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Plasticity is an inherent feature of cancer stem cells (CSCs) and regulates the balance of key processes required at different stages of breast cancer progression, including epithelial-to-mesenchymal transition (EMT) versus mesenchymal-to-epithelial transition (MET), and glycolysis versus oxidative phosphorylation. Understanding the key factors that regulate the switch between these processes could lead to novel therapeutic strategies that limit tumor progression. We found that aldehyde dehydrogenase 1A3 (ALDH1A3) regulates these cancer-promoting processes and the abundance of the two distinct breast CSC populations defined by high ALDH activity and CD24 - CD44 + cell surface expression. While ALDH1A3 increases ALDH + breast cancer cells, it inversely suppresses the CD24 - CD44 + population by retinoic acid signaling-mediated gene expression changes. This switch in CSC populations induced by ALDH1A3 was paired with decreased migration but increased invasion and an intermediate EMT phenotype. We also demonstrate that ALDH1A3 increases oxidative phosphorylation and decreases glycolysis and reactive oxygen species (ROS). The effects of ALDH1A3 reduction were countered with the glycolysis inhibitor 2-deoxy-D-glucose (2DG). In cell culture and tumor xenograft models, 2DG suppresses the increase in the CD24 - CD44 + population and ROS induced by ALDH1A3 knockdown. Combined inhibition of ALDH1A3 and glycolysis best reduces breast tumor growth and tumor-initiating cells, suggesting that the combination of targeting ALDH1A3 and glycolysis has therapeutic potential for limiting CSCs and tumor progression. Together, these findings identify ALDH1A3 as a key regulator of processes required for breast cancer progression and depletion of ALDH1A3 makes breast cancer cells more susceptible to glycolysis inhibition., (© 2024. The Author(s).)

Metastasis is responsible for the majority of cancer-related fatalities. We previously identified specific cancer cell populations responsible for metastatic events which are cytokeratin-14 (CK14) and E-cadherin positive in luminal tumors, and E-cadherin and vimentin positive in triple-negative tumors. Since cancer cells evolve within a complex ecosystem comprised of immune cells and stromal cells, we sought to decipher the spatial interactions of these aggressive cancer cell populations within the tumor microenvironment (TME). We used imaging mass cytometry to detect 36 proteins in tumor microarrays containing paired primary and metastatic lesions from luminal or triple-negative breast cancers (TNBC), resulting in a dataset of 1,477,337 annotated cells. Focusing on metastasis-initiating cell populations, we observed close proximity to specific fibroblast and macrophage subtypes, a relationship maintained between primary and metastatic tumors. Notably, high CK14 in luminal cancer cells and high vimentin in TNBC cells correlated with close proximity to specific macrophage subtypes (CD163 int CD206 int PDL1 int HLA-DR + or PDL1 high ARG1 high ). Our in-depth spatial analysis demonstrates that metastasis-initiating cancer cells consistently colocalizes with distinct cell populations within the TME, suggesting a role for these cell-cell interactions in promoting metastasis., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Interest in the use of proteolysis-targeting chimeras (PROTACs) in cancer therapy has increased in recent years. Targeting bromodomain and extra terminal domain (BET) proteins, especially bromodomain-containing protein 4 (BRD4), has shown inhibitory effects on basal-like breast cancer (BLBC). However, the bioavailability of BRD4 PROTACs is restricted by their non-selective biodegradability and low tumor-targeting ability. We demonstrated that 6b (BRD4 PROTAC) suppresses BLBC cell growth by targeting BRD4, but not BRD2 and BRD3, for cereblon (CRBN)-mediated ubiquitination and proteasomal degradation. Compound 6b also inhibited expression of Krüppel-like factor 5 (KLF5) transcription factor, a key oncoprotein in BLBC, controlled by BRD4-mediated super-enhancers. Moreover, 6b inhibited HCC1806 tumor growth in a xenograft mouse model. The combination of 6b and KLF5 inhibitors showed additive effects on BLBC. These results suggest that BRD4-specific PROTAC can effectively inhibit BLBC by downregulating KLF5, and that 6b has potential as a novel therapeutic drug for BLBC., (© 2024. The Author(s).)

Breast cancer (BC) remains the second leading cause of cancer-related mortalities in women. Resistance to hormone therapies such as tamoxifen, an estrogen receptor (ER) inhibitor, is a major hurdle in the treatment of BC. Enhancer of zeste homolog 2 (EZH2), the methyltransferase component of the Polycomb repressive complex 2 (PRC2), has been implicated in tamoxifen resistance. Evidence suggests that EZH2 often functions noncanonically, in a methyltransferase-independent manner, as a transcription coactivator through interacting with oncogenic transcription factors. Unlike methyltransferase inhibitors, proteolysis targeting chimeras (PROTAC) can suppress both activating and repressive functions of EZH2. Here, we find that EZH2 PROTACs, MS177 and MS8815, effectively inhibited the growth of BC cells, including those with acquired tamoxifen resistance, to a much greater degree when compared to methyltransferase inhibitors. Mechanistically, EZH2 associates with forkhead box M1 (FOXM1) and binds to the promoters of FOXM1 target genes. EZH2 PROTACs induce degradation of both EZH2 and FOXM1, leading to reduced expression of target genes involved in cell cycle progression and tamoxifen resistance. Together, this study supports that EZH2-targeted PROTACs represent a promising avenue of research for the future treatment of BC, including in the setting of tamoxifen resistance., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Solid tumours have abnormally high intracellular [Na + ]. The activity of various Na + channels may underlie this Na + accumulation. Voltage-gated Na + channels (VGSCs) have been shown to be functionally active in cancer cell lines, where they promote invasion. However, the mechanisms involved, and clinical relevance, are incompletely understood. Here, we show that protein expression of the Na v 1.5 VGSC subtype strongly correlates with increased metastasis and shortened cancer-specific survival in breast cancer patients. In addition, VGSCs are functionally active in patient-derived breast tumour cells, cell lines, and cancer-associated fibroblasts. Knockdown of Na v 1.5 in a mouse model of breast cancer suppresses expression of invasion-regulating genes. Na v 1.5 activity increases ATP demand and glycolysis in breast cancer cells, likely by upregulating activity of the Na + /K + ATPase, thus promoting H + production and extracellular acidification. The pH of murine xenograft tumours is lower at the periphery than in the core, in regions of higher proliferation and lower apoptosis. In turn, acidic extracellular pH elevates persistent Na + influx through Na v 1.5 into breast cancer cells. Together, these findings show positive feedback between extracellular acidification and the movement of Na + into cancer cells which can facilitate invasion. These results highlight the clinical significance of Na v 1.5 activity as a potentiator of breast cancer metastasis and provide further evidence supporting the use of VGSC inhibitors in cancer treatment., (© 2024. The Author(s).)

