Search

Rationale: SUMOylation regulates a plethora of biological processes, and its inhibitors are currently under investigation in clinical trials as anticancer agents. Thus, identifying new targets with site-specific SUMOylation and defining their biological functions will not only provide new mechanistic insights into the SUMOylation signaling but also open an avenue for developing new strategy for cancer therapy. MORC family CW-type zinc finger 2 (MORC2) is a newly identified chromatin-remodeling enzyme with an emerging role in the DNA damage response (DDR), but its regulatory mechanism remains enigmatic. Methods: In vivo and in vitro SUMOylation assays were used to determine the SUMOylation levels of MORC2. Overexpression and knockdown of SUMO-associated enzymes were used to detect their effects on MORC2 SUMOylation. The effect of dynamic MORC2 SUMOylation on the sensitivity of breast cancer cells to chemotherapeutic drugs was examined through in vitro and in vivo functional assays. Immunoprecipitation, GST pull-down, MNase, and chromatin segregation assays were used to explore the underlying mechanisms. Results: Here, we report that MORC2 is modified by small ubiquitin-like modifier 1 (SUMO1) and SUMO2/3 at lysine 767 (K767) in a SUMO-interacting motif dependent manner. MORC2 SUMOylation is induced by SUMO E3 ligase tripartite motif containing 28 (TRIM28) and reversed by deSUMOylase sentrin-specific protease 1 (SENP1). Intriguingly, SUMOylation of MORC2 is decreased at the early stage of DNA damage induced by chemotherapeutic drugs that attenuate the interaction of MORC2 with TRIM28. MORC2 deSUMOylation induces transient chromatin relaxation to enable efficient DNA repair. At the relatively late stage of DNA damage, MORC2 SUMOylation is restored, and SUMOylated MORC2 interacts with protein kinase CSK21 (casein kinase II subunit alpha), which in turn phosphorylates DNA-PKcs (DNA-dependent protein kinase catalytic subunit), thus promoting DNA repair. Notably, expression of a SUMOylation-deficient mutant MORC2 or administration of SUMO inhibitor enhances the sensitivity of breast cancer cells to DNA-damaging chemotherapeutic drugs. Conclusions: Collectively, these findings uncover a novel regulatory mechanism of MORC2 by SUMOylation and reveal the intricate dynamics of MORC2 SUMOylation important for proper DDR. We also propose a promising strategy to sensitize MORC2-driven breast tumors to chemotherapeutic drugs by inhibition of the SUMO pathway., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Aims: MORC family CW-type zinc finger 2 (MORC2), a GHKL-type ATPase, is aberrantly upregulated in multiple types of human tumors with profound effects on cancer aggressiveness, therapeutic resistance, and clinical outcome, thus making it an attractive drug target for anticancer therapy. However, the antagonists of MORC2 have not yet been documented., Methods and Results: We report that MORC2 is a relatively stable protein, and the N-terminal homodimerization but not ATP binding and hydrolysis is crucial for its stability through immunoblotting analysis and Quantitative real-time PCR. The N-terminal but not C-terminal inhibitors of heat shock protein 90 (HSP90) destabilize MORC2 in multiple cancer cell lines, and strikingly, this process is independent on HSP90. Mechanistical investigations revealed that HSP90 N-terminal inhibitors disrupt MORC2 homodimer formation without affecting its ATPase activities, and promote its lysosomal degradation through the chaperone-mediated autophagy pathway. Consequently, HSP90 inhibitor 17-AAG effectively blocks the growth and metastatic potential of MORC2-expressing breast cancer cells both in vitro and in vivo, and these noted effects are not due to HSP90 inhibition., Conclusion: We uncover a previously unknown role for HSP90 N-terminal inhibitors in promoting MORC2 degradation in a HSP90-indepentent manner and support the potential application of these inhibitors for treating MORC2-overexpressing tumors, even those with low or absent HSP90 expression. These results also provide new clue for further design of novel small-molecule inhibitors of MORC2 for anticancer therapeutic application., (© 2022 The Authors. Clinical and Translational Medicine published by John Wiley & Sons Australia, Ltd on behalf of Shanghai Institute of Clinical Bioinformatics.)

