SGCE promotes breast cancer stemness by promoting the transcription of FGF-BP1 by Sp1.

Breast cancer stem cells are mainly responsible for poor prognosis, especially in triple-negative breast cancer (TNBC). In a previous study, we demonstrated that ε-Sarcoglycan (SGCE), a type Ⅰ single-transmembrane protein, is a potential oncogene that promotes TNBC stemness by stabilizing EGFR. Here, we further found that SGCE depletion reduces breast cancer stem cells, partially through inhibiting the transcription of FGF-BP1, a secreted oncoprotein. Mechanistically, we demonstrate that SGCE could interact with the specific protein 1 transcription factor and translocate into the nucleus, which leads to an increase in the transcription of FGF-BP1, and the secreted FBF-BP1 activates FGF-FGFR signaling to promote cancer cell stemness. The novel SGCE-Sp1-FGF-BP1 axis provides novel potential candidate diagnostic markers and therapeutic targets for TNBC.

Angiopoietin-1 promotes triple-negative breast cancer cell proliferation by upregulating carboxypeptidase A4.

Angiopoietin-1 (ANG1) is a pro-angiogenic regulator that contributes to the progression of solid tumors by stimulating the proliferation, migration and tube formation of vascular endothelial cells, as well as the renewal and stability of blood vessels. However, the functions and mechanisms of ANG1 in triple-negative breast cancer (TNBC) are unclear. The clinical sample database shows that a higher level of ANG1 in TNBC is associated with poor prognosis compared to non-TNBC. In addition, knockdown of ANG1 inhibits TNBC cell proliferation and induces cell cycle G1 phase arrest and apoptosis. Overexpression of ANG1 promotes tumor growth in nude mice. Mechanistically, ANG1 promotes TNBC by upregulating carboxypeptidase A4 (CPA4) expression. Overall, the ANG1-CPA4 axis can be a therapeutic target for TNBC.

USP3 promotes breast cancer cell proliferation by deubiquitinating KLF5.

The Krüppel-like factor 5 (KLF5) transcription factor is highly expressed in basal type breast cancer and promotes breast cancer cell proliferation, survival, migration, and tumorigenesis. KLF5 protein stability is regulated by ubiquitination. In this study, ubiquitin-specific protease 3 (USP3) was identified as a new KLF5 deubiquitinase by genome-wide siRNA library screening. We demonstrated that USP3 interacts with KLF5 and stabilizes KLF5 via deubiquitination. USP3 knockdown inhibits breast cancer cell proliferation in vitro and tumorigenesis in vivo, which can be partially rescued by ectopic expression of KLF5. Furthermore, we observed a positive correlation between USP3 and KLF5 protein expression levels in human breast cancer samples. These findings suggest that USP3 is a new KLF5 deubiquitinase and that USP3 may represent a potential therapeutic target for breast cancer.

CUL7 promotes cancer cell survival through promoting Caspase-8 ubiquitination.

The Cullin 7 (CUL7) gene encodes a member of the cullin family of E3 ubiquitin ligases. Accumulated evidence suggests that CUL7 is oncogenic. However, the mechanism by which CUL7 improves cancer cell survival has not been fully elucidated. Here, we reported that CUL7 confers anti-apoptotic functions by interacting with Caspase-8. CUL7 prevents Caspase-8 activation by promoting Caspase-8 modification with non-degradative polyubiquitin chains at K215. CUL7 knockdown sensitized cancer cells to TRAIL-induced apoptosis in vitro and in nude mice. These results suggest that CUL7 limits extrinsic apoptotic signaling by promoting Caspase-8 ubiquitination.

Krüpple-like factor 5 is essential for mammary gland development and tumorigenesis.