Aberrant cell proliferation is a hallmark of cancer, including breast cancer. Here, we show that USP27X is required for cell proliferation and tumorigenesis in breast cancer. We identify a PIM2-USP27X regulator of MYC signaling axis whose activity is an important contributor to the tumor biology of breast cancer. PIM2 phosphorylates USP27X, and promotes its deubiquitylation activity for MYC, which promotes its protein stability and leads to increase HK2-mediated aerobic glycolysis in breast cancer. Moreover, the PIM2-USP27X-MYC axis is also validated in PIM2-knockout mice. Taken together, these findings show a PIM2-USP27X-MYC signaling axis as a new potential target for breast cancer treatment., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Pyruvate kinase M2 (PKM2) is a central metabolic enzyme driving the Warburg effect in tumor growth. Previous investigations have demonstrated that PKM2 is subject to O-linked β-N-acetylglucosamine (O-GlcNAc) modification, which is a nutrient-sensitive post-translational modification. Here we found that unc-51 like autophagy activating kinase 1 (ULK1), a glucose-sensitive kinase, interacts with PKM2 and phosphorylates PKM2 at Ser333. Ser333 phosphorylation antagonizes PKM2 O-GlcNAcylation, promotes its tetramer formation and enzymatic activity, and decreases its nuclear localization. As PKM2 is known to have a nuclear role in regulating c-Myc, we also show that PKM2-S333 phosphorylation inhibits c-Myc expression. By downregulating glucose consumption and lactate production, PKM2 pS333 attenuates the Warburg effect. Through mouse xenograft assays, we demonstrate that the phospho-deficient PKM2-S333A mutant promotes tumor growth in vivo. In conclusion, we identified a ULK1-PKM2-c-Myc axis in inhibiting breast cancer, and a glucose-sensitive phosphorylation of PKM2 in modulating the Warburg effect., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Epigenetic regulation established during development to maintain patterns of transcriptional expression and silencing for metabolism and other fundamental cell processes can be reprogrammed in cancer, providing a molecular mechanism for persistent alterations in phenotype. Metabolic deregulation and reprogramming are thus an emerging hallmark of cancer with opportunities for molecular classification as a critical preliminary step for precision therapeutic intervention. Yet, acquisition of therapy resistance against most conventional treatment regimens coupled with tumor relapse, continue to pose unsolved problems for precision healthcare, as exemplified in breast cancer where existing data informs both cancer genotype and phenotype. Furthermore, epigenetic reprograming of the metabolic milieu of cancer cells is among the most crucial determinants of therapeutic resistance and cancer relapse. Importantly, subtype-specific epigenetic-metabolic interplay profoundly affects malignant transformation, resistance to chemotherapy, and response to targeted therapies. In this review, we therefore prismatically dissect interconnected epigenetic and metabolic regulatory pathways and then integrate them into an observable cancer metabolism-therapy-resistance axis that may inform clinical intervention. Optimally coupling genome-wide analysis with an understanding of metabolic elements, epigenetic reprogramming, and their integration by metabolic profiling may decode missing molecular mechanisms at the level of individual tumors. The proposed approach of linking metabolic biochemistry back to genotype, epigenetics, and phenotype for specific tumors and their microenvironment may thus enable successful mechanistic targeting of epigenetic modifiers and oncometabolites despite tumor metabolic heterogeneity., (© 2024. The Author(s).)

Regulatory T cells (Tregs) prevent autoimmunity and contribute to cancer progression. They exert contact-dependent inhibition of immune cells through the production of active transforming growth factor-β1 (TGF-β1). However, the absence of a specific surface marker makes inhibiting the production of active TGF-β1 to specifically deplete human Tregs but not other cell types a challenge. TGF-β1 in an inactive form binds to Tregs membrane protein Glycoprotein A Repetitions Predominant (GARP) and then activates it via an unknown mechanism. Here, we demonstrated that tumour necrosis factor receptor-associated factor 3 interacting protein 3 (TRAF3IP3) in the Treg lysosome is involved in this activation mechanism. Using a novel naphthalenelactam-platinum-based anticancer drug (NPt), we developed a new synergistic effect by suppressing ATP-binding cassette subfamily B member 9 (ABCB9) and TRAF3IP3-mediated divergent lysosomal metabolic programs in tumors and human Tregs to block the production of active GARP/TGF-β1 for remodeling the tumor microenvironment. Mechanistically, NPt is stored in Treg lysosome to inhibit TRAF3IP3-meditated GARP/TGF-β1 complex activation to specifically deplete Tregs. In addition, by promoting the expression of ABCB9 in lysosome membrane, NPt inhibits SARA/p-SMAD2/3 through CHRD-induced TGF-β1 signaling pathway. In addition to expose a previously undefined divergent lysosomal metabolic program-meditated GARP/TGF-β1 complex blockade by exploring the inherent metabolic plasticity, NPt may serve as a therapeutic tool to boost unrecognized Treg-based immune responses to infection or cancer via a mechanism distinct from traditional platinum drugs and currently available immune-modulatory antibodies., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

The deubiquitinase OTUB1, implicated as a potential oncogene in various tumors, lacks clarity in its regulatory mechanism in tumor progression. Our study investigated the effects and underlying mechanisms of OTUB1 on the breast cancer cell cycle and proliferation in IFNγ stimulation. Loss of OTUB1 abrogated IFNγ-induced cell cycle arrest by regulating p27 protein expression, whereas OTUB1 overexpression significantly enhanced p27 expression even without IFNγ treatment. Tyr26 phosphorylation residue of OTUB1 directly bound to p27, modulating its post-translational expression. Furthermore, we identified crucial lysine residues (K134, K153, and K163) for p27 ubiquitination. Src downregulation reduced OTUB1 and p27 expression, suggesting that IFNγ-induced cell cycle arrest is mediated by the Src-OTUB1-p27 signaling pathway. Our findings highlight the pivotal role of OTUB1 in IFNγ-induced p27 expression and cell cycle arrest, offering therapeutic implications., (© 2024. The Author(s).)

The loss of intercellular adhesion molecule E-cadherin is a hallmark of the epithelial-mesenchymal transition (EMT), during which tumor cells transition into an invasive phenotype. Accordingly, E-cadherin has long been considered a tumor suppressor gene; however, E-cadherin expression is paradoxically correlated with breast cancer survival rates. Using novel multi-compartment organoids and multiple in vivo models, we show that E-cadherin promotes a hyper-proliferative phenotype in breast cancer cells via interaction with the transmembrane receptor EGFR. The E-cad and EGFR interaction results in activation of the MEK/ERK signaling pathway, leading to a significant increase in proliferation via activation of transcription factors, including c-Fos. Pharmacological inhibition of MEK activity in E-cadherin positive breast cancer significantly decreases both tumor growth and macro-metastasis in vivo. This work provides evidence for a novel role of E-cadherin in breast tumor progression and identifies a new target to treat hyper-proliferative E-cadherin-positive breast tumors, thus providing the foundation to utilize E-cadherin as a biomarker for specific therapeutic success., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Advanced breast cancers represent a major therapeutic challenge due to their refractoriness to treatment. Cancer-associated fibroblasts (CAFs) are the most abundant constituents of the tumor microenvironment and have been linked to most hallmarks of cancer. However, the influence of CAFs on therapeutic outcome remains largely unchartered. Here, we reveal that spatial coincidence of abundant CAF infiltration with malignant cells was associated with reduced estrogen receptor (ER)-α expression and activity in luminal breast tumors. Notably, CAFs mediated estrogen-independent tumor growth by selectively regulating ER-α signaling. Whereas most prototypical estrogen-responsive genes were suppressed, CAFs maintained gene expression related to therapeutic resistance, basal-like differentiation, and invasion. A functional drug screen in co-cultures identified effector pathways involved in the CAF-induced regulation of ER-α signaling. Among these, the Transforming Growth Factor-β and the Janus kinase signaling cascades were validated as actionable targets to counteract the CAF-induced modulation of ER-α activity. Finally, genes that were downregulated in cancer cells by CAFs were predictive of poor response to endocrine treatment. In conclusion, our work reveals that CAFs directly control the luminal breast cancer phenotype by selectively modulating ER-α expression and transcriptional function, and further proposes novel targets to disrupt the crosstalk between CAFs and tumor cells to reinstate treatment response to endocrine therapy in patients., (© 2024. The Author(s).)

