SHOC2 mediates the drug-resistance of triple-negative breast cancer cells to everolimus.

Aberrant activation of the mTOR pathway is a characteristic alteration in triple-negative breast cancer, but the mTOR pathway inhibitor everolimus is not effective for the triple-negative breast cancer (TNBC) patients. Presently, we showed that the activation of ERK pathway was an important mechanism of resistance to everolimus in TNBC cells in this study. SHOC2, a key protein mediating the Ras-Raf-ERK pathway, could act as a scaffolding protein to facilitate the activation of the pathway by mediating the interaction of key components of the pathway. Our results showed that everolimus activated the Raf-ERK pathway by promoting the interaction between SHOC2 and c-Raf and that knockdown of SHOC2 significantly inhibited the Raf-ERK pathway induced by everolimus. We further demonstrated that SHOC2 expression levels were closely related to the sensitivity of TNBC cells to everolimus and that interference with SHOC2 expression in combination with everolimus had significant effects on the cell cycle progression and apoptosis in vitro experiments. Western blotting analysis showed that cell cycle regulators and apoptosis-related proteins were significantly altered by the combination treatment. Xenograft model also demonstrated that knockdown of SHOC2 significantly increased the sensitivity of tumor to everolimus in nude mice. In conclusion, our study showed that SHOC2 is a key factor in regulating the sensitivity of TNBC cells to everolimus and that combined therapy may be a more effective therapeutic approach for TNBC patients.

Crouzon syndrome complicated with binocular strabismus and extraocular muscle fibrosis: a case report.

BACKGROUND: Crouzon syndrome, a rare genetic disorder characterized by premature closure of coronal sutures, results in skull and facial deformities along with abnormal brain and ocular development. CASE PRESENTATION: Here, we report a case of a 27-year-old ethnic han male patient who presented with complex binocular strabismus secondary to Crouzon syndrome. At the time of surgery, extraocular muscles were found to be fibrotic and results of the pathological examination revealed degeneration of muscle fibers, which were replaced by adipose tissue. The entire exome sequencing DNA testing indicated that the patient and his father possessed the fibroblast growth factor receptor 2 (FGFR2) gene c.G812T:p.G271V heterozygous mutation. Binocular strabismus corrective surgery was performed in this patient with a satisfactory outcome. CONCLUSIONS: This case demonstrates that Crouzon syndrome patients can show an FGFR2 gene c.G812T:p.G271V mutation and display clinical symptoms such as extraocular muscle fibrosis, exotropia, exophthalmos, and a pointed head deformity.

19q13.12 KRAB zinc-finger protein ZNF383 represses p53 signaling pathway by interacting with p53.

As one of the most important tumor suppressors, the activity of p53 is precisely regulated. However, the mechanism of p53 regulation is still being elucidated and new regulatory molecules for p53 have also been frequently identified. Our previous works revealed that two members of the KRAB zinc-finger protein (KZFP) family Apak and PISA, which are located on human 19q13.12, participated in the regulation of p53 signaling pathway. KZFPs genes are mainly amplified via tandem in situ duplication during evolution, which indicates that similar sequences and functions may be conserved in evolutionarily and physically close KZFPs. Here, we revealed that ZNF383, another member of the KZFPs mapped at 19q13.12, could inhibit p53-mediated apoptosis and the activation of IFN-ß pathway by decreasing the H3K36me2 level at p53's binding sites and the attenuating the binding of p53 to its target genes. We further explored the effect of other KZFPs clustered on 19q13.12 on p53, and found that 85% of these KZFPs exerted p53-repressive activity. Intriguingly, an acidic amino acid-enriched sequence, the SAcL motif in the zinc-finger domains of these KZFPs, was found to be critical for p53 binding. Taken together, our findings revealed the KZFPs cluster located at 19q13.12 not only was involved in p53 regulation but also exhibited different features in the selective regulation of p53 and functional mechanisms, and for the first time, confirmed a motif in KZFPs that mediates the interaction of KZFPs and p53. These results would enrich the knowledge on the role, sequence features, and functional mechanisms of the KZFP family in p53 regulation.