Krüpple-like factor 5 (KLF5) is required for the development of the embryo and multiple organs, such as the lung and intestine. KLF5 plays a pro-proliferative and oncogenic role in several carcinomas, including breast cancer. However, its role in normal mammary gland development and oncogenesis has not been elucidated in vivo. In this study, we used mammary gland-specific Klf5 conditional knockout mice derived by mating Klf5-LoxP and MMTV-Cre mice. The genetic ablation of Klf5 suppresses mammary gland ductal elongation and lobuloalveolar formation. Klf5 deficiency inhibits mammary epithelial cell proliferation, survival, and stem cell maintenance. Klf5 promotes mammary stemness, at least partially, by directly promoting the transcription of Slug. Finally, Klf5 depletion suppressed PyMT-induced mammary gland tumor cell stemness, tumor initiation, and growth in vivo. Slug also mediated these functions of Klf5 in vivo. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

miR-153 inhibits the migration and the tube formation of endothelial cells by blocking the paracrine of angiopoietin 1 in breast cancer cells.

The sprouting of endothelial cells is the first step of tumor angiogenesis. Our previous study suggests that miR-153 suppresses breast tumor angiogenesis partially through targeting hypoxia-induced factor (HIF1α). In this study, we demonstrated that miR-153 also suppresses the migration and the tube formation of endothelial cells through directly targeting angiopoietin 1 (ANG1) in breast cancer cells. There was a negative correlation between miR-153 and ANG1 levels in breast cancer. miR-153 blocked the expression and secretion of ANG1 in breast cancer cells through binding to ANG1 mRNA. Conditioned medium from the breast cancer cell, MCF7, treated with miR-153 had no effect on the proliferation of HUVECs, but significantly inhibited the migration and tube formation of HUVECs, which could be rescued by overexpression of ANG1. In addition, miR-153 also directly inhibited the proliferation and migration of MCF7 through downregulation of ANG1. These findings suggest that miR-153 suppresses the activity of tumor cells and the migration and tube formation of endothelial cells by silencing ANG1.

CC chemokine receptor 7 promotes triple-negative breast cancer growth and metastasis.

Metastasis is the leading cause of breast cancer-related death. Chemokine (C-C motif) receptor 7 (CCR7) plays important roles in breast cancer metastasis. However, the role of CCR7 in triple-negative breast cancer (TNBC) has not been fully elucidated. In this study, we found that CCR7 is highly expressed in both TNBC cell lines and breast cancer tissues. CCR7 was knocked down by shRNA in 4T1 and MDA-MB-231, two TNBC cell lines, and we found that the depletion of CCR7 significantly decreased TNBC cell proliferation, migration and invasion in vitro. Furthermore, we confirmed that the knockdown of CCR7 reduced the distant metastasis of 4T1 cells in an orthotopic mouse model. Proteomic analysis in 4T1 cells indicated that several signaling pathways such as epithelial cell adhesion molecule might contribute to CCR7's function in breast cancer metastasis. Our results suggest that CCR7 promotes TNBC metastasis and may serve as a target for breast cancer diagnosis and treatment.

Transforming growth factor-beta increases breast cancer stem cell population partially through upregulating PMEPA1 expression.

The prostate transmembrane protein, androgen-induced 1 (PMEPA1) has been previously shown to promote solid malignancies in a variety of cancers, but the role and mechanisms of PMEPA1 in breast cancer has not been fully addressed. Here, we found that PMEPA1 was upregulated in breast cancer cell lines as well as in a set of clinical invasive breast ductal carcinomas. Interestingly, depletion of PMEPA1 decreased breast cancer stem cell (CSC)-enriched populations, while ectopic overexpression of PMEPA1 increased breast CSC-enriched populations. Furthermore, transforming growth factor-ß (TGF-ß) treatment was also found to upregulate PMEPA1 expression and the CSC-enriched populations in triple-negative breast cancer cell lines. TGF-ß-induced PMEPA1 expression partially contributed to TGF-ß-induced breast CSC maintenance. These findings suggest that TGF-ß-PMEPA1 axis might provide new diagnosis and therapeutic targets for breast cancer treatment.