Breast cancer is one of the major malignant tumors among women worldwide. Long noncoding RNAs (lncRNAs) have been documented as significant modulators in the development and progression of various cancers; however, the contribution of lncRNAs to breast cancer remains largely unknown. In this study, we found a novel lncRNA (NONHSAT137675) whose expression was significantly increased in the breast cancer tissues. We named the novel lncRNA as lncRNA PRBC (PABPC1-related lncRNA in breast cancer) and identified it as a key lncRNA associated with breast cancer progression and prognosis. Functional analysis displayed that lncRNA PRBC could promote autophagy and progression of breast cancer. Mechanistically, we verified that lncRNA PRBC physically interacted with PABPC1 through RIP assay, and PABPC1 overexpression could reverse the inhibiting effect of lncRNA PRBC knockdown on the malignant behaviors in breast cancer cells. Knockdown of lncRNA PRBC interfered the translocation of PABPC1 from nucleus to cytoplasm as indicated by western blot and IF assays. Significantly, the cytoplasmic location of PABPC1 was required for the interaction between PABPC1 and AGO2, which could be enhanced by lncRNA PRBC overexpression, leading to strengthened recruitment of mRNA to RNA-induced silencing complex (RISC) and thus reinforcing the inhibition efficiency of miRNAs. In general, lncRNA PRBC played a critical role in malignant progression of breast cancer by inducing the cytoplasmic translocation of PABPC1 to further regulate the function of downstream miRNAs. This study provides novel insight on the molecular mechanism of breast cancer progression, and lncRNA PRBC might be a promising therapeutic target and prognostic predictor for breast cancer., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Transient early endosome (EE)-mitochondria interactions can mediate mitochondrial iron translocation, but the associated mechanisms are still elusive. We showed that Divalent Metal Transporter 1 (DMT1) sustains mitochondrial iron translocation via EE-mitochondria interactions in triple-negative MDA-MB-231, but not in luminal A T47D breast cancer cells. DMT1 silencing increases labile iron pool (LIP) levels and activates PINK1/Parkin-dependent mitophagy in MDA-MB-231 cells. Mitochondrial bioenergetics and the iron-associated protein profile were altered by DMT1 silencing and rescued by DMT1 re-expression. Transcriptomic profiles upon DMT1 silencing are strikingly different between 2D and 3D culture conditions, suggesting that the environment context is crucial for the DMT1 knockout phenotype observed in MDA-MB-231 cells. Lastly, in vivo lung metastasis assay revealed that DMT1 silencing promoted the outgrowth of lung metastatic nodules in both human and murine models of triple-negative breast cancer cells. These findings reveal a DMT1-dependent pathway connecting EE-mitochondria interactions to mitochondrial iron translocation and metastatic fitness of breast cancer cells., (© 2024. The Author(s).)

The over-activation of ERα signaling is regarded as the major driver for luminal breast cancers, which could be effective controlled via selective estrogen receptor modulators (SERM), such as tamoxifen. The endocrine resistance is still a challenge for breast cancer treatment, while recently studies implicate the post-translational modification on ERα play important roles in endocrine resistance. The stability of ERα protein and ERα transcriptome are subject to a balance between E3 ubiquitin ligases and deubiquitinases. Through deubiquitinases siRNA library screening, we discover PSMD14 as a critical deubiquitinase for ERα signaling and breast cancer progression. PSMD14 could facilitate breast cancer progression through ERα signaling in vitro and in vivo, while pharmaceutical inhibition of PSMD14 via Thiolutin could block the tumorigenesis in breast cancer. In endocrine resistant models, PSMD14 inhibition could de-stabilize the resistant form of ERα (Y537S) and restore tamoxifen sensitivity. Molecular studies reveal that PSMD14 could inhibition K48-linked poly-ubiquitination on ERα, facilitate ERα transcriptome. Interestingly, ChIP assay shows that ERα could bind to the promoter region of PSMD14 and facilitate its gene transcription, which indicates PSMD14 is both the upstream modulator and downstream target for ERα signaling in breast cancer. In general, we identified a novel positive feedback loop between PSMD14 and ERα signaling in breast cancer progression, while blockade of PSMD14 could be a plausible strategy for luminal breast cancer., (© 2023. The Author(s).)

Chemotherapy resistance represents a major cause of therapeutic failure and mortality in cancer patients. Mesenchymal stromal cells (MSCs), an integral component of tumor microenvironment, are known to promote drug resistance. However, the detailed mechanisms remain to be elucidated. Here, we found that MSCs confer breast cancer resistance to doxorubicin by diminishing its intratumoral accumulation. Hyaluronan (HA), a major extracellular matrix (ECM) product of MSCs, was found to mediate the chemoresistant effect. The chemoresistant effect of MSCs was abrogated when hyaluronic acid synthase 2 (HAS2) was depleted or inhibited. Exogenous HA also protected tumor grafts from doxorubicin. Molecular dynamics simulation analysis indicates that HA can bind with doxorubicin, mainly via hydrophobic and hydrogen bonds, and thus reduce its entry into breast cancer cells. This mechanism is distinct from the reported chemoresistant effect of HA via its receptor on cell surface. High HA serum levels were also found to be positively associated with chemoresistance in breast cancer patients. Our findings indicate that the HA-doxorubicin binding dynamics can confer cancer cells chemoresistance. Reducing HA may enhance chemotherapy efficacy., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

Hormone receptor-positive, HER2-negative advanced breast cancers exhibit high sensitivity to CDK4/6 inhibitors such as palbociclib. However, most patients inevitably develop resistance, thus identification of new actionable therapeutic targets to overcome the recurrent disease is an urgent need. Immunohistochemical studies of tissue microarray revealed increased activation of non-receptor tyrosine kinase, ACK1 (also known as TNK2) in most of the breast cancer subtypes, independent of their hormone receptor status. Chromatin immunoprecipitation studies demonstrated that the nuclear target of activated ACK1, pY88-H4 epigenetic marks, were deposited at cell cycle genes, CCNB1, CCNB2 and CDC20, which in turn initiated their efficient transcription. Pharmacological inhibition of ACK1 using its inhibitor, (R)-9b dampened CCNB1, CCNB2 and CDC20 expression, caused G2/M arrest, culminating in regression of palbociclib-resistant breast tumor growth. Further, (R)-9b suppressed expression of CXCR4 receptor, which resulted in significant impairment of metastasis of breast cancer cells to lung. Overall, our pre-clinical data identifies activated ACK1 as an oncogene that epigenetically controls the cell cycle genes governing the G2/M transition in breast cancer cells. ACK1 inhibitor, (R)-9b could be a novel therapeutic option for the breast cancer patients that have developed resistance to CDK4/6 inhibitors., (© 2023. The Author(s).)

Exploring the relationship between various neurotransmitters and breast cancer cell growth has revealed their likely centrality to improving breast cancer treatment. Neurotransmitters play a key role in breast cancer biology through their effects on the cell cycle, epithelial mesenchymal transition, angiogenesis, inflammation, the tumor microenvironment and other pathways. Neurotransmitters and their receptors are vital to the initiation, progression and drug resistance of cancer and progress in our biological understanding may point the way to lower-cost and lower-risk antitumor therapeutic strategies. This review discusses multiple neurotransmitters in the context of breast cancer. It also discusses risk factors, repurposing of pharmaceuticals impacting neurotransmitter pathways, and the opportunity for better integrated models that encompass exercise, the intestinal microbiome, and other non-pharmacologic considerations. Neurotransmitters' role in breast cancer should no longer be ignored; it may appear to complicate the molecular picture but the ubiquity of neurotransmitters and their wide-ranging impacts provide an organizing framework upon which further understanding and progress against breast cancer can be based., (© 2023. The Author(s).)