Gene Signatures and Cancer-Immune Phenotypes Based on m6A Regulators in Breast Cancer.

The N6-methyladenosine (m6A) has been considered as a new layer of epitranscriptomic regulation on mRNA processing, stability, and translation. However, potential roles of m6A RNA methylation modification in tumor immune microenvironment (TIME) of breast cancer are yet fully understood. In this study, we comprehensively evaluated the genetic variations and transcript expressions of 15 m6A regulators in 1,079 breast cancer samples from the Cancer Genome Atlas (TCGA) database. We validated major regulators had significantly differential mRNA and protein expression in tumor tissue compared to normal tissues from 39 pairs of clinical breast cancer samples with different molecular subtypes, and especially high expression of m6A readers YTHDF1 and YTHDF3 predicted poor survival. Two clusters of breast cancer patients identified by the 15 m6A regulators' pattern showed distinct overall survival, immune activation status, and immune cell infiltration, and clinical samples confirmed the diversity of lymphocytic infiltration. The profiles of these two clusters accorded with that of two classical cancer-immune phenotypes, immune-excluded and immune-inflamed phenotypes, it suggested that m6A regulators-based patterns might serve as crucial mediators of TIME in breast cancer. Moreover, the m6A phenotype-related gene signatures could also be survival predictor in breast cancer. Therefore, comprehensive evaluation of tumor m6A modification pattern will contribute to enhance our understanding of the characterization of immune cell infiltration in the tumor microenvironment and promote the responsiveness of breast cancer to immunotherapy.

miR-520h Stimulates Drug Resistance to Paclitaxel by Targeting the OTUD3-PTEN Axis in Breast Cancer.

MicroRNAs (miRNAs) have been identified as negative posttranscriptional regulators of target genes and are involved directly in the pathological processes of tumors, including drug resistance. However, the exact function of miR-520h in breast cancer remains poorly understood. The aim of this study was to investigate the molecular mechanisms of miR-520h in paclitaxel resistance in the MCF-7 breast cancer cell line. Ectopic expression of miR-520h could promote the proliferation of breast cancer cells and inhibit paclitaxel-induced cell apoptosis. Inhibiting the expression of miR-520h could enhance the sensitivity to paclitaxel in paclitaxel-resistant MCF-7/Taxol cells. Furthermore, luciferase reporter assays showed that OTUD3 was a direct target of miR-520h. OTUD3 plays a necessary role in the paclitaxel resistance effect of miR-520h, and cotreatment with a miR-520h inhibitor and OTUD3 overexpression significantly enhanced MCF-7 cell sensitivity to paclitaxel. Moreover, miR-520h substantially inhibited the protein expression of PTEN via OTUD3 and subsequently affected downstream p-AKT pathway activity. In a clinical study, we also found that high miR-520h expression was associated with more aggressive pathological characteristic and poor prognosis. Therefore, our findings showed that miR-520h targeted the OTUD3-PTEN axis to drive paclitaxel resistance, and this miR might be an important potential target for breast cancer treatment.

Tumor suppressor OTUD3 induces growth inhibition and apoptosis by directly deubiquitinating and stabilizing p53 in invasive breast carcinoma cells.