MORC family CW-type zinc finger 2 (MORC2) is a newly identified chromatin-remodeling enzyme involved in DNA damage response and gene transcription, and its dysregulation has been linked with Charcot-Marie-Tooth disease, neurodevelopmental disorder, and cancer. Despite its functional importance, how MORC2 is regulated remains enigmatic. Here, we report that MORC2 is O-GlcNAcylated by O-GlcNAc transferase (OGT) at threonine 556. Mutation of this site or pharmacological inhibition of OGT impairs MORC2-mediated breast cancer cell migration and invasion in vitro and lung colonization in vivo. Moreover, transforming growth factor-β1 (TGF-β1) induces MORC2 O-GlcNAcylation through enhancing the stability of glutamine-fructose-6-phosphate aminotransferase (GFAT), the rate-limiting enzyme for producing the sugar donor for OGT. O-GlcNAcylated MORC2 is required for transcriptional activation of TGF-β1 target genes connective tissue growth factor (CTGF) and snail family transcriptional repressor 1 (SNAIL). In support of these observations, knockdown of GFAT, SNAIL or CTGF compromises TGF-β1-induced, MORC2 O-GlcNAcylation-mediated breast cancer cell migration and invasion. Clinically, high expression of OGT, MORC2, SNAIL, and CTGF in breast tumors is associated with poor patient prognosis. Collectively, these findings uncover a previously unrecognized mechanistic role for MORC2 O-GlcNAcylation in breast cancer progression and provide evidence for targeting MORC2-dependent breast cancer through blocking its O-GlcNAcylation., (© 2021. The Author(s).)

Ring finger protein 144A (RNF144A), a poorly characterized member of the RING-in-between-RING family of E3 ubiquitin ligases, is an emerging tumor suppressor, but its underlying mechanism remains largely elusive. To address this issue, we used Affymetrix GeneChip Human Transcriptome Array 2.0 to profile gene expression in MDA-MB-231 cells stably expressing empty vector pCDH and Flag-RNF144A, and found that 128 genes were differentially expressed between pCDH- and RNF144A-expressing cells with fold change over 1.5. We further demonstrated that RNF144A negatively regulated the protein and mRNA levels of glial maturation factor γ (GMFG). Mechanistical investigations revealed that transcription factor YY1 transcriptionally activated GMFG expression, and RNF144A interacted with YY1 and promoted its ubiquitination-dependent degradation, thus blocking YY1-induced GMFG expression. Functional rescue assays showed that ectopic expression of RNF144A suppressed the proliferative, migratory, and invasive potential of breast cancer cells, and the noted effects were partially restored by re-expression of GMFG in RNF144A-overexpressing breast cancer cells. Collectively, these findings reveal that RNF144A negatively regulates GMFG expression by targeting YY1 for proteasomal degradation, thus inhibiting the proliferation, migration, and invasion of breast cancer cells., (© 2022. The Author(s).)

The remarkable advances in next-generation sequencing technology have enabled the wide usage of sequencing as a clinical tool. To promote the advance of precision oncology for breast cancer in China, here we report a large-scale prospective clinical sequencing program using the Fudan-BC panel, and comprehensively analyze the clinical and genomic characteristics of Chinese breast cancer. The mutational landscape of 1,134 breast cancers reveals that the most significant differences between Chinese and Western patients occurred in the hormone receptor positive, human epidermal growth factor receptor 2 negative breast cancer subtype. Mutations in p53 and Hippo signaling pathways are more prevalent, and 2 mutually exclusive and 9 co-occurring patterns exist among 9 oncogenic pathways in our cohort. Further preclinical investigation partially suggests that NF2 loss-of-function mutations can be sensitive to a Hippo-targeted strategy. We establish a public database (Fudan Portal) and a precision medicine knowledge base for data exchange and interpretation. Collectively, our study presents a leading approach to Chinese precision oncology treatment and reveals potentially actionable mutations in breast cancer.