Coiled-coil domain containing 3 (CCDC3) was previously shown to regulate liver lipid metabolism as a secretory protein. Here, we report an unexpected intracellular role of CCDC3 as a tumor suppressor in breast cancer (BrC). Bioinformatics datasets analysis showed that CCDC3 is under-expressed in BrCs, while its higher levels are correlated with higher overall survival and lower relapse of cancer patients, and CCDC3 is positively correlated with p53 and its target genes. Ectopic CCDC3 markedly suppressed proliferation, colony formation, and xenograft tumor growth by augmenting p53 activity in BrC cells. Depletion of endogenous CCDC3 by CRISPR-Cas9 increased proliferation and drug resistance of BrC cells by alleviating 5-Fluorouracil (5-FU)-induced p53 level and activity. Mechanistically, CCDC3 bound to the C-termini of p53 and MDM2, consequently stabilizing p53 in the nucleus and impairing MDM2 recruitment of p53 to the 26S proteosome without inhibiting p53 ubiquitination. p53 induced CCDC3 expression by binding to its promoter in BrC cells. Our results unveil a unique mechanism underlying CCDC3 activation of p53 in a positive feedback fashion to suppress BrC growth., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Tamoxifen is a first-line therapeutic drug for oestrogen-receptor positive breast cancer; however, like other therapeutics, its clinical use is limited by acquired resistance. Tamoxifen-resistant cells have demonstrated enhanced aerobic glycolysis; however, the mechanisms underlying this upregulation remain unclear. Here, we demonstrated that G-protein coupled oestrogen receptor (GPER) was involved in the upregulation of aerobic glycolysis via induction of hypoxia-inducible factor-1α (HIF-1α) expression and transcriptional activity in tamoxifen-resistant cells. Additionally, GPER stabilized HIF-1α through inhibiting its hydroxylation and ubiquitin-mediated degradation, which were associated with upregulation of C-terminal hydrolase-L1 (UCH-L1), downregulation of prolyl hydroxylase 2 (PHD2) and von Hippel-Lindau tumour suppressor protein (pVHL), induction of HIF-1α/UCH-L1 interaction, and suppression of HIF-1α/PHD2-pVHL association. The GPER/HIF-1α axis was functionally responsible for regulating tamoxifen sensitivity both in vitro and in vivo. Moreover, there was a positive correlation between GPER and HIF-1α expression in clinical breast cancer tissues, and high levels of GPER combined with nuclear HIF-1α indicated poor overall survival. High levels of the GPER/HIF-1α axis were also correlated with shorter relapse-free survival in patients receiving tamoxifen. Hence, our findings support a critical role of GPER/HIF-1α axis in the regulation of aerobic glycolysis in tamoxifen-resistant cells, offering a potential therapeutic target for tamoxifen-resistant breast cancer., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Mutations in the estrogen receptor (ESR1) gene are common in ER-positive breast cancer patients who progress on endocrine therapies. Most mutations localise to just three residues at, or near, the C-terminal helix 12 of the hormone binding domain, at leucine-536, tyrosine-537 and aspartate-538. To investigate these mutations, we have used CRISPR-Cas9 mediated genome engineering to generate a comprehensive set of isogenic mutant breast cancer cell lines. Our results confirm that L536R, Y537C, Y537N, Y537S and D538G mutations confer estrogen-independent growth in breast cancer cells. Growth assays show mutation-specific reductions in sensitivities to drugs representing three classes of clinical anti-estrogens. These differential mutation- and drug-selectivity profiles have implications for treatment choices following clinical emergence of ER mutations. Our results further suggest that mutant expression levels may be determinants of the degree of resistance to some anti-estrogens. Differential gene expression analysis demonstrates up-regulation of estrogen-responsive genes, as expected, but also reveals that enrichment for interferon-regulated gene expression is a common feature of all mutations. Finally, a new gene signature developed from the gene expression profiles in ER mutant cells predicts clinical response in breast cancer patients with ER mutations., (© 2022. The Author(s).)

Most basal-like breast cancers (BLBCs) are triple-negative breast cancers (TNBCs), which is associated with high malignancy, high rate of recurrence and distant metastasis, and poor prognosis among all types of breast cancer. However, there are currently no effective therapies for BLBC. Furthermore, chemoresistance limits the therapeutic options for BLBC treatment. In this study, we screen out protein activator of the interferon-induced protein kinase (PACT) as an essential gene in BLBC metastasis. We find that high PACT expression level was associated with poor prognosis among BLBC patients. In vivo and in vitro investigations indicated that PACT could regulate BLBC metastasis by interacting with SUMO-conjugating enzyme Ubc9 to stimulate the SUMOylation and thus consequently the activation of Rac1. BLBC patients receiving chemotherapy presents poorer prognosis with PACT high expression, and PACT disruption sensitizes experimental mammary tumor metastases to chemotherapy, thus providing insights to consider PACT as a potential therapeutic target to overcome acquired chemoresistance in BLBC., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

The acquisition of novel detrimental cellular properties following exposure to cytotoxic drugs leads to aggressive and metastatic tumors that often translates into an incurable disease. While the bulk of the primary tumor is eliminated upon exposure to chemotherapeutic treatment, residual cancer cells and non-transformed cells within the host can engage a stable cell cycle exit program named senescence. Senescent cells secrete a distinct set of pro-inflammatory factors, collectively termed the senescence-associated secretory phenotype (SASP). Upon exposure to the SASP, cancer cells undergo cellular plasticity resulting in increased proliferation, migration and epithelial-to-mesenchymal transition. The molecular mechanisms by which the SASP regulates these pro-tumorigenic features are poorly understood. Here, we report that breast cancer cells exposed to the SASP strongly upregulate Lipocalin-2 (LCN2). Furthermore, we demonstrate that LCN2 is critical for SASP-induced increased migration in breast cancer cells, and its inactivation potentiates the response to chemotherapeutic treatment in mouse models of breast cancer. Finally, we show that neoadjuvant chemotherapy treatment leads to LCN2 upregulation in residual human breast tumors, and correlates with worse overall survival. These findings provide the foundation for targeting LCN2 as an adjuvant therapeutic approach to prevent the emergence of aggressive tumors following chemotherapy., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

The management of metastatic estrogen receptor (ER) positive HER2 negative breast cancer (ER+) has improved; however, therapeutic resistance and disease progression emerges in majority of cases. Using unbiased approaches, as expected PI3K and MTOR inhibitors emerge as potent inhibitors to delay proliferation of ER+ models harboring PIK3CA mutations. However, the cytostatic efficacy of these drugs is hindered due to marginal impact on the expression of cyclin D1. Different combination approaches involving the inhibition of ER pathway or cell cycle result in durable growth arrest via RB activation and subsequent inhibition of CDK2 activity. However, cell cycle alterations due to RB loss or ectopic CDK4/cyclin D1 activation yields resistance to these cytostatic combination treatments. To define means to counter resistance to targeted therapies imparted with RB loss; complementary drug screens were performed with RB-deleted isogenic cell lines. In this setting, RB loss renders ER+ breast cancer models more vulnerable to drugs that target DNA replication and mitosis. Pairwise combinations using these classes of drugs defines greater selectivity for RB deficiency. The combination of AURK and WEE1 inhibitors, yields synergistic cell death selectively in RB-deleted ER+ breast cancer cells via apoptosis and yields profound disease control in vivo. Through unbiased efforts the XIAP/CIAP inhibitor birinapant was identified as a novel RB-selective agent. Birinapant further enhances the cytotoxic effect of chemotherapies and targeted therapies used in the treatment of ER+ breast cancer models selectively in the RB-deficient setting. Using organoid culture and xenograft models, we demonstrate the highly selective use of birinapant based combinations for the treatment of RB-deficient tumors. Together, these data illustrate the critical role of RB-pathway in response to many agents used to treat ER+ breast cancer, whilst informing new therapeutic approaches that could be deployed against resistant disease., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

X-linked inhibitor of apoptosis-associated factor 1 (XAF1) is a pro-apoptotic tumor suppressor that is frequently inactivated in multiple human cancers. However, its candidacy as a suppressor in the pathogenesis of breast cancer remains undefined. Here, we report that XAF1 acts as a molecular switch in estrogen (E2)-mediated cell-fate decisions favoring apoptosis over cell proliferation. XAF1 promoter hypermethylation is observed predominantly in estrogen receptor α (ERα)-positive versus ERα-negative tumor cells and associated with attenuated apoptotic response to E2. XAF1 is activated by E2 through a G protein-coupled estrogen receptor-mediated non-genomic pathway and induces ERα degradation and apoptosis while it is repressed by ERα for E2 stimulation of cell proliferation. The XAF1-ERα mutual antagonism dictates the outcomes of E2 signaling and its alteration is linked to the development of E2-resistant tumors. Mechanistically, XAF1 destabilizes ERα through the assembly of breast cancer-associated gene 1 (BRCA1)-mediated destruction complex. XAF1 interacts with ERα and BRCA1 via the zinc finger (ZF) domains 5/6 and 4, respectively, and the mutants lacking either of these domains fail to drive ERα ubiquitination and apoptosis. E2-induced regression of XAF1 +/+ tumors is abolished by XAF1 depletion while XAF1 -/- tumors recover E2 response by XAF1 restoration. XAF1 and ERα expression show an inverse correlation in primary breast tumors, and XAF1 expression is associated with the overall survival of patients with ERα-positive but not ERα-negative cancer. Together, this study uncovers an important role for the XAF1-ERα antagonism as a linchpin to govern E2-mediated cell-fate decisions, illuminating the mechanistic consequence of XAF1 alteration in breast tumorigenesis., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