BACKGROUND: P53 pathway inactivation plays an important role in the process of breast cancer tumorigenesis. Post-translational protein modification abnormalities have been confirmed to be an important mechanism underlying inactivation of p53. Numerous deubiquitinating enzymes are aberrantly expressed in breast cancer, and a few deubiquitination enzymes can deubiquitinate and stabilize p53. Here, we report that ovarian tumor (OTU) deubiquitinase 3 (OTUD3) is a deubiquitylase of p53 in breast carcinoma (BC). METHODS: Correlations between the mRNA expression levels of OTUD3, TP53 and PTEN and the prognosis of BC were assessed with the Kaplan-Meier Plotter tool. OTUD3 protein expression in 80 pairs of specimens in our cohort was examined by immunohistochemistry and western blotting. The relationship among OTUD3, p53, and p21 proteins was analyzed. Half-life analysis and ubiquitylation assay were performed to elucidate the molecular mechanism by which OTUD3 stabilizes p53. The interaction between OTUD3 and p53 in BC cells was verified by a co-immunoprecipitation assay and GST pulldown experiments. MTS assay for proliferation detection, detection of apoptosis induced by cisplatin and colony formation assay were employed to investigate the functional effects of OTUD3 on breast cancer cells. RESULTS: OTUD3 downregulation is correlated with a poor prognosis in BC patients. OTUD3 expression is decreased in breast cancer tissues and not associated with the histological grade. OTUD3 also inhibits cell proliferation and clone formation and increases the sensitivity of BC cells to apoptosis induced by chemotherapy drugs. Reduced OTUD3 expression accompanied by decreased p53 abundance is correlated with human breast cancer progression. Ectopic expression of wild-type OTUD3, but not its catalytically inactive mutant, stabilizes and activates p53. Mechanistically, OTUD3 interacts directly with p53 through the amino-terminal OTU region. Finally, OTUD3 protects p53 from murine double minute 2 (Mdm2)-mediated ubiquitination and degradation, enabling the deubiquitination of p53 in BC cells. CONCLUSIONS: In summary, we found that OTUD3 may be a potential therapeutic target for restoring p53 function in breast cancer cells and suggest that the OTUD3-p53 signaling axis may play a critical role in tumor suppression.

The Novel Transcription Factor CREB3L4 Contributes to the Progression of Human Breast Carcinoma.

Breast carcinoma(BC)is the most common cancer type among females globally. Understanding the molecular pathways that trigger the development of BC is crucial for both prevention and treatment. As such, the role of transcription factors (TFs) in the development of BC is a focal point in this field. CREB3s play a critical role in initiating the unfolded protein response (UPR); however, the role of CREB3 family members in breast cancer development remains largely unknown. Here, we mined the ONCOMINE database for the transcriptional data of CREB3s in patients with BC. Then, the regulatory functions of a novel TF, CREB3L4, were investigated. CREB3L4 knockdown in MDA-MB-231 and MCF-7 cells suppressed proliferation and promoted apoptosis and cell cycle arrest. ChIP assays confirmed that CREB3L4 can directly bind to the PCNA promoter region, suggesting that the PCNA protein may be functionally downstream of CREB3L4. Additionally, the expression level of CREB3L4 was assessed using our cohort. CREB3L4 is upregulated in breast cancer tissues and is significantly associated with histological grade and tumour size (P = 0.001 and P < 0.001, respectively). Furthermore, PCNA expression was upregulated in breast cancer tissues and positively correlated with CREB3L4. In summary, CREB3L4 may play an important role in the progression of human BC and may serve as a therapeutic target.

SHOC2 is associated with the survival of breast cancer cells and has prognostic value for patients with breast cancer.

SHOC2 leucine rich repeat scaffold protein (SHOC2) has been identified as a positive regulator of the Ras pathway; however, the function of SHOC2 in breast cancer has rarely been explored. The current study investigated the effects of SHOC2 on breast cancer cell growth and evaluated its prognostic value in patients with breast cancer. The effects of SHOC2 on MCF­7 and MDA­MB­231 breast cancer cells were studied using short hairpin RNA. In total, 120 pairs of formalin­fixed, paraffin­embedded breast cancer tissue specimens were compared to normal tissue using immunohistochemical staining. SHOC2 knockdown significantly inhibited MCF­7 and MDA­MB­231 breast cancer cell proliferation, and induced cell apoptosis and cell cycle arrest. Additionally, the RAS­MAPK/PI3K pathway was inhibited by SHOC2 knockdown. In a clinical study, the results revealed that high SHOC2 expression was associated with more aggressive clinical characteristics of breast cancer. Moreover, Kaplan­Meier and Cox regression analyses indicated that SHOC2 expression was an independent prognostic factor for survival, suggesting that increased SHOC2 expression predicted a worse overall survival. This indicated that SHOC2 knockdown could affect breast cancer cell survival, and SHOC2 upregulation may be associated with a poor prognosis in patients with breast cancer.

Loss of TIPE2 Has Opposing Effects on the Pathogenesis of Autoimmune Diseases.

