor cluster formation and block cluster-mediated metastases.

Genetic alterations and aberrant gene expression trigger malignant tumors. Tumor suppressor p53 is the most altered gene in human cancers. p53 induces apoptosis by promoting pro-apoptotic genes in response to DNA damage. Protein kinase C delta (PKCδ) also induces apoptosis via various mechanisms including modification of p53. The PKCδ-dependent apoptotic mechanism has been extensively studied; however, the transcriptional regulation of PKCδ remains obscure. The current study demonstrates the transcriptional regulation of PKCδ by p53 upon genotoxic stress. The p53-binding site in the promoter region of PKCδ was detected by the ChIP-sequencing assay. Notably, the expression of PKCδ was increased upon DNA damage, which is required for the stabilization of p53. More importantly, targeting single guide RNA-driven dead Cas9 to the p53-binding site of PKCδ disturbed p53-promoted PKCδ expression and suppressed apoptosis following DNA damage. Thus, our findings suggest that the transcriptional regulation of PKCδ is controlled by p53 in a positive feedback mechanism to induce apoptosis in response to DNA damage.

The tumor suppressor p53 is mutated in more than half of human cancers. Recent evidence has revealed that p53 not only regulates apoptosis but also regulates necrotic/necroptotic cell death via the mitochondria. The regulation of apoptosis by p53 is tightly connected to the mitochondrial outer membrane permeabilization and the induction of and interaction with Bcl-2 family members. Interestingly, p53-mediated regulation of necrosis/necroptosis is correlated with mitochondrial permeabilization pore opening via interactions with CypD and Drp1. This review discusses the p53-regulating molecules that induce apoptosis or necrosis/necroptosis via the mitochondria.

The antiapoptotic protein Bcl-2 is overexpressed in human cancers, and confers resistance to antitumor agents in cancer cells. Bcl-2 is negatively regulated by the tumor suppressor p53 in response to DNA damage during apoptotic cell death. However, this molecular mechanism remains unclear. The available evidence indicates that miR-1915 represses Bcl-2 expression at the post-transcriptional level in human colorectal carcinoma cells, which is correlated with drug resistance. Here, we show that p53 controls miR-1915 expression in response to DNA damage. Induction of p53 affects the expression of precursor and mature, but not primary, miR-1915. Inhibition of miR-1915 abrogates downregulation of Bcl-2 expression following treatment with genotoxin. These findings demonstrate that p53 negatively regulates Bcl-2 expression by targeting miR-1915 processing from primary into precursor miRNA. Taken together, the findings of the current study reveal a novel mechanism whereby p53 negatively modulates Bcl-2 by controlling miR-1915.

p53 and PKCδ are tumor suppressors that execute apoptotic mechanisms in response to various cellular stresses. p53 is a transcription factor that is frequently mutated in human cancers; it regulates apoptosis in transcription-dependent and -independent ways in response to genotoxic stresses. PKCδ is a serine/threonine protein kinase and mutated in human cancers. Available evidence shows that PKCδ activates p53 by direct and/or indirect mechanisms. Moreover, PKCδ is also implicated in the transcriptional regulation of p53 in response to DNA damage. Recent findings demonstrated that p53, in turn, binds onto the PKCδ promoter and induces its expression upon DNA damage to facilitate apoptosis. Both p53 and PKCδ are associated with the apoptotic mechanisms in the mitochondria by regulating Bcl-2 family proteins to provide mitochondrial outer membrane permeabilization. This review discusses the crosstalk between p53 and PKCδ in the context of apoptotic cell death and cancer therapy.

The cellular response to genotoxic stress is multifaceted in nature. Following DNA damage, the tumor suppressor gene p53 activates and plays critical roles in cell cycle arrest, activation of DNA repair and in the event of irreparable damage, induction of apoptosis. The breakdown of apoptosis causes the accumulation of mutant cells. The elucidation of the mechanism for the p53-dependent apoptosis will be crucial in applying the strategy for cancer patients. However, the mechanism of p53-dependent apoptosis remains largely unclear. Here, we carried out ChIP followed by massively parallel DNA sequencing assay (ChIP-seq) to uncover mechanisms of apoptosis. Using ChIP-seq, we identified PDCD6 as a novel p53-responsive gene. We determined putative p53-binding sites that are important for p53 regulation in response to DNA damage in the promoter region of PDCD6. Knockdown of PDCD6 suppressed p53-dependent apoptosis. We also observed that cytochrome c release and the cleavage of PARP by caspase-3 were suppressed by depletion of PDCD6. We further observed that PDCD6 localizes in the nucleus in response to DNA damage. We identified the nuclear localization signal of PDCD6 and, importantly, the nuclear accumulation of PDCD6 significantly induced apoptosis after genotoxic stress. Therefore, we conclude that a novel p53-responsive gene PDCD6 is accumulated in the nucleus and induces apoptosis in response to DNA damage.