The endocytic adaptor protein Numb acts as a tumor suppressor through downregulation of oncogenic pathways in multiple cancer types. The identification of splicing alterations giving rise to changes in Numb protein isoform expression indicate that Numb also has tumor promoting activity, though the underlying mechanisms are unknown. Here we report that NUMB exon 9 inclusion, which results in production of a protein isoform with an additional 49 amino acids, is a feature of multiple cancer types including all subtypes of breast cancer and correlates with worse progression-free survival. Specific deletion of exon 9-included Numb isoforms (Exon9in) from breast cancer cells reduced cell growth and prevents spontaneous lung metastasis in a mouse model. Quantitative proteome profiling showed that loss of Exon9in causes downregulation of membrane receptors and adhesion molecules, as well as proteins involved in extracellular matrix organization and the epithelial-mesenchymal transition (EMT) state. In addition, exon 9 deletion caused remodeling of the endocytic network, decreased ITGβ5 surface localization, cell spreading on vitronectin and downstream signaling to ERK and SRC. Together these observations suggest that Exon9in isoform expression disrupts the endocytic trafficking functions of Numb, resulting in increased surface expression of ITGβ5 as well as other plasma membrane proteins to promote cell adhesion, EMT, and tumor metastasis., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Dual-specificity mitogen-activated protein kinase phosphatase-1 (MKP-1/DUSP1/CL-100) has been documented to promote breast cancer cell survival and chemoresistance. MKP-1 is an unstable protein that is ubiquitinated and degraded via the ubiquitin-proteasome system. However, it is not clear how MKP-1 protein stability is regulated in breast cancer. In this study, we performed a genome-wide siRNA library screen of deubiquitinases (DUBs) and identified STAMBPL1 as an MKP-1 DUB in breast cancer cells. STAMBPL1 interacts with MKP-1 and stabilizes MKP-1 via deubiquitination. Both STAMBPL1 and MKP-1 depletion sensitize breast cancer cells to cisplatin in vitro and in vivo, and ectopic overexpression of MKP-1 partially rescues STAMBPL1 depletion-induced cisplatin sensitivity. Furthermore, STAMBPL1 and MKP-1 depletion increased breast cancer sensitivity to cisplatin by increasing the phosphorylation and activation of c-Jun N-terminal protein kinase (JNK). Collectively, our findings not only identify STAMBPL1 as an MKP-1 DUB but also reveal a critical mechanism that regulates MKP-1 expression in breast cancer. Our findings indicate that the STAMBPL1/MKP-1 axis represents a potential therapeutic target in breast cancer., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Cancer stem cells (CSC) are the major obstacle for cancer therapy in clinic. Exosomes are one type of vesicles that containing circular RNA (circRNAs) involved in cell-cell communication. However, the roles of breast CSC (BCSC) exosomes are still unclear, and the purpose of the study was to investigate breast cancer cell metabolism reprogramming by circRNAs from BCSC exosomes. The circRNA array was performed in the exosomes secreted from spheroids of MDA-231 cells. circCARM1 was higher in BCSC exosomes than it in the parent breast cancer cells. Further investigation demonstrated that BCSC exosomes circCARM1 played an important role in breast cancer cell glycolysis by miR-1252-5p/PFKFB2. In a conclusion, BCSC exosome-derived circCARM1 played an important role in breast cancer cell glycolysis by sponging miR-1252-5p which regulated PFKFB2 expression., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Cancer stem cells (CSCs) drive tumor initiation, progression, metastasis, and drug resistance. We report here that programmed cell death ligand 1 (PD-L1) is constitutively expressed in cancer cells to maintain and expand CSC through a novel mechanism in addition to promoting cancer cell immune evasion. We discovered that PD-L1 interacts with receptor Frizzled 6 to activate β-catenin signaling and increase β-catenin-targeted gene expression, such as a putative stem cell marker leucine-rich-repeat-containing G-protein-coupled receptor 5. Blockage of PD-L1 function, using a specific small hairpin RNA or a specific antibody, inhibits disease progression by reducing the CSC population in both colorectal and breast tumors. Moreover, β-catenin conversely regulates PD-L1 expression through a β-catenin complex binding site in the PD-L1 promoter. Our discoveries reveal that besides assistant tumor cell immune escaping, PD-L1 and β-catenin signaling form a positive feedback loop to promote cancer progression through CSC maintenance and expansion., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

Nischarin has been demonstrated to have tumor suppressor functions. In this review, we comprehensively discuss up to date information about Nischarin. In addition, this paper aims to report the prognostic value, clinical relevance, and biological significance of the Nischarin gene (NISCH) in breast cancer (BCa) patients using the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) and The Cancer Genome Atlas (TCGA) datasets. We evaluated NISCH gene expression and its correlation to patient survival, baseline expression, and expression variation based on age groups, tumor stage, tumor size, tumor grade, and lymph node status in different subtypes of BCa. Since NISCH has been extensively reported to inhibit EMT and cancer cell migration, we also checked for the correlation between NISCH and EMT genes in addition to the correlation between NISCH and cell migration genes. Our results indicate that NISCH is a tumor suppressor that plays a critical role in BCa initiation, progression, and tumor development. We find that there is a higher level of NISCH expression in normal breast tissues compared to breast cancer tissues. Also, aggressive subtypes of breast cancers, such as the triple negative/basal category, have decreased levels of NISCH as the disease progresses. Finally, we report that NISCH is inversely correlated with many EMT and cancer cell migration genes in BCa. Interestingly, we identified a significant negative correlation between NISCH expression and its methylation in breast cancer patients. Overall, the goal of this report is to establish a strong clinical basis for further investigation into the cellular, molecular, and physiological roles of NISCH in BCa. Ultimately, NISCH gene expression might be clinically harnessed as a biomarker or predictor of invasiveness and metastasis in BCa., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

Ectodysplasin A receptor (EDAR) is a death receptor in the Tumour Necrosis Factor Receptor (TNFR) superfamily with roles in the development of hair follicles, teeth and cutaneous glands. Here we report that human Oestrogen Receptor (ER) negative breast carcinomas which display squamous differentiation express EDAR strongly. Using a mouse model with a high Edar copy number, we show that elevated EDAR signalling results in a high incidence of mammary tumours in breeding female mice. These tumours resemble the EDAR-high human tumours in that they are characterised by a lack of oestrogen receptor expression, contain extensive squamous metaplasia, and display strong β-catenin transcriptional activity. In the mouse model, all of the tumours carry somatic deletions of the third exon of the CTNNB1 gene that encodes β-catenin. Deletion of this exon yields unconstrained β-catenin signalling activity. We also demonstrate that β-catenin activity is required for transformed cell growth, showing that increased EDAR signalling creates an environment in which β-catenin activity can readily promote tumourigenesis. Together, this work identifies a novel death receptor oncogene in breast cancer, whose mechanism of transformation is based on the interaction between the WNT and Ectodysplasin A (EDA) pathways., (© 2021. The Author(s).)