Aberrant activation of estrogen signaling through three ESR (estrogen receptor) subtypes, termed ESR1/ERα, ESR2/ERβ, and GPER1 (G protein-coupled estrogen receptor 1), is implicated in breast cancer pathogenesis and progression. Antiestrogens tamoxifen (TAM) and fulvestrant (FUL) are effective for treatment of ESR1-positive breast tumors, but development of resistance represents a major clinical challenge. However, the molecular mechanisms behind these events remain largely unknown. Here, we report that 17β-estradiol (E2), TAM, and FUL stabilize MORC2 (MORC family CW-type zinc finger 2), an emerging oncoprotein in human cancer, in a GPER1-dependent manner. Mechanistically, GPER1 activates PRKACA (protein kinase cAMP-activated catalytic subunit alpha), which in turn phosphorylates MORC2 at threonine 582 (T582). Phosphorylated MORC2 decreases its interaction with HSPA8 (heat shock protein family A [Hsp70] member 8) and LAMP2A (lysosomal associated membrane protein 2A), two core components of the chaperone-mediated autophagy (CMA) machinery, thus protecting MORC2 from lysosomal degradation by CMA. Functionally, knockdown of MORC2 attenuates E2-induced cell proliferation and enhances cellular sensitivity to TAM and FUL. Moreover, introduction of wild-type MORC2, but not its phosphorylation-lacking mutant (T582A), in MORC2-depleted cells restores resistance to antiestrogens. Clinically, the phosphorylation levels of MORC2 at T582 are elevated in breast tumors from patients undergoing recurrence after TAM treatment. Together, these findings delineate a phosphorylation-dependent mechanism for MORC2 stabilization in response to estrogen and antiestrogens via blocking CMA-mediated lysosomal degradation and uncover a dual role for MORC2 in both estrogen-induced proliferation and resistance to antiestrogen therapies of breast cancer cells., Abbreviations: 4-OHT: 4-hydroxytamoxifen; Baf A1: bafilomycin A 1 ; CMA: chaperone-mediated autophagy; E2: 17β-estradiol; ESR: estrogen receptor; FUL: fulvestrant; GPER1: G protein-coupled estrogen receptor 1; HSPA8: heat shock protein family A (Hsp70) member 8; LAMP2A: lysosomal associated membrane protein 2A; MORC2: MORC family CW-type zinc finger 2; PRKACA: protein kinase cAMP-activated catalytic subunit alpha; TAM: tamoxifen; VCL: vinculin.

Background: Estrogen receptor-positive (ER+) breast cancers represent approximately two-thirds of all breast cancers and have a sustained risk of late disease recurrence. Cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitors have shown significant efficacy in ER+ breast cancer. However, their effects are still limited by drug resistance. In this study, we aim to explore the role of long noncoding RNA TROJAN in ER+ breast cancer., Methods: The expression level of TROJAN in breast cancer tissue and cell lines was determined by quantitative real-time PCR. In vitro and in vivo assays as well as patient derived organoid were preformed to explore the phenotype of TROJAN in ER+ breast cancer. The TROJAN-NKRF-CDK2 axis were screened and validated by RNA pull-down, mass spectrometry, RNA immunoprecipitation, microarray, dual-luciferase reporter and chromatin immunoprecipitation assays., Results: Herein, we showed that TROJAN was highly expressed in ER+ breast cancer. TROJAN promoted cell proliferation and resistance to a CDK4/6 inhibitor and was associated with poor survival in ER+ breast cancer. TROJAN can bind to NKRF and inhibit its interaction with RELA, upregulating the expression of CDK2. The inhibition of TROJAN abolished the activity of CDK2, reversing the resistance to CDK4/6 inhibitor. A TROJAN antisense oligonucleotide sensitized breast cancer cells and organoid to the CDK4/6 inhibitor palbociclib both in vitro and in vivo., Conclusions: TROJAN promotes ER+ breast cancer proliferation and is a potential target for reversing CDK4/6 inhibitor resistance.

