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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.)

The internal tandem duplication of the FMS-like tyrosine kinase 3 (FLT3-ITD) is one of the most frequent genetic alterations in acute myeloid leukemia (AML). Limited and transient clinical benefit of FLT3 kinase inhibitors (FLT3i) emphasizes the need for alternative therapeutic options for this subset of myeloid malignancies. Herein, we showed that FLT3-ITD mutant (FLT3-ITD + ) AML cells were susceptible toward inhibitors of DHODH, a rate-limiting enzyme of de novo pyrimidine biosynthesis. Genetic and pharmacological blockade of DHODH triggered downregulation of FLT3-ITD protein, subsequently suppressed activation of downstream ERK and STAT5, and promoted cell death of FLT3-ITD + AML cells. Mechanistically, DHODH blockade triggered autophagy-mediated FLT3-ITD degradation via inactivating mTOR, a potent autophagy repressor. Notably, blockade of DHODH synergized with an FDA-approved FLT3i quizartinib in significantly impairing the growth of FLT3-ITD + AML cells and improving tumor-bearing mice survival. We further demonstrated that DHODH blockade exhibited profound anti-proliferation effect on quizartinib-resistant cells in vitro and in vivo. In summary, this study demonstrates that the induction of degradation of FLT3-ITD protein by DHODH blockade may offer a promising therapeutic strategy for AML patients harboring FLT3-ITD mutation., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

Lung cancer is the leading cause of cancer-related death worldwide. KRAS mutations are the most common oncogenic alterations found in lung cancer. Unfortunately, treating KRAS-mutant lung adenocarcinoma (ADC) remains a major oncotherapeutic challenge. Here, we used both autochthonous and transplantable KRAS-mutant tumor models to investigate the role of tumor-derived CUL4B in KRAS-driven lung cancers. We showed that knockout or knockdown of CUL4B promotes lung ADC growth and progression in both models. Mechanistically, CUL4B directly binds to the promoter of Cxcl2 and epigenetically represses its transcription. CUL4B deletion increases the expression of CXCL2, which binds to CXCR2 on myeloid-derived suppressor cells (MDSCs) and promotes their migration to the tumor microenvironment. Targeting of MDSCs significantly delayed the growth of CUL4B knockdown KRAS-mutant tumors. Collectively, our study provides mechanistic insights into the novel tumor suppressor-like functions of CUL4B in regulating KRAS-driven lung tumor development., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

N 6 -Methyladenosine (m 6 A) is the most prevalent epigenetic RNA modification and is vital in regulating malignancies. The roles of m 6 A modifiers on noncoding RNAs have not been fully investigated in esophageal cancer. By screening all m 6 A modifiers, ALKBH5 was the most potent member related to patient outcomes and suppressing esophageal cancer malignancy in cell and animal models. It demethylated pri-miR-194-2 and inhibited miR-194-2 biogenesis through an m 6 A/DGCR8-dependent manner. RAI1, previously considered as a circadian clock transcriptional regulator, was the main target of miR-194-2. It enhanced transcription of Hippo pathway upstream genes by binding to their 3'UTR and suppressed YAP/TAZ nuclear translocation. The ALKBH5/miR-194-2/RAI1 axis was also validated in clinical samples. In addition, the increased malignancy by low ALKBH5 was abolished by the YAP inhibitor verteporfin. Our findings uncover a critical role of ALKBH5 in miRNAs biogenesis and provide novel insight for developing treatment strategies in esophageal cancer., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

CUL4B, which acts as a scaffold protein in CUL4B-RING ubiquitin ligase (CRL4B) complexes, participates in a variety of biological processes. Previous studies have shown that CUL4B is often overexpressed and exhibits oncogenic activities in a variety of solid tumors. However, the roles and the underlying mechanisms of CUL4B in bladder cancer (BC) were poorly understood. Here, we showed that CUL4B levels were overexpressed and positively correlated with the malignancy of BC, and CUL4B could confer BC cells increased motility, invasiveness, stemness, and chemoresistance. The PIK3CA/AKT pathway was identified as a critical downstream mediator of CUL4B-driven oncogenicity in BC cells. Furthermore, we demonstrated that CRL4B epigenetically repressed the transcription of miR-372/373, via catalyzing monoubiquitination of H2AK119 at the gene cluster encoding miR-372/373, leading to upregulation of PIK3CA and activation of AKT. Our findings thus establish a critical role for the CUL4B-miR-372/373-PIK3CA/AKT axis in the pathogenesis of BC and have important prognostic and therapeutic implications in BC.