Kruppel-like factor 5 transcription factor promotes microsomal prostaglandin E2 synthase 1 gene transcription in breast cancer.

The KLF5 (Krüppel-like factor 5) transcription factor is specifically expressed in a subset of estrogen receptor α-negative breast cancers. Although KLF5 promotes breast cancer cell cycle progression, survival, and tumorigenesis, the mechanism by which KLF5 promotes breast cancer is still not entirely understood. Here, we demonstrate that mPGES1, encoding microsomal prostaglandin E2 synthase 1 (mPGES1), is a KLF5 direct downstream target gene. KLF5 overexpression or knockdown positively altered the levels of mPGES1 mRNA and protein in multiple breast cell lines. 12-O-Tetradecanoylphorbol-13-acetate induced the expression of both KLF5 and mPGES1 in dosage- and time-dependent manners. The induction of KLF5 was essential for 12-O-tetradecanoylphorbol-13-acetate to induce mPGES1 expression. Additionally, KLF5 bound to the mPGES1 gene proximal promoter and activated its transcription. Both KLF5 and mPGES1 promoted prostaglandin E2 production; regulated p21, p27, and Survivin downstream gene expression; and likewise stimulated cell proliferation. Overexpression of mPGES1 partially rescued the KLF5 knockdown-induced downstream gene expression changes and growth arrest in MCF10A cells. Finally, we demonstrate that the expression of mPGES1 was positively correlated with the estrogen receptor α/progesterone receptor/HER2 triple-negative status. These findings suggest that mPGES1 is a target gene of KLF5, making it a new biomarker and a potential therapeutic target for triple-negative breast cancers.

Classification based on pruning and double covered rule sets for the internet of things applications.

The Internet of things (IOT) is a hot issue in recent years. It accumulates large amounts of data by IOT users, which is a great challenge to mining useful knowledge from IOT. Classification is an effective strategy which can predict the need of users in IOT. However, many traditional rule-based classifiers cannot guarantee that all instances can be covered by at least two classification rules. Thus, these algorithms cannot achieve high accuracy in some datasets. In this paper, we propose a new rule-based classification, CDCR-P (Classification based on the Pruning and Double Covered Rule sets). CDCR-P can induce two different rule sets A and B. Every instance in training set can be covered by at least one rule not only in rule set A, but also in rule set B. In order to improve the quality of rule set B, we take measure to prune the length of rules in rule set B. Our experimental results indicate that, CDCR-P not only is feasible, but also it can achieve high accuracy.

WWOX suppresses KLF5 expression and breast cancer cell growth.

The WW domain-containing oxidoreductase (WWOX) is a tumor suppressor in a variety of cancers, including breast cancer. Reduced WWOX expression is associated with the basal-like subtype and a relatively poor disease-free survival rate among breast cancer patients. Though several WWOX partners have been identified, the functional mechanisms of WWOX's role in cancers have not been fully addressed to date. In the current study, we found WWOX suppresses expression of KLF5-an oncogenic transcription factor-at protein level, and suppresses cancer cell proliferation in both bladder and breast cancer cell lines. Furthermore, we demonstrated that WWOX physically interacts with KLF5 via the former's WW domains and the latter's PY motifs. Interestingly, we found the expression of WWOX negatively correlates with KLF5 expression in a panel of breast cancer cell lines. Taken together, we conjecture that WWOX may suppress cancer cell proliferation partially by reducing the expression of KLF5.

YAP promotes breast cell proliferation and survival partially through stabilizing the KLF5 transcription factor.