MORC family CW-type zinc finger 2 (MORC2) is an oncogenic chromatin-remodeling enzyme with an emerging role in DNA repair. Here, we report a novel function for MORC2 in cell-cycle checkpoint control through an acetylation-dependent mechanism. MORC2 is acetylated by the acetyltransferase NAT10 at lysine 767 (K767Ac) and this process is counteracted by the deacetylase SIRT2 under unperturbed conditions. DNA-damaging chemotherapeutic agents and ionizing radiation stimulate MORC2 K767Ac through enhancing the interaction between MORC2 and NAT10. Notably, acetylated MORC2 binds to histone H3 phosphorylation at threonine 11 (H3T11P) and is essential for DNA damage-induced reduction of H3T11P and transcriptional repression of its downstream target genes CDK1 and Cyclin B1, thus contributing to DNA damage-induced G2 checkpoint activation. Chemical inhibition or depletion of NAT10 or expression of an acetylation-defective MORC2 (K767R) forces cells to pass through G2 checkpoint, resulting in hypersensitivity to DNA-damaging agents. Moreover, MORC2 acetylation levels are associated with elevated NAT10 expression in clinical breast tumor samples. Together, these findings uncover a previously unrecognized role for MORC2 in regulating DNA damage-induced G2 checkpoint through NAT10-mediated acetylation and provide a potential therapeutic strategy to sensitize breast cancer cells to DNA-damaging chemotherapy and radiotherapy by targeting NAT10., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Rationale : Chromodomain Y-like 2 (CDYL2) is a member of the CDY gene family involved in spermatogenesis, but its role in human cancer has not been reported. Analyses of publicly available databases demonstrate that CDYL2 is abundantly expressed in breast tumors. However, whether CDYL2 is involved in breast cancer progression remains unknown. Methods : Quantitative real-time PCR and immunoblotting assays were used to determine the expression levels of CDYL2 transcript variants in breast cancer cell lines and primary breast tumors. The effect of CDYL2 transcript variants on the malignant phenotypes of breast cancer cells was examined through in vitro and in vivo assays. Immunofluorescent staining, RNA-seq, ATAC-seq, and ChIP-qPCR were used to investigate the underlying mechanisms behind the aforementioned observations. Results : Here we show that CDYL2 generated four transcript variants, named CDYL2a-CDYL2d. CDYL2a and CDYL2b were the predominant variants expressed in breast cancer cell lines and breast tumors and exerted strikingly discrete functions in breast cancer growth and metastasis. CDYL2a was upregulated in the majority of the breast cancer cell lines and tumors, and promoted breast cancer cell proliferation, colony formation in vitro , and tumorigenesis in xenografts. In contrast, CDYL2b was mainly expressed in luminal- and HER2-positive types of breast cancer cell lines and tumors, and suppressed the migratory, invasive, and metastatic potential of breast cancer cells in vitro and in vivo . Mechanistically, CDYL2a partially localized to SC35-positive nuclear speckles and promoted alternative splicing of a subset of target genes, including FIP1L1, NKTR, and ADD3 by exon skipping. Elimination of full-length FIP1L1, NKTR, and ADD3 rescued the impaired cell proliferation through CDYL2a depletion. In contrast, CDYL2b localized to heterochromatin and transcriptionally repressed several metastasis-promoting genes, including HPSE, HLA-F, and SELL. Restoration of HPSE, HLA-F, or SELL expression in CDYL2b-overexpressing cells attenuated the ability of CDYL2b to suppress breast cancer cell migration and invasion. Conclusions : Collectively, these findings establish an isoform-specific function of CDYL2 in breast cancer development and progression and highlight that pharmacological inhibition of the CDYL2a, but not the CDYL2b, isoform may be an effective strategy for breast cancer therapy., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Deregulation of E3 ubiquitin ligases is intimately implicated in breast cancer pathogenesis and progression, but the underlying mechanisms still remain elusive. Here we report that RING finger protein 144A (RNF144A), a poorly characterized member of the RING-in-between-RING family of E3 ubiquitin ligases, functions as a tumor suppressor in breast cancer. RNF144A was  downregulated in a subset of primary breast tumors and restoration of RNF144A suppressed breast cancer cell proliferation, colony formation, migration, invasion in vitro, tumor growth, and lung metastasis in vivo. In contrast, knockdown of RNF144A promoted malignant phenotypes of breast cancer cells. Quantitative proteomics and biochemical analysis revealed that RNF144A interacted with and targeted heat-shock protein family A member 2 (HSPA2), a putative oncoprotein that is frequently upregulated in human cancer and promotes tumor growth and progression, for ubiquitination and degradation. Notably, the ligase activity-defective mutants of RNF144A impaired its ability to induce ubiquitination and degradation of HSPA2, and to suppress breast cancer cell proliferation, migration, and invasion as compared with its wild-type counterpart. Moreover, RNF144A-mediated suppression of breast cancer cell proliferation, migration, and invasion was rescued by ectopic HSPA2 expression. Clinically, low RNF144A and high HSPA2 expression in breast cancer patients was correlated with aggressive clinicopathological characteristics and decreased overall and disease-free survival. Collectively, these findings reveal a previously unappreciated role for RNF144A in suppression of breast cancer growth and metastasis, and identify RNF144A as the first, to our knowledge, E3 ubiquitin ligase for HSPA2 in human cancer.