Mitochondrial pyruvate carrier 1 (MPC-1) appears to be a tumor suppressor. In this study, we determined the regulation of MPC-1 expression by Lysine demethylase 5A (KDM5A) and critical impact of this novel KDM5A/MPC-1 signaling on PDA progression. TCGA database, paired PDA and adjacent normal pancreatic tissues, PDA tissue array and cell lines were used to determine the levels of MPC-1 and KDM5A expression, and their relationship with the clinicopathologic characteristics and overall survival (OS) of PDA patients. Both in vitro and in vivo models were used to determine biologic impacts of MPC-1 and KDM5A on PDA and mitochondrial pyruvate metabolism, and the mechanism underling reduced MPC-1 expression in PDA. The expression of MPC-1 was decreased in PDA cell lines and tissues, and negatively associated with tumor poorer differentiation, lymph nodes metastasis, higher TNM stages, and patients' overall survival (OS). Functional analysis revealed that restored expression of MPC-1 suppressed the growth, invasion, migration, stemness and tumorigenicity. Re-expression of MPC-1 stimulated the mitochondrial pyruvate metabolism and inhibited glycolysis, while MPC-1-specific inhibitor UK5099 attenuated these effects. Furthermore, KDM5A bound directly to MPC-1 promoter region and transcriptionally suppressed the expression of MPC-1 via demethylation H3K4. Consistently, KDM5A expression was elevated in PDA and promoted PDA cell proliferation in vitro and tumor growth in vivo via suppressing the expression of MPC-1. The expression of KDM5A was inversely correlated with that of MPC-1 in PDA. KDM5A/MPC-1 signaling promoted PDA growth, invasion, migration, and stemness via inhibiting mitochondrial pyruvate metabolism. Targeting KDM5A/MPC-1 signaling may be an effective therapeutic strategy for PDA.

Elk-1, a c-Fos protooncogene regulator, which belongs to the ETS-domain family of transcriptional factors, plays an important role in the induction of immediate early gene expression in response to a variety of extracellular signals. In this study, we demonstrate for the first time the in vitro and in vivo interaction of Elk-1 with BRCA1 splice variants BRCA1a and BRCA1b using GST-pull down assays, co-imunoprecipitations/Western blot analysis of cell extracts from breast cancer cells and mammalian two-hybrid assays. We have localized the BRCA1 interaction domain of Elk-1 protein to the conserved ETS domain, a motif involved in DNA binding and protein-protein interactions. We also observed binding of BRCA1 proteins to other ETS-domain transcription factors SAP1, ETS-1, ERG-2 and Fli-1 but not to Elk-1 splice variant DeltaElk-1 and c-Fos protooncogene. Both BRCA1a and BRCA1b splice variants function as growth suppressors of human breast cancer cells. Interestingly, our studies reveal that although both Elk-1 and SAP-1 are highly homologous members of a subfamily of ETS domain proteins called ternary complex factors, it is only Elk-1 but not SAP-1 that can augment the growth suppressive function of BRCA1a/1b proteins in breast cancer cells. Thus Elk-1 could be a potential downstream target of BRCA1 in its growth control pathway. Furthermore, we have observed inhibition of c-Fos promoter activity in BRCA1a transfected stable breast cancer cells and over expression of BRCA1a/1b attenuates MEK-induced SRE activation in vivo. These results demonstrate for the first time a link between the growth suppressive function of BRCA1a/1b proteins and signal transduction pathway involving Elk-1 protein. All these results taken together suggest that one of the mechanisms by which BRCA1a/1b proteins function as growth/tumor suppressors is through inhibition of the expression of Elk-1 target genes like c-Fos.