The Yes-associated protein (YAP), an oncoprotein in the Hippo tumor suppressor pathway, regulates tumorigenesis and has been found in a variety of tumors, including breast, ovarian, and hepatocellular cancers. Although YAP functions through its WW domains, the YAP WW domain-binding partners have not yet been completely determined. With this study, we demonstrate that YAP functions partially through its binding to KLF5, a transcription factor that promotes breast cell proliferation and survival. YAP interacted with the KLF5 PY motif through its WW domains, preventing the E3 ubiquitin ligase WWP1 from ubiquitinating KLF5. Overexpression of the wild-type YAP but not the WW domain-mutated YAP up-regulated KLF5 protein levels and mRNA expression levels of KLF5 downstream target genes, including FGFBP1 (alias FGF-BP) and ITGB2. In addition, knockdown of YAP decreased expression levels of KLF5, FGF-BP, and ITGB2. Depletion of either YAP or KLF5 decreased breast cell proliferation and survival in MCF10A and SW527 breast cell lines, and stable knockdown of either YAP or KLF5 suppressed SW527 xenograft growth in mice. The YAP upstream kinase LATS1 suppressed the KLF5-FGF-BP axis, as well as cell growth through YAP signaling. Both YAP and KLF5 are coexpressed in estrogen receptor ERα-negative breast cell lines. These findings suggest that KLF5 could be an important transcription factor partner for YAP and may contribute to the Hippo pathway.

RNF126-Mediated MRE11 Ubiquitination Activates the DNA Damage Response and Confers Resistance of Triple-Negative Breast Cancer to Radiotherapy.

Triple-negative breast cancer (TNBC) has higher molecular heterogeneity and metastatic potential and the poorest prognosis. Because of limited therapeutics against TNBC, irradiation (IR) therapy is still a common treatment option for patients with lymph nodes or brain metastasis. Thus, it is urgent to develop strategies to enhance the sensitivity of TNBC tumors to low-dose IR. Here, the authors report that E3 ubiquitin ligase Ring finger protein 126 (RNF126) is important for IR-induced ATR-CHK1 pathway activation to enhance DNA damage repair (DDR). Mechanistically, RNF126 physically associates with the MRE11-RAD50-NBS1 (MRN) complex and ubiquitinates MRE11 at K339 and K480 to increase its DNA exonuclease activity, subsequent RPA binding, and ATR phosphorylation, promoting sustained DDR in a homologous recombination repair-prone manner. Accordingly, depletion of RNF126 leads to increased genomic instability and radiation sensitivity in both TNBC cells and mice. Furthermore, it is found that RNF126 expression is induced by IR activating the HER2-AKT-NF-κB pathway and targeting RNF126 expression with dihydroartemisinin significantly improves the sensitivity of TNBC tumors in the brain to IR treatment in vivo. Together, these results reveal that RNF126-mediated MRE11 ubiquitination is a critical regulator of the DDR, which provides a promising target for improving the sensitivity of TNBC to radiotherapy.

TAZ antagonizes the WWP1-mediated KLF5 degradation and promotes breast cell proliferation and tumorigenesis.

Krüppel-like factor 5 (KLF5) is a PY motif-containing transcription factor promoting breast cell proliferation. The KLF5 protein is rapidly degraded through the proteasome after ubiquitination by E3 ubiquitin ligases, such as WWP1 and SCF(Fbw7). In this study, we demonstrated that a transcriptional co-activator with the PDZ-binding motif (TAZ) upregulated the KLF5 expression through antagonizing the WWP1-, but not Fbw7-, mediated KLF5 ubiquitination and degradation. TAZ interacted with KLF5 through the WW domain of TAZ and the PY motif of KLF5, which is the binding site for WWP1. TAZ inhibited WWP1-KLF5 protein interaction and WWP1-mediated KLF5 ubiquitination and degradation in a WW domain-dependent manner. Overexpression of TAZ upregulated the protein levels of KLF5 and FGF-BP, which is a well-established KLF5 target gene. In addition, depletion of TAZ in both 184A1 and HCC1937 breast cells downregulated protein levels of KLF5 and FGF-BP and inhibited cell growth. Furthermore, stable depletion of either TAZ or KLF5 significantly suppressed HCC1937 xenograft growth in immunodeficient mice. Knockdown of LATS1, a TAZ upstream inhibitory kinase, up-regulated the protein levels of KLF5 and FGF-BP in 184A1 and promoted cell growth through TAZ. Finally, both KLF5 and TAZ were co-expressed in a subset of estrogen receptor α-negative breast cell lines. These results, for the first time, suggest that TAZ promotes breast cell growth partially through protecting KLF5 from WWP1-mediated degradation and enhancing KLF5's activities.