The study aimed to investigate the regulatory effect of miR-146a in proliferation, invasion and migration of breast cancer and its possible mechanism via NM23-H1. The expression levels of miR-146a in breast cancer with different pathological classification were significantly increased, while the expression levels of NM23-H1 were significantly decreased, which were closely correlated. Double luciferase reporter gene was used to verify the target regulatory relationship between miR-146 and NM23-H1 on a human breast cancer cell line. miR-146a was closely related to the proliferation and metastasis of breast cancer. miR-146a also promoted the growth of breast cancer in vivo via targeting NM23-H1. In conclusion, miR-146 can promote the proliferation and invasion of breast cancer by targeting NM23-H1., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.)

The E3 ubiquitin ligase ring finger protein 115 (RNF115) is overexpressed in more than half of human breast tumors and is implicated in the pathogenesis and progression of breast cancer. However, the mechanism behind RNF115 overexpression in breast tumors remains largely unknown. Here we report that ubiquitin-specific protease 9X (USP9X), a substrate-specific deubiquitinating enzyme, stabilizes RNF115 and thereby regulates its biological functions in breast cancer cells. Immunoprecipitation and GST pull-down assays showed that USP9X interacted with RNF115. Depletion of RNF115 by siRNAs or overexpression of RNF115 did not significantly affect USP9X expression. In contrast, knockdown of USP9X in breast cancer cells by siRNAs reduced RNF115 protein abundance, which was partially restored following treatment with proteasome inhibitor MG-132. Moreover, depletion of USP9X reduced the half-life of RNF115 and increased its ubiquitination. Conversely, overexpression of USP9X resulted in an accumulation of RNF115 protein, accompanied by a decrease in its ubiquitination. RNF115 mRNA levels were unaffected by overexpression or knockdown of USP9X. Furthermore, USP9X protein expression levels correlated positively with RNF115 in breast cancer cell lines and breast tumor samples. Importantly, reintroduction of RNF115 in USP9X-depleted cells partially rescued the reduced proliferation, migration, and invasion of breast cancer cells by USP9X knockdown. Collectively, these findings indicate that USP9X is a stabilizer of RNF115 protein and that the USP9X-RNF115 signaling axis is implicated in the breast cancer malignant phenotype., (© 2019 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

The molecular underpinnings behind malignant progression of breast cancer from a localized lesion to an invasive and ultimately metastatic disease are incompletely understood. Here, we report that F-box only protein 22 (FBXO22) plays a dual role in mammary tumorigenesis and metastasis. FBXO22 was upregulated in primary breast tumors and promoted cell proliferation and colony formation in vitro and xenograft tumorigenicity in vivo Surprisingly, FBXO22 suppressed epithelial-mesenchymal transition (EMT), cell motility, and invasiveness in vitro and metastatic lung colonization in vivo Clinical data showed that expression levels of FBXO22 were associated with favorable clinical outcomes, supporting the notion that metastasis, rather than primary cancer, is the major determinant of the mortality of patients with breast cancer. Mechanistic investigations further revealed that FBXO22 elicits its antimetastatic effects by targeting SNAIL, a master regulator of EMT and breast cancer metastasis, for ubiquitin-mediated proteasomal degradation in a glycogen synthase kinase 3β phosphorylation-dependent manner. Importantly, expression of SNAIL rescued FBXO22-mediated suppression of EMT, cell migration, and invasion. A patient-derived tryptophan-to-arginine mutation at residue 52 (W52R) within the F-box domain impaired FBXO22 binding to the SKP1-Cullin1 complex and blocked FBXO22-mediated SNAIL degradation, thus abrogating the ability of FBXO22 to suppress cell migration, invasion, and metastasis. Collectively, these findings uncover an unexpected dual role for FBXO22 in mammary tumorigenesis and metastatic progression and delineate the mechanism of an oncogenic mutation of FBXO22 in breast cancer progression. Significance: These findings highlight the paradoxical roles of FBXO22 in breast cancer, as it promotes breast tumor cell proliferation but prevents EMT and metastasis. Cancer Res; 78(18); 5274-86. ©2018 AACR ., (©2018 American Association for Cancer Research.)