Autoimmune diseases are a physiological state wherein immune responses are directed against and damage the body's own tissues. Cytokines secreted by infiltrated inflammatory cells contribute to the pathogenesis of autoimmune diseases. TIPE2, one of the four family members of Tumor necrosis factor-α induced protein-8 (TNFAIP8), is a negative regulator of innate and adaptive immunity and plays essential roles in the maintenance of immune tolerance. However, studies on the role of TIPE2 during the development of autoimmune diseases have generated contradictory results. In the current study, we sought to determine the role of TIPE2 during the development of IMQ-induced psoriasis and Experimental Autoimmune Uveitis (EAU) in mice. Our study revealed that, while TIPE2-deficiency alleviates psoriasis, it exacerbates the development of EAU. Further studies demonstrated that, although TIPE2-deficient T cells produced more IL-17A, they do not migrate efficiently to the local inflammatory site, i.e., the skin. This in turn led to the decreased IL-17A production in the skin and consequently reduced the severity of psoriasis in TIPE2-deficient mice. However, although TIPE2-deficient T cells still produced more IL-17A in EAU model, they migrate into the inflamed eye as efficient as TIPE2-sufficient T cells, and consequently exacerbates the development of EAU in TIPE2-deficient mice. Taken together, these results indicate that TIPE2 may either promote or suppress autoimmunity depending on the specific inflammatory microenvironment in different types of autoimmune diseases.

TIPE2 in dendritic cells inhibits the induction of pTregs in the gut mucosa.

Dendritic cells (DCs) are professional antigen-presenting cells. The main function of DCs is to process antigen and present it to the T cells to induce T cell immunity. In addition to their function as potent stimulators of adaptive immunity, DCs are also crucial for maintaining immunological tolerance through the induction of peripheral regulatory T cells. Tumor necrosis factor-α-induced protein 8-2 (Tumor necrosis factor-α induced protein-8-like 2, TNFAIP8L2 or TIPE2) was expressed primarily by immune cells and maintains immune tolerance through the negative regulation of innate and adaptive immune responses. Previous studies indicate that TIPE2 in DCs may inhibit the innate immune response to RNA. However, the role of TIPE2 in DCs in the induction of peripheral tolerance remains unknown. Our current study showed that Tipe2-deficient DCs are more immature under homeostatic condition and consequently promote the induction of peripheral Tregs in the gut mucosa. Mechanistic studies revealed that TIPE2 promotes the expression of DC maturation markers CD80 and CD86 through the activation of PI3K-PKCδ-MAPK signaling pathway during the differentiation of DCs. Taken together, these results indicate that, in addition to acting as a negative regulator of pathogen-induced immune response, TIPE2 in DCs is also capable of promoting immune response under homeostatic condition through the suppression of peripheral tolerance.

miR-340 Alleviates Psoriasis in Mice through Direct Targeting of IL-17A.

Th17 cell is a well-known lineage of CD4+ effector Th cells that selectively produce IL-17A and play critical roles during the pathogenesis of autoimmune disease. A microRNA (miRNA) is a small noncoding RNA molecule that functions in posttranscriptional regulation of gene expression. Recently, an increasing number of studies have demonstrated that multiple miRNAs are dysregulated in patients with various autoimmune diseases and mediate autoimmune disease pathologic condition at least in part through the regulation of Th17 response. However, among the few miRNAs identified so far that play possible roles in the differentiation of Th17 cells, they all regulate the Th17 response through targeting negative or positive regulators of Th17 differentiation. In the current study, we sought to identify new miRNAs that can directly regulate the expression of IL-17A, the most important cytokine produced by Th17 cells. Our results showed that the 3' untranslated region of mouse IL-17A can act as a negative regulatory element to downregulate gene expression. Further study revealed that miR-340 can specifically bind to the 3' untranslated region of mouse IL-17A and downregulate the expression of endogenous IL-17A. More importantly, we demonstrated that treatment with miR-340 alleviates the clinical severity of imiquimod-induced psoriasis in mice through the downregulation of IL-17A. These data indicate that miR-340 may be a useful therapeutic target for the treatment of psoriasis and other IL-17A-mediated autoimmune diseases.