The WWP1 ubiquitin E3 ligase increases TRAIL resistance in breast cancer.

WW domain containing E3 ubiquitin protein ligase 1 (WWP1) is an HECT domain-containing E3 ligase regulating apoptosis. The WWP1 gene is frequently amplified and overexpressed in estrogen receptor α (ERα)-positive breast cancer. Inhibition of WWP1 by siRNA induced apoptosis in MCF7 and HCC1500. In our study, we demonstrate that WWP1 depletion by siRNA activated the extrinsic apoptotic pathway. WWP1 depletion-induced apoptosis was rescued by the overexpression of the wild-type WWP1 but not the E3 ligase inactive WWP1-C890A mutant in MCF7 cells. In contrast, WWP1-C890A enhanced apoptosis, suggesting that the E3 ligase activity is required for WWP1 to promote cell survival. The expression levels of WWP1 in four breast cancer cell lines were specifically correlated with the tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) resistance, but not TNFα and doxorubicin resistance. Both WWP1 depletion and dominant negative WWP1 overexpression increased the TRAIL-induced caspase-8 recruitment and apoptosis although WWP1 did not regulate FLIP and death receptor levels. Depletion of the initial caspase-8 blocked WWP1 inhibition-induced apoptosis in MCF7. These findings suggest that inhibition of WWP1 may be combined with TRAIL to suppress ERα-positive breast cancer cell survival.

PMEPA1 promotes androgen receptor-negative prostate cell proliferation through suppressing the Smad3/4-c-Myc-p21 Cip1 signaling pathway.

The PMEPA1 gene has been shown to suppress the androgen receptor (AR) and TGFß signaling pathways and is abnormally expressed in prostate tumours. However, the role and mechanism action of PMEPA1 in AR-negative prostate cancer are unclear. Here, we demonstrate that inhibition of PMEPA1 suppresses AR-negative RWPE1 and PC-3 prostate cell proliferation through up-regulating the p21 transcription. Additionally, PMEPA1 overexpression suppresses the p21 expression and promotes cell proliferation. PMEPA1 is induced by TGFß as a negative feedback loop to suppress Smad3 phosphorylation and nuclear translocation; up-regulates c-Myc; down-regulates p21; and promotes PC-3 cell proliferation. The PMEPA1 functions depend on its Smad2/3 binding motif. Consistently, depletion of Smad3/4, but not Smad2, blocks PMEPA1's functions of regulating c-Myc and p21. Importantly, stable depletion of PMEPA1 in PC-3 inhibits xenograft growth. Finally, we found that PMEPA1 is overexpressed in a subset of prostate cancer cell lines and tumours. These findings suggest that PMEPA1 may promote AR-negative prostate cancer cell proliferation through p21.

STAMBPL1 promotes breast cancer cell resistance to cisplatin partially by stabilizing MKP-1 expression.

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.

Human fibroblasts facilitate the generation of iPSCs-derived mammary-like organoids.