L-A03 is a dihydroartemisinin derivative and exerts distinct anti-tumor activity in vitro. Previous studies showed that induction of autophagy and deficiency in nitric oxide (NO) generation contributed to apoptotic cell death in L-A03-treated MCF-7 cells. However, the detailed mechanism is still unclear. In this study, the role of mitogen-activated protein kinases (MAPKs) in this apoptotic process was investigated. L-A03 (7.5-30 μM) selectively inhibited the activation of c-Jun N-terminal protein kinase (JNK) with no significant effect on extracellular signal related kinase (ERK) and p38. In addition, the possible mechanism of interaction between JNK and L-A03 was also investigated by molecular docking. In the presence of SP600125, a specific JNK inhibitor, induction of autophagy and apoptosis with L-A03 at 15 μM were elevated, but NO generation was attenuated, indicating that JNK inactivation is essential for apoptotic cell death. Interestingly, autophagy induction and NO generation did not affect the activation of JNK, demonstrating that JNK is upstream to autophagy and NO. Taken together, L-A03-induced JNK inactivation enhances autophagic and apoptotic cell death, but represses the generation of NO. This study provides a new insight on the mechanism of L-A03-induced cell death by targeting JNK., (Copyright © 2018 Elsevier Masson SAS. All rights reserved.)

Alternative splicing (AS) and its regulation play critical roles in cancer, yet the dysregulation of AS and its molecular bases in breast cancer development have not yet been elucidated. Using an in vivo CRISPR screen targeting RNA-binding proteins, we identified PHD finger protein 5A (PHF5A) as a key splicing factor involved in tumor progression. PHF5A expression was frequently upregulated in breast cancer and correlated with poor survival, and knockdown of PHF5A significantly suppressed cell proliferation, migration, and tumor formation. PHF5A was required for SF3b spliceosome stability and linked the complex to histones, and the PHF5A-SF3b complex modulated AS changes in apoptotic signaling. In addition, expression of a short truncated FAS-activated serine/threonine kinase (FASTK) protein was increased after PHF5A ablation and facilitated Fas-mediated apoptosis. This PHF5A-modulated FASTK-AS axis was widely present in breast cancer specimens, particularly those of the triple-negative subtype. Taken together, our findings reveal that PHF5A serves as an epigenetic suppressor of apoptosis and thus provides a mechanistic basis for breast cancer progression and may be a valuable therapeutic target. Significance: This study provides an epigenetic mechanistic basis for the aggressive biology of breast cancer and identifies a translatable therapeutic target. Cancer Res; 78(12); 3190-206. ©2018 AACR ., (©2018 American Association for Cancer Research.)

Emerging evidence shows that ring finger protein 144A (RNF144A), a poorly characterized member of the Ring-between-Ring (RBR) family of E3 ubiquitin ligases, is a potential tumor suppressor gene. However, its regulatory mechanism in breast cancer remains undefined. Here, we report that RNF144A promoter contains a putative CpG island and the methylation levels of RNF144A promoter are higher in primary breast tumors than those in normal breast tissues. Consistently, RNF144A promoter methylation levels are associated with its transcriptional silencing in breast cancer cells, and treatment with DNA methylation inhibitor 5-Aza-2-deoxycytidine (AZA) reactivates RNF144A expression in cells with RNF144A promoter hypermethylation. Furthermore, genetic knockdown or pharmacological inhibition of endogenous methyl-CpG-binding domain 4 (MBD4) results in increased RNF144A expression. These findings suggest that RNF144A is epigenetically silenced in breast cancer cells by promoter hypermethylation and MBD4., (© 2018 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.)

Distant metastasis to specific target organs is responsible for over 90% of breast cancer-related deaths, but the underlying molecular mechanism is unclear. Mounting evidence suggests that the interplay between breast cancer cells and the target organ microenvironment is the key determinant of organ-specific metastasis of this lethal disease. Here, we highlight new findings and concepts concerning the emerging role of the tumor microenvironment in breast cancer metastasis; we also discuss potential therapeutic intervention strategies aimed at targeting components of the tumor microenvironment.