Erratum: MicroRNA-122 ameliorates corneal allograft rejection through the downregulation of its target CPEB1.

[This corrects the article DOI: 10.1038/cddiscovery.2017.21.].

MicroRNA-122 ameliorates corneal allograft rejection through the downregulation of its target CPEB1.

Transplant rejection is a major cause of corneal transplantation failure. MicroRNAs (miRNAs) are a family of small RNAs that regulates gene expression in a sequence-specific manner. miRNAs have recently been shown to have important roles in human organ transplantation, but reports of miRNAs directly associated with corneal transplantation rejection remain limited. To investigate the role of miRNAs during corneal allograft rejection, we established a mouse penetrating keratoplasty model and used microarrays to screen for differentially expressed miRNAs. Our results revealed that the expression of miR-122 was significantly decreased in the allogeneic group. Consistent with this result, the expression of cytoplasmic polyadenylation element-binding protein-1 (CPEB1), a direct target of miR-122, was significantly increased. Further analysis demonstrated that miR-122 inhibited inflammatory cytokine-induced apoptosis in corneal keratocytes through the downregulation of its target CPEB1. We also found that increased miR-122 expression significantly reduced the risk of corneal transplantation rejection. Thus, our results indicate that miR-122 is an important miRNA associated with corneal graft rejection and can be used as a therapeutic target for the prevention of immune rejection after keratoplasty.

Negative Immune Regulator TIPE2 Promotes M2 Macrophage Differentiation through the Activation of PI3K-AKT Signaling Pathway.

Macrophages play important roles in the regulation of the innate and adaptive immune responses. Classically activated macrophages and alternatively activated macrophages are the two major forms of macrophages and have opposing functionalities. Tumor necrosis factor-α-induced protein 8-2 is expressed primarily by immune cells and negatively regulates type 1 innate and adaptive immune responses to maintain immune tolerance. While previous studies indicate that TIPE2 promotes M2 but inhibits M1 macrophage differentiation, the underlying molecular mechanism by which TIPE2 promotes M2 macrophage differentiation remains unclear. Our current study shows that TIPE2-deficient bone-marrow cells are defective in IL-4 induced M2 macrophage differentiation in vitro. Mechanistic studies revealed that TIPE2 promotes phosphoinositide metabolism and the activation of the down-stream AKT signaling pathway, which in turn leads to the expression of markers specific for M2 macrophages. In addition, our results showed that Tipe2-deficiency does not affect the activation of the JAK-STAT6 signaling pathway that also plays an important role during M2 macrophage differentiation. Taken together, these results indicate that TIPE2 promotes M2 macrophage differentiation through the activation of PI3K-AKT signaling pathway, and may play an important role during the resolution of inflammation, parasite control, as well as tissue repair.

Treating psoriasis by targeting its susceptibility gene Rel.

Psoriasis is a chronic inflammatory disorder of the skin. Accumulating evidence indicates that the Rel gene, a member of the NF-κB family, is a risk factor for the disease. We sought to investigate whether psoriasis can be prevented by directly targeting the Rel gene transcript, i.e., the c-Rel mRNA. Using chemically-modified c-Rel specific siRNA (siRel) and poly(ethylene glycol)-b-poly(l-lysine)-b-poly(l-leucine) (PEG-PLL-PLLeu) micelles, we successfully knocked down the expression of c-Rel, and showed that the expression of cytokine IL-23, a direct target of c-Rel that can drive the development of IL-17-producing T cells, was markedly inhibited. More importantly, treating mice with siRel not only prevented but also ameliorated imiquimod (IMQ)-induced psoriasis. Mechanistic studies showed that siRel treatment down-regulated the expression of multiple inflammatory cytokines. Taken together, these results indicate that the susceptibility gene Rel can be targeted to treat and prevent psoriasis.

[Analysis of correlation between breast cancer molecular subtypes and prognosis of patients receiving breast-conserving therapy].

OBJECTIVE: To evaluate the effects of molecular subtypes on the