Metastasis-initiating cells (MICs) display stem cell-like features, cause metastatic recurrences and defy chemotherapy, which leads to patients' demise. Here we show that prostate and breast cancer patients harbor contingents of tumor cells with high expression of CX3CR1, OCT4a (POU5F1), and NANOG. Impairing CX3CR1 expression or signaling hampered the formation of tumor spheroids by cell lines from which we isolated small subsets co-expressing CX3CR1 and stemness-related markers, similarly to patients' tumors. These rare CX3CR1 High cells show transcriptomic profiles enriched in pathways that regulate pluripotency and endowed with metastasis-initiating behavior in murine models. Cancer cells lacking these features (CX3CR1 Low ) were capable of re-acquiring CX3CR1-associated features over time, implying that MICs can continuously emerge from non-stem cancer cells. CX3CR1 expression also conferred resistance to docetaxel, and prolonged treatment with docetaxel selected CX3CR1 High phenotypes with de-enriched transcriptomic profiles for apoptotic pathways. These findings nominate CX3CR1 as a novel marker of stem-like tumor cells and provide conceptual ground for future development of approaches targeting CX3CR1 signaling and (re)expression as therapeutic means to prevent or contain metastasis initiation., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Epidemiological evidence has indicated an inverse association between selenium status and various types of cancer, including breast cancer. Selenoproteins are the primary mediators of selenium effects in human health. We have previously reported loss of heterozygosity in breast tumor samples of the gene for one of the selenoproteins, SELENOF. The function of SELENOF remains unclear and whether SELENOF levels impact breast cancer risk or outcome is unknown. The mining of breast cancer patient databases revealed that SELENOF mRNA is significantly lower in late-stage tumor samples and lower levels of SELENOF also predict poor patient outcome from breast cancer. Genetically manipulating SELENOF in human breast cancer cells or in the murine mammary gland by overexpression, silencing or knockout impacted cell viability by affecting both proliferation and cell death. Restoring SELENOF can attenuate a number of aggressive cancer phenotypes in breast cancer cells, including clonogenic survival, and enhance the response to drugs or radiation used in breast cancer therapy. Importantly, enhancing SELENOF expression reduced in vivo tumor growth in a murine xenograft model of breast cancer. These data indicate that SELENOF is a new tumor suppressor in breast cancer., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

Emerging evidence shows the association between nuclear envelope and tumor progression, however, the functional contributions of specific constituents of the nuclear envelope remain largely unclear. We found that the expression level of transmembrane protein 201 (TMEM201), an integral inner nuclear membrane protein of unknown function, was significantly elevated in invasive breast cancer and predicted poor breast cancer prognosis. We showed that TMEM201, as a positive modulator, was both necessary and sufficient to regulate the migration and invasion of breast cancer cells in vitro and in vivo. Mechanistically, RNA-sequencing analysis and validation showed that TMEM201 deficiency inhibited epithelial-to-mesenchymal transition and transforming growth factor-β signaling. Finally, we showed that TMEM201 physically interacted with SMAD2/3 and was required for the phosphorylation of SMAD2/3, nuclear translocation and transcriptional activation of the TGFβ. Thus, we demonstrated that specific inner nuclear membrane component mediated signal-dependent transcriptional effects to control breast cancer metastasis., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

STING (Stimulator of Interferon Genes) is an endoplasmic reticulum-anchored adaptor of the innate immunity best known to trigger pro-inflammatory cytokine expression in response to pathogen infection. In cancer, this canonical pathway can be activated by intrinsic or drug-induced genomic instability, potentiating antitumor immune responses. Here we report that STING downregulation decreases cell survival and increases sensitivity to genotoxic treatment in a panel of breast cancer cell lines in a cell-autonomous manner. STING silencing impaired DNA Damage Response (53BP1) foci formation and increased DNA break accumulation. These newly identified properties were found to be independent of STING partner cGAS and of its canonical pro-inflammatory pathway. STING was shown to partially localize at the inner nuclear membrane in a variety of breast cancer cell models and clinical tumor samples. Interactomics analysis of nuclear STING identified several proteins of the DNA Damage Response, including the three proteins of the DNA-PK complex, further supporting a role of STING in the regulation of genomic stability. In breast and ovarian cancer patients that received adjuvant chemotherapy, high STING expression is associated with increased risk of relapse. In summary, this study highlights an alternative, non-canonical tumor-promoting role of STING that opposes its well-documented function in tumor immunosurveillance., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

ISG15 is an ubiquitin-like modifier that is associated with reduced survival rates in breast cancer patients. The mechanism by which ISG15 achieves this however remains elusive. We demonstrate that modification of Rab GDP-Dissociation Inhibitor Beta (GDI2) by ISG15 (ISGylation) alters endocytic recycling of the EGF receptor (EGFR) in non-interferon stimulated cells using CRISPR-knock out models for ISGylation. By regulating EGFR trafficking, ISGylation enhances EGFR recycling and sustains Akt-signalling. We further show that Akt signalling positively correlates with levels of ISG15 and its E2-ligase in basal breast cancer cohorts, confirming the link between ISGylation and Akt signalling in human tumours. Persistent and enhanced Akt activation explains the more aggressive tumour behaviour observed in human breast cancers. We show that ISGylation can act as a driver of tumour progression rather than merely being a bystander., (© 2021. The Author(s).)

Complete blockade of the HER2 protein itself and HER signaling network is critical to achieving effective HER2-targeted therapies. Despite the success of HER2-targeted therapies, the diseases will relapse in a significant fraction of patients with HER2 + breast cancers. How to improve the therapeutic efficacy of existing HER2-targeted agents remains an unmet clinical need. Here, we uncover a role of Melatonin in diminishing HER2-mediated signaling by destruction of HER2 protein. Mechanistically, Melatonin treatment attenuated the protective effect of the HSP90 chaperone complex on its client protein HER2, triggering ubiquitylation and subsequent endocytic lysosomal degradation of HER2. The inhibitory effect of Melatonin on HER2 signaling substantially enhanced the cytotoxic effects of the pan-HER inhibitor Neratinib in HER2 + breast cancer cells. Lastly, we demonstrate that dual inhibition of HER2 by combined use of Melatonin and Neratinib effectively blocked the growth of HER2 + breast tumor xenografts in vivo. Our findings shed light on the potential use of Melatonin in a novel dual HER2 blockade strategy for HER2 + breast cancer treatment., (© 2021. The Author(s).)

Canonical Wnt/β-catenin signaling is an established regulator of cellular state and its critical contributions to tumor initiation, malignant tumor progression and metastasis formation have been demonstrated in various cancer types. Here, we investigated how the binding of β-catenin to the transcriptional coactivators B-cell CLL/lymphoma 9 (Bcl9) and Bcl9-Like (Bcl9L) affected mammary gland carcinogenesis in the MMTV-PyMT transgenic mouse model of metastatic breast cancer. Conditional knockout of both Bcl9 and Bcl9L resulted into tumor cell death. In contrast, disrupting the interaction of Bcl9/Bcl9L with β-catenin, either by deletion of their HD2 domains or by a point mutation in the N-terminal domain of β-catenin (D164A), diminished primary tumor growth and tumor cell proliferation and reduced tumor cell invasion and lung metastasis. In comparison, the disruption of HD1 domain-mediated binding of Bcl9/Bcl9L to Pygopus had only moderate effects. Interestingly, interfering with the β-catenin-Bcl9/Bcl9L-Pygo chain of adapters only partially impaired the transcriptional response of mammary tumor cells to Wnt3a and TGFβ treatments. Together, the results indicate that Bcl9/Bcl9L modulate but are not critically required for canonical Wnt signaling in its contribution to breast cancer growth and malignant progression, a notion consistent with the "just-right" hypothesis of Wnt-driven tumor progression., (© 2021. The Author(s).)