Since the initial recognition of a mechanistic role of p21-activated kinase 1 (PAK1) in breast cancer invasion, PAK1 has emerged as one of the widely overexpressed or hyperactivated kinases in human cancer at-large, allowing the PAK family to make in-roads in cancer biology, tumorigenesis, and cancer therapeutics. Much of our current understanding of the PAK family in cancer progression relates to a central role of the PAK family in the integration of cancer-promoting signals from cell membrane receptors as well as function as a key nexus-modifier of complex, cytoplasmic signaling network. Another core aspect of PAK signaling that highlights its importance in cancer progression is through PAK's central role in the cross talk with signaling and interacting proteins, as well as PAK's position as a key player in the phosphorylation of effector substrates to engage downstream components that ultimately leads to the development cancerous phenotypes. Here we provide a comprehensive review of the recent advances in PAK cancer research and its downstream substrates in the context of invasion, nuclear signaling and localization, gene expression, and DNA damage response. We discuss how a deeper understanding of PAK1's pathobiology over the years has widened research interest to the PAK family and human cancer, and positioning the PAK family as a promising cancer therapeutic target either alone or in combination with other therapies. With many landmark findings and leaps in the progress of PAK cancer research since the infancy of this field nearly 20 years ago, we also discuss postulated advances in the coming decade as the PAK family continues to shape the future of oncobiology., (© 2016 Elsevier Inc. All rights reserved.)

Triple-negative breast cancer (TNBC) is characterized by the lack of expression of estrogen receptor-α (ER-α), progesterone receptor (PR), and human epidermal growth factor receptor-2 (HER-2). However, pathways responsible for downregulation of therapeutic receptors, as well as subsequent aggressiveness, remain unknown. In this study, we discovered that lactoferrin (Lf) efficiently downregulates levels of ER-α, PR, and HER-2 in a proteasome-dependent manner in breast cancer cells, and it accounts for the loss of responsiveness to ER- or HER-2-targeted therapies. Furthermore, we found that lactoferrin increases migration and invasiveness of both non-TNBC and TNBC cell lines. We discovered that lactoferrin directly stimulates the transcription of endothelin-1 (ET-1), a secreted proinvasive polypeptide that acts through a specific receptor, ET(A)R, leading to secretion of the bioactive ET-1 peptide. Interestingly, a therapeutic ET-1 receptor-antagonist blocked lactoferrin-dependent motility and invasiveness of breast cancer cells. The physiologic significance of this newly discovered Lf-ET-1 axis in the manifestation of TNBC phenotypes is revealed by elevated plasma and tissue lactoferrin and ET-1 levels in patients with TNBC compared with those in ER(+) cases. These findings describe the first physiologically relevant polypeptide as a functional determinant in downregulating all three therapeutic receptors in breast cancer, which uses another secreted ET-1 system to confer invasiveness. Results presented in this article provide proof-of-principle evidence in support of the therapeutic effectiveness of ET-1 receptor antagonist to completely block the lactoferrin-induced motility and invasiveness of the TNBC as well as non-TNBC cells, and thus, open a remarkable opportunity to treat TNBC by targeting the Lf-ET-1 axis using an approved developmental drug.

A novel highly metastatic MDA-MB-231HM cells, derived from MDA-MB-231, was established in our institute. RT-PCR, real-time PCR and Western blot showed that AF1Q gene was differentially expressed between highly metastatic MDA-MB-231HM cells and its parental MDA-MB-231 cells. However, its molecular mechanisms in breast cancer metastasis remain to be characterized. To investigate the effects of AF1Q on the progression of human breast cancer cells, in the present study, recombinant expression plasmid vectors of the human AF1Q gene was transfected into MDA-MB-231 cells. We demonstrated that AF1Q overexpression enhanced the in vitro proliferation and invasive potential of breast cancer cells. Focused microarray analyses showed that 22 genes were differentially expressed between AF1Q transfected cells and its parental counterparts. Integrin alpha3, accompanied by up-regulation of Ets-1 and MMP-2, significantly enhanced the in vitro invasive potential of human breast cancer cells mediated by AF1Q. Estrogen-responsive ring finger protein gene (EFP), also played a role in the enhancement of in vitro proliferation of human breast cancer cells mediated by AF1Q, accompanied by down-regulation of 14-3-3delta. The association was ERalpha independent. These results were further demonstrated by RNA interference (RNAi) experiment in vitro. In in vivo study, we also demonstrated that AF1Q transfected breast cancer cells grew much faster and had more pulmonary metastases than vector-transfected or its parental counterparts. On the contrary, AF1Q knockdown cells grew slower and had less pulmonary metastasis. Similar effects of AF1Q on integrin alpha3, Ets-1, MMP-2, EFP, and 14-3-3delta expression observed in vitro studies were also found in the in vivo study. Taken together, these results provide functional evidences that overexpression of AF1Q leads to a more progression in human breast cancer, at least in part, through regulating the integrin alpha3, Ets-1, MMP-2, EFP, and 14-3-3delta expression.