Tumor suppressor p53 plays a pivotal role in cell cycle arrest, DNA repair, and apoptosis in response to DNA damage. Promoter selectivity of p53 depends mainly on post-translational modification. Notably, the apoptotic function of p53 is related to its phosphorylation at serine-46 (ser46) to promote pro-apoptotic genes. However, little is known about the pro-apoptotic genes induced by Ser46 phosphorylation. Our research achieved to investigate the pro-apoptotic genes induced by p53 in a phospho-ser46-specific manner using microarray and ChIP sequencing in human cancer cell lines. As a result, palmdelphin (PALMD), an isoform of paralemmin protein, was strongly elicited from the phosphorylation of ser46. The mRNA and protein expression of PALMD increased only in wild type p53 transfected cells, but not in ser46-mutated cells. Importantly, PALMD moved to the nucleus in response to DNA damage and the apoptotic function of PALMD was tightly exerted with localization into nucleus. Interestingly, down-regulation of PALMD by siRNA resulted in necroptosis-like cell death through ATP depletion. Moreover, we found vimentin as a PALMD interacting protein and the depletion of vimentin increased PALMD level to accelerate apoptosis. These results demonstrate that p53 regulates cell death fate (apoptosis or necroptosis-like cell death) through promoting PALMD expression in a phospho-ser46-specific manner in response to DNA damage. Citation Format: Nurmaa Khund Dashzeveg, Kiyotsugu Yoshida. The phosphorylation of p53 at serine 46 is essential to induce cell death through palmdelphin in response to DNA damage. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 24. doi:10.1158/1538-7445.AM2015-24

The tumor suppressor gene p53 regulates apoptosis in response to DNA damage. Promoter selectivity of p53 depends on mainly its phosphorylation. Particularly, the phosphorylation at serine-46 of p53 is indispensable in promoting pro-apoptotic genes that are, however, poorly determined. In the current study, we identified palmdelphin as a pro-apoptotic gene induced by p53 in a phosphorylated serine-46-specific manner. Upregulation of palmdelphin was observed in wild-type p53-transfected cells, but not in serine-46-mutated cells. Expression of palmdelphin was induced by p53 in response to DNA damage. In turn, palmdelphin induced apoptosis. Intriguingly, downregulation of palmdelphin resulted in necroptosis-like cell death via ATP depletion. Upon DNA damage, palmdelphin dominantly accumulated in the nucleus to induce apoptosis. These findings define palmdelphin as a target of serine-46-phosphorylated p53 that controls cell death in response to DNA damage.

The tumor suppressor p53 regulates apoptosis in response to DNA damage. Promoter selectivity of p53 depends mainly on its phosphorylation. Notably, the phosphorylation at serine-46 (Ser46) of p53 is indispensable to promote pro-apoptotic genes. However, little is known about the pro-apoptotic genes induced by Ser46 phosphorylation of p53. In our study, we aimed to investigate pro-apoptotic genes induced by p53 using microarray analysis and ChIP sequencing assay. Although microarray analysis became common tool in molecular biology, novelty of our research is combining microarray analysis with ChIP sequencing assay to discover direct target of p53 in Ser46 specific manner. Analyze of microarray showed specific up-regulation of 2723 genes in wild type p53 (wt-p53). Alternatively, 1338 genes were interacted directly with only wt-p53 but not with mutant p53 (Ser46 is substituted with alanine, p53S46A) in ChIP sequencing assay. The comparison of two assays, microarray analysis and ChIP sequencing assay, investigated thirty genes in relation with Ser46 phosphorylation of tumor suppressor p53. Those thirty genes were clarified by real time RT-PCR and the genes strongly elicited were chosen further study. The research will be continued with more clarification and apoptotic function of candidate genes in response to DNA damage will be investigated. We hope that the discovery of pro-apoptotic genes induced by p53 will convey the important role in cancer research and therapy. Acknowledgement: This project is partly supported by Global Center of Excellence (GCOE) program at TMDU. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4950. doi:1538-7445.AM2012-4950