The ARID1A gene, which encodes a subunit of the SWI/SNF chromatin remodeling complex, has been found to be frequently mutated in many human cancer types. However, the function and mechanism of ARID1A in cancer metastasis are still unclear. Here, we show that knockdown of ARID1A increases the ability of breast cancer cells to proliferate, migrate, invade, and metastasize in vivo. The ARID1A-related SWI/SNF complex binds to the second exon of CDH1 and negatively modulates the expression of E-cadherin/CDH1 by recruiting the transcriptional repressor ZEB2 to the CDH1 promoter and excluding the presence of RNA polymerase II. The silencing of CDH1 attenuated the migration, invasion, and metastasis of breast cancer cells in which ARID1A was silenced. ARID1A depletion increased the intracellular enzymatic processing of E-cadherin and the production of C-terminal fragment 2 (CTF2) of E-cadherin, which stabilized β-catenin by competing for binding to the phosphorylation and degradation complex of β-catenin. The matrix metalloproteinase inhibitor GM6001 inhibited the production of CTF2. In zebrafish and nude mice, ARID1A silencing or CTF2 overexpression activated β-catenin signaling and promoted migration/invasion and metastasis of cancer cells in vivo. The inhibitors GM6001, BB94, and ICG-001 suppressed the migration and invasion of cancer cells with ARID1A-deficiency. Our findings provide novel insights into the mechanism of ARID1A metastasis and offer a scientific basis for targeted therapy of ARID1A-deficient cancer cells., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

Breast cancer is the most frequently diagnosed cancer among women worldwide. Though advances in diagnosis and treatment have prolonged overall survival (OS) for patients with breast cancer, metastasis remains the major obstacles to improved survival for breast cancer patients. The existence of breast cancer stem cells (BCSCs) is a major reason underlying cancer metastasis and recurrence. Therefore, understanding the molecular pathways sustaining BCSC properties and targeting BCSCs will ultimately improve breast cancer treatments. In this study, we found that activation of β-Catenin directly regulated CCL2 expression at the transcriptional level, and in turn promoted macrophages infiltration and M2 polarization. Moreover, macrophages co-cultured with breast cancer cells showed a significant increase in CCL2 expression and promoted β-Catenin-induced BCSCs properties, whereas depletion of CCL2 by adding neutralizing antibodies suppressed BSCSs properties. In addition, we found that β-Catenin-mediated CCL2 secretion recruited macrophages into tumor microenvironment and promoted breast cancer growth and metastasis in vivo. Clinically, we observed a significant positive correlation between β-Catenin, CCL2 and CD163 expression, and increased expression of β-Catenin, CCL2 and CD163 predicted poor prognosis in breast cancer. Furthermore, pharmacological inhibition of CCR2 and β-Catenin synergistically suppressed BCSC properties and breast cancer growth. Collectively, our findings suggested that β-Catenin-mediated CCL2 secretion forms a paracrine feedback loop between breast cancer cells and macrophages, which in turn promotes BCSC properties and supports breast cancer growth and metastasis. Targeting β-Catenin/CCL2 signaling might be an effective strategy for breast cancer therapy., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

Metastatic breast cancer causes most breast cancer-associated deaths, especially in triple negative breast cancers (TNBC). The metastatic drivers of TNBCs are still poorly understood, and effective treatment non-existent. Here we reveal that the presence of Aurora-A Kinase (AURKA) in the nucleus and metastatic dissemination are molecularly connected through HIF1 (Hypoxia-Inducible Factor-1) signaling. Nuclear AURKA activates transcription of "hypoxia-induced genes" under normoxic conditions (pseudohypoxia) and without upregulation of oxygen-sensitive HIF1A subunit. We uncover that AURKA preferentially binds to HIF1B and co-localizes with the HIF complex on DNA. The mass-spectrometry analysis of the AURKA complex further confirmed the presence of CBP and p300 along with other TFIIB/RNApol II components. Importantly, the expression of multiple HIF-dependent genes induced by nuclear AURKA (N-AURKA), including migration/invasion, survival/death, and stemness, promote early cancer dissemination. These results indicate that nuclear, but not cytoplasmic, AURKA is a novel driver of early metastasis. Analysis of clinical tumor specimens revealed a correlation between N-AURKA presence and decreased patient survival. Our results establish a mechanistic link between two critical pathways in cancer metastasis, identifying nuclear AURKA as a crucial upstream regulator of the HIF1 transcription complex and a target for anti-metastatic therapy., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

Metastatic recurrence is still a major challenge in breast cancer treatment, but the underlying mechanisms remain unclear. Here, we report that a small adaptor protein, SH3BGRL, is upregulated in the majority of breast cancer patients, especially elevated in those with metastatic relapse, indicating it as a marker for the poor prognosis of breast cancer. Physiologically, SH3BGRL can multifunctionally promote breast cancer cell tumorigenicity, migration, invasiveness, and efficient lung colonization in nude mice. Mechanistically, SH3BGRL downregulates the acting-binding protein profilin 1 (PFN1) by accelerating the translation of the PFN1 E3 ligase, STUB1 via SH3BGRL interaction with ribosomal proteins, or/and enhancing the interaction of PFN1 with STUB1 to accelerate PFN1 degradation. Loss of PFN1 consequently contributes to downstream multiple activations of AKT, NF- k B, and WNT signaling pathways. In contrast, the forced expression of compensatory PFN1 in SH3BGRL-high cells efficiently neutralizes SH3BGRL-induced metastasis and tumorigenesis with PTEN upregulation and PI3K-AKT signaling inactivation. Clinical analysis validates that SH3BGRL expression is negatively correlated with PFN1 and PTEN levels, but positively to the activations of AKT, NF- k B, and WNT signaling pathways in breast patient tissues. Our results thus suggest that SH3BGRL is a valuable prognostic factor and a potential therapeutic target for preventing breast cancer progression and metastasis., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

Despite advances in breast cancer treatment, residual disease driven by dormant tumor cells continues to be a significant clinical problem. Leukemia inhibitory factor receptor (LIFR) promotes a dormancy phenotype in breast cancer cells and LIFR loss is correlated with poor patient survival. Herein, we demonstrate that histone deacetylase inhibitors (HDACi), which are in phase III clinical trials for breast cancer, epigenetically induced LIFR and activated a pro-dormancy program in breast cancer cells. HDACi slowed breast cancer cell proliferation and reduced primary tumor growth. Primary breast tumors from HDACi-treated patients had increased LIFR levels and reduced proliferation rates compared to pre-treatment levels. Recent Phase II clinical trial data studying entinostat and azacitidine in metastatic breast cancer revealed that induction of several pro-dormancy genes post-treatment was associated with prolonged patient survival. Together, these findings suggest HDACi as a potential therapeutic avenue to promote dormancy, prevent recurrence, and improve patient outcomes in breast cancer., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

Intercellular mechanisms by which the stromal microenvironment contributes to solid tumor progression and targeted therapy resistance remain poorly understood, presenting significant clinical hurdles. PEAK1 (Pseudopodium-Enriched Atypical Kinase One) is an actin cytoskeleton- and focal adhesion-associated pseudokinase that promotes cell state plasticity and cancer metastasis by mediating growth factor-integrin signaling crosstalk. Here, we determined that stromal PEAK1 expression predicts poor outcomes in HER2-positive breast cancers high in SNAI2 expression and enriched for MSC content. Specifically, we identified that the fibroblastic stroma in HER2-positive breast cancer patient tissue stains positive for both nuclear SNAI2 and cytoplasmic PEAK1. Furthermore, mesenchymal stem cells (MSCs) and cancer-associated fibroblasts (CAFs) express high PEAK1 protein levels and potentiate tumorigenesis, lapatinib resistance and metastasis of HER2-positive breast cancer cells in a PEAK1-dependent manner. Analysis of PEAK1-dependent secreted factors from MSCs revealed INHBA/activin-A as a necessary factor in the conditioned media of PEAK1-expressing MSCs that promotes lapatinib resistance. Single-cell CycIF analysis of MSC-breast cancer cell co-cultures identified enrichment of p-Akt high /p-gH2AX low , MCL1 high /p-gH2AX low and GRP78 high /VIM high breast cancer cell subpopulations by the presence of PEAK1-expressing MSCs and lapatinib treatment. Bioinformatic analyses on a PEAK1-centric stroma-tumor cell gene set and follow-up immunostaining of co-cultures predict targeting antiapoptotic and stress pathways as a means to improve targeted therapy responses and patient outcomes in HER2-positive breast cancer and other stroma-rich malignancies. These data provide the first evidence that PEAK1 promotes tumorigenic phenotypes through a previously unrecognized SNAI2-PEAK1-INHBA stromal cell axis., (© 2021. The Author(s).)