Inherited mutations in the breast and ovarian cancer susceptibility gene BRCA1 are associated with high risk for developing breast and ovarian cancers. Several studies link BRCA1 to transcriptional regulation, DNA repair, apoptosis and growth/tumor suppression. BRCA1 associates with p53 and stimulates transcription in both p53 dependent and p53-independent manners. BRCA1 splice variants BRCA1a (p110) and BRCA1b (p100) associates with CBP/p300 co-activators. Here we show that BRCA1a and BRCA1b proteins stimulate p53-dependent transcription from the p21WAF1/CIP1 promoter. In addition, the C-terminal second BRCA1 (BRCT) domain is sufficient for p53 mediated transactivation of the p21 promoter. Previous studies emphasized the importance of the BRCT domain, which shows homology with p53 binding protein (53BP1), in transcriptional activation, growth inhibition and tumor suppression. Our findings demonstrate an additional function for this domain in protein-protein interaction and co-activation of p53. We also found that BRCA1a and BRCA1b proteins interact with p53 in vitro and in vivo. The p53 interaction domain of BRCA1a/1b maps, in vitro, to the second BRCT domain (aa 1760-1863). The BRCT domain binds to the central domain of p53 which is required for sequence specific DNA binding. These results demonstrate for the first time the presence of a second p53 interaction domain in BRCA1 proteins and suggests that BRCA1a and BRCA1b proteins, like BRCA1, function as p53 co-activators. This BRCT domain also binds in vitro to CBP. These results suggest that one of the mechanisms by which BRCA1 proteins function is through recruitment of CBP/p300 associated HAT/FAT activity for acetylation of p53 to specific promoters resulting in transcriptional activation.

Elk-1, an ets related gene codes for at least two splice variants Elk-1, which regulates c-fos transcription and deltaElk-1, both of which function as transcriptional activators. To investigate the role of Elk-1 and deltaElk-1 proteins in apoptosis; we have developed rat fibroblast cell lines and human breast cancer cell lines expressing Elk-1 and deltaElk-1. The expression of Elk-1 and deltaElk-1 proteins in the Elk-1/deltaElk-1 transfectants were analysed by immunofluorescence, immunohistochemistry, and Western blot analysis. The Elk-1 unlike deltaElk-1 transfectants showed a shortened and flattened morphology compared to the parental cells. We have found that calcium ionophore treatment of Rat-1 Elk-1, MCF-7 Elk-1, Rat-1 deltaElk-1 and MCF-7 deltaElk-1 transfectants resulted in programmed cell death. These results indicate that constitutive expression of Elk-1 and deltaElk-1 proteins triggers apoptosis in Rat-1 fibroblasts and breast cancer cells when treated with calcium ionophore.

BRCA1, a familial breast and ovarian cancer susceptibility gene encodes nuclear phosphoproteins that function as tumor suppressors in human breast cancer cells. Previously, we have shown that overexpression of a BRCA1 splice variant BRCA1a accelerates apoptosis in human breast cancer cells. In an attempt to determine whether the subcellular localization of BRCA1 is cell cycle regulated, we have studied the subcellular distribution of BRCA1 in asynchronous and growth arrested normal, breast and ovarian cancer cells using different BRCA1 antibodies by immunofluorescence and immunohistochemical staining. Upon serum starvation of NIH3T3, some breast and ovarian cancer cells, most of the BRCA1 protein redistributed to the nucleus revealing a new type of regulation that may modulate the activity of BRCA1 gene. We have also characterized two new variant BRCA1 proteins (BRCA1a/p110 and BRCA1b/ p100) which are phosphoproteins containing phosphotyrosine. Immunofluorescence and Western blotting analysis indicate cytoplasmic and nuclear localization of BRCA1a and BRCA1b proteins. To elucidate the biological function of BRCA1, we created a bacterial fusion protein of glutathione-transferase (GST) and BRCA1 zinc finger domain and detected two cellular proteins with molecular weights of approximately 32 and 65 kD, one of which contains phosphotyrosine designated p32 and p65 BRCA1 interacting proteins (BIP) that specifically interact with BRCA1. Western blot analysis of BIP with cyclins/CDKs and E2F antisera indicated association with cdc2, cdk2, cdk4, cyclin B, cyclin D, cyclin A and E2F-4 but not with cdk3, cdk5, cdk6, E2F-1, E2F-2, E2F-3, E2F-5 and cyclin E. Furthermore, we have also demonstrated a direct interaction of in vitro translated BRCA1a and BRCA1b proteins with recombinant cyclin A, cyclin B1, cyclin D1, cdc2, cdk2 and E2F fusion proteins in vitro. Taken together these results seem to suggest that BRCA1 could be an important negative regulator of cell cycle that functions through interaction with E2F transcriptional factors and phosphorylation by cyclins/cdk complexes with the zinc ring finger functioning as a major protein-protein interaction domain. If the interactions we observe in vitro is also seen in vivo then it may be possible that lack or impaired binding of the disrupted BRCA1 proteins to E2F, cyclins/CDKs in patients with mutations in the zinc finger domain could deprive the cell of an important mechanism for braking cell proliferation leading to the development of breast and ovarian cancers.