BACKGROUND: Breast cancer is the most common malignancy in women worldwide, and its treatment largely depends on mastectomy. Patients after mastectomy suffer from crippled body image, self-esteem, and quality of life. Post-mastectomy breast reconstruction can improve patients' psychosocial health. Although silicone and fat have been widely used for breast reconstruction, they have remarkable limitations. Our study aimed to establish an improved method for breast reconstruction from human-induced pluripotent stem cells (iPSCs). METHODS: We used a two-step procedure to induce mammary-like organoids (MLOs) from iPSCs and applied transcriptome sequencing to analyze the gene expression profiles during the development process from embryoid bodies (mEBs) to MLOs. Moreover, we evaluated the in vitro effect of fibroblasts cell line HFF (human foreskin fibroblasts) on the size and morphology of MLOs and explored the in vivo effect of HFF on regeneration rate of MLOs. RESULTS: MLOs had a similar gene expression profile and morphogenesis as the normal mammary glands. Furthermore, the addition of HFF increases the branching ratio and organoid diameters and facilitates the formation of multiple cell layers duct-like structures in MLOs in vitro. Finally, orthotopical transplantation of the MLOs to cleared mammary gland fad pad of NSG mice showed that HFF increases the formation of mammary gland-like structures. CONCLUSIONS: Fibroblasts facilitate iPSC-derived MLOs to generate mammary gland-like structures in both in vitro and in vivo conditions. Our findings lay a foundation for breast reconstruction by using iPSCs.

YB-1 is a positive regulator of KLF5 transcription factor in basal-like breast cancer.

Y-box binding protein 1 (YB-1) is a well-known oncogene highly expressed in various cancers, including basal-like breast cancer (BLBC). Beyond its role as a transcription factor, YB-1 is newly defined as an epigenetic regulator involving RNA 5-methylcytosine. However, its specific targets and pro-cancer functions are poorly defined. Here, based on clinical database, we demonstrate a positive correlation between Kruppel-like factor 5 (KLF5) and YB-1 expression in breast cancer patients, but a negative correlation with that of Dachshund homolog 1 (DACH1). Mechanistically, YB-1 enhances KLF5 expression not only through transcriptional activation that can be inhibited by DACH1, but also by stabilizing KLF5 mRNA in a RNA 5-methylcytosine modification-dependent manner. Additionally, ribosomal S6 kinase 2 (RSK2) mediated YB-1 phosphorylation at Ser102 promotes YB-1/KLF5 transcriptional complex formation, which co-regulates the expression of BLBC specific genes, Keratin 16 (KRT16) and lymphocyte antigen 6 family member D (Ly6D), to promote cancer cell proliferation. The RSK inhibitor, LJH685, suppressed BLBC cell tumourigenesis in vivo by disturbing YB-1-KLF5 axis. Our data suggest that YB-1 positively regulates KLF5 at multiple levels to promote BLBC progression. The novel RSK2-YB-1-KLF5-KRT16/Ly6D axis provides candidate diagnostic markers and therapeutic targets for BLBC.

A feedforward circuit between KLF5 and lncRNA KPRT4 contributes to basal-like breast cancer.

Basal-like breast cancer (BLBC) is the most aggressive subtype of breast cancer with a poor prognosis. Long noncoding RNAs (lncRNAs) play critical roles in human cancers. Krüppel-like Factor 5 (KLF5) is a key oncogenic transcription factor in BLBC. However, the underlying mechanism of mutual regulation between KLF5 and lncRNA remains largely unknown. Here, we demonstrate that lncRNA KPRT4 promotes BLBC cell proliferation in vitro and in vivo. Mechanistically, KLF5 directly binds to the promoter of KPRT4 to promote KPRT4 transcription. Reciprocally, KPRT4 recruits the YB-1 transcription factor to the KLF5 promoter by interacting with YB-1 at its 5' domain and forming an RNA-DNA-DNA triplex structure at its 3' domain, resulting in enhanced transcription of KLF5 and ultimately establishing a feedforward circuit to promote cell proliferation. Moreover, the antisense oligonucleotide (ASO)-based therapy targeting KPRT4 substantially attenuated tumor growth in vivo. Clinically, the expression levels of YB-1, KLF5 and KPRT4 are positively correlated in clinical breast specimens. Together, our data suggest that KPRT4 is a major molecule for BLBC progression and that the feedforward circuit between KLF5 and KPRT4 may represent a potential therapeutic target in BLBC.