Introduction: The molecular mechanisms involved in breast cancer metastasis still remain unclear to date. In our previous study, differential expression of peroxiredoxin 6 was found between the highly metastatic MDA-MB-435HM cells and their parental counterparts, MDA-MB-435 cells. In this study, we investigated the effects of peroxiredoxin 6 on the proliferation and metastatic potential of human breast cancer cells and their potential mechanism., Methods: Expression of peroxiredoxin 6 in the highly metastatic MDA-MB-231HM cells was investigated by RT-PCR, real-time PCR and western blot. A recombinant expression plasmid of the human peroxiredoxin 6 gene was constructed and transfected into MDA-MB-231 and MDA-MB-435 cells. The effects of peroxiredoxin 6 on the proliferation and invasion of MDA-MB-231 and MDA-MB-435 cells were investigated by the Cell Counting Kit-8 method, colony-formation assay, adhesion assay, flow cytometry and invasion assay in vitro. miRNA was used to downregulate the expression of peroxiredoxin 6. Genes related to the invasion and metastasis of cancer were determined by RT-PCR, real-time PCR and western blot. The tumorigenicity and spontaneously metastatic capability regulated by peroxiredoxin 6 were determined using an orthotopic xenograft tumor model in athymic mice., Results: Overexpression of peroxiredoxin 6 in MDA-MB-231HM cells compared with their parental counterparts was confirmed. Upregulation of peroxiredoxin 6 enhanced the in vitro proliferation and invasion of breast cancer cells. The enhancement was associated with decreasing levels of tissue inhibitor of matrix metalloproteinase (TIMP)-2 and increasing levels of the urokinase-type plasminogen activator receptor (uPAR), Ets-1 (E26 transformation-specific-1), matrix metalloproteinase (MMP)-9 and RhoC (ras homolog gene family, member C) expression. The results were further demonstrated by RNA interference experiments in vitro. In an in vivo study, we also demonstrated that peroxiredoxin 6-transfected breast cancer cells grew much faster and had more pulmonary metastases than control cells. By contrast, peroxiredoxin 6 knockdown breast cancer cells grew more slowly and had fewer pulmonary metastases. Effects similar to those of peroxiredoxin 6 on the uPAR, Ets-1, MMP-9, RhoC and TIMP-2 expression observed in in vitro studies were found in the in vivo study., Conclusion: Overexpression of peroxiredoxin 6 leads to a more invasive phenotype and metastatic potential in human breast cancer, at least in part, through regulation of the levels of uPAR, Ets-1, MMP-9, RhoC and TIMP-2 expression.

To study the molecular mechanisms underlying breast cancer metastasis, gene expression profile analysis was performed on two well-established breast cancer cell lines with high and low metastatic potentials: MDA-MB-435HM and MDA-MB-435LM. The analysis was conducted using cDNA microarrays containing 8000 genes. Of 60 differentially expressed genes, ALL1-fused gene from chromosome 1q (AF1Q), a putative oncogene not described previously in breast cancer, was identified and found to be over-expressed in MDA-MB-435HM cells compared with MDA-MB-435LM cells. The results indicate that AF1Q may play an important role in breast cancer metastasis. To test this hypothesis, we generated an AF1Q high-expression cell line by stable transfection of AF1Q cDNA into MDA-MB-435LM cells. Results showed that over-expression of AF1Q led to a marked increase in the invasive and metastatic potential of MDA-MB-435LM cells in vitro and in vivo, accompanied by the up-regulation of matrix metalloproteinase-2 (MMP-2), MMP-9, transcription factor Ets-1, and RhoC expression in both mRNA and protein levels. Consistent with this observation, reduced AF1Q expression in MDA-MB-435HM cells by small interfering RNA (siRNA) resulted in a significant decrease in the invasive potential of MDA-MB-435HM cells in vitro and in the protein expression of MMP-2, MMP-9, Ets-1, and RhoC, compared with either parental or non-silencing control cells. These data provide functional evidence that oncogene AF1Q may be a novel mediator of metastasis promotion in human breast cancer through regulation of the MMP pathway and RhoC expression.