Metastasis is the main cause of death in breast cancer patients. The initial step of metastasis is invadopodia-mediated extracellular matrix (ECM) degradation, which enables local breast tumor cells to invade surrounding tissues. However, the molecular mechanism underlying invadopodia-mediated metastasis remains largely unknown. Here we found that the RNA-binding protein Musashi1 (Msi1) exhibited elevated expression in invasive breast tumors and promoted lung metastasis of mammary cancer cells. Suppression of Msi1 reduced invadopodia formation in mammary cancer cells. Furthermore, Msi1 deficiency decreased the expression and activity of Mmp9, an important enzyme in ECM degradation. Mechanistically, Msi1 directly suppressed Timp3, an endogenous inhibitor of Mmp9. In clinical breast cancer specimens, TIMP3 and MSI1 levels were significantly inversely correlated both in normal breast tissue and breast cancer tissues and associated with overall survival in breast cancer patients. Taken together, our findings demonstrate that the MSI1-TIMP3-MMP9 cascade is critical for invadopodia-mediated onset of metastasis in breast cancer, providing novel insights into a promising therapeutic strategy for breast cancer metastasis., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

Membrane Type 1 Matrix Metalloprotease (MT1-MMP) contributes to the invasive progression of breast cancers by degrading extracellular matrix tissues. Nucleoside diphosphate kinase, NME1/NM23-H1, has been identified as a metastasis suppressor; however, its contribution to local invasion in breast cancer is not known. Here, we report that NME1 is up-regulated in ductal carcinoma in situ (DCIS) as compared to normal breast epithelial tissues. NME1 levels drop in microinvasive and invasive components of breast tumor cells relative to synchronous DCIS foci. We find a strong anti-correlation between NME1 and plasma membrane MT1-MMP levels in the invasive components of breast tumors, particularly in aggressive histological grade III and triple-negative breast cancers. Knockout of NME1 accelerates the invasive transition of breast tumors in the intraductal xenograft model. At the mechanistic level, we find that MT1-MMP, NME1 and dynamin-2, a GTPase known to require GTP production by NME1 for its membrane fission activity in the endocytic pathway, interact in clathrin-coated vesicles at the plasma membrane. Loss of NME1 function increases MT1-MMP surface levels by inhibiting endocytic clearance. As a consequence, the ECM degradation and invasive potentials of breast cancer cells are enhanced. This study identifies the down-modulation of NME1 as a potent driver of the in situ-to invasive transition during breast cancer progression.

The Ubiquitin-Specific Protease 22 (USP22) is a deubiquitinating subunit of the mammalian SAGA transcriptional co-activating complex. USP22 was identified as a member of the so-called "death-from-cancer" signature predicting therapy failure in cancer patients. However, the importance and functional role of USP22 in different types and subtypes of cancer remain largely unknown. In the present study, we leveraged human cell lines and genetic mouse models to investigate the role of USP22 in HER2-driven breast cancer (HER2 + -BC) and demonstrate for the first time that USP22 is required for the tumorigenic properties in murine and human HER2 + -BC models. To get insight into the underlying mechanisms, we performed transcriptome-wide gene expression analyses and identified the Unfolded Protein Response (UPR) as a pathway deregulated upon USP22 loss. The UPR is normally induced upon extrinsic or intrinsic stresses that can promote cell survival and recovery if shortly activated or programmed cell death if activated for an extended period. Strikingly, we found that USP22 actively suppresses UPR induction in HER2 + -BC cells by stabilizing the major endoplasmic reticulum (ER) chaperone HSPA5. Consistently, loss of USP22 renders tumor cells more sensitive to apoptosis and significantly increases the efficiency of therapies targeting the ER folding capacity. Together, our data suggest that therapeutic strategies targeting USP22 activity may sensitize tumor cells to UPR induction and could provide a novel, effective approach to treat HER2 + -BC.

The heat shock protein GRP78 typically resides in the endoplasmic reticulum in normal tissues, but it has been shown to be expressed on the cell surface of several cancer cells, and some stem cells, where it can act as a signaling molecule by not-yet-fully defined mechanisms. Although cell surface GRP78 (sGRP78) has emerged as an attractive chemotherapeutic target, understanding how sGRP78 is functioning in cancer has been complicated by the fact that sGRP78 can function in a cell-context dependent manner, with a diverse array of reported binding partners, to regulate a variety of cellular responses. We had previously shown that sGRP78 was important in regulating pluripotent stem cell (PSC) functions, and hypothesized that embryonic-like mechanisms of GRP78 were critical to regulating aggressive breast cancer cell functions. Here, using proteomics we identify Dermcidin (DCD) as a novel sGRP78 binding partner common to both PSCs and breast cancer cells. We show that GRP78 and DCD cooperate to regulate stem cell and cancer cell migration that is dependent on the cell surface functions of these proteins. Finally, we identify Wnt/β-catenin signaling, a critical pathway in stem cell and cancer cell biology, as an important downstream intermediate in regulating this migration phenotype.

Estrogen receptor alpha (ER)-positive breast cancer is commonly treated with endocrine therapies, including antiestrogens that bind and inhibit ER activity, and aromatase inhibitors that suppress estrogen biosynthesis to inhibit estrogen-dependent ER activity. Paradoxically, treatment with estrogens such as 17b-estradiol can also be effective against ER+ breast cancer. Despite the known efficacy of estrogen therapy, the lack of a predictive biomarker of response and understanding of the mechanism of action have contributed to its limited clinical use. Herein, we demonstrate that ER overexpression confers resistance to estrogen deprivation through ER activation in human ER+ breast cancer cells and xenografts grown in mice. However, ER overexpression and the associated high levels of ER transcriptional activation converted 17b-estradiol from a growth-promoter to a growth-suppressor, offering a targetable therapeutic vulnerability and a potential means of identifying patients likely to benefit from estrogen therapy. Since ER+ breast cancer cells and tumors ultimately developed resistance to continuous estrogen deprivation or continuous 17b-estradiol treatment, we tested schedules of alternating treatments. Oscillation of ER activity through cycling of 17b-estradiol and estrogen deprivation provided long-term control of patient-derived xenografts, offering a novel endocrine-only strategy to manage ER+ breast cancer.

Mitochondrial oxidative phosphorylation (OXPHOS) is a vital regulator of tumor metastasis. However, the mechanisms governing OXPHOS to facilitate tumor metastasis remain unclear. In this study, we discovered that arginine 21(R21) and lysine 108 (K108) of mitochondrial ribosomal protein S23 (MRPS23) was methylated by the protein arginine methyltransferase 7 (PRMT7) and SET-domain-containing protein 6 (SETD6), respectively. R21 methylation accelerated the poly-ubiquitin-dependent degradation of MRPS23 to a low level. The MRPS23 degradation inhibited OXPHOS with elevated mtROS level, which consequently increased breast cancer cell invasion and metastasis. In contrast, K108 methylation increased MRPS23 stability, and K108 methylation coordinated with R21 methylation to maintain a low level of MRPS23, which was in favor of supporting breast cancer cell survival through regulating OXPHOS. Consistently, R21 and K108 methylation was correlated with malignant breast carcinoma. Significantly, our findings unveil a unique mechanism of controlling OXPHOS by arginine and lysine methylation and point to the impact of the PRMT7-SETD6-MRPS23 axis during breast cancer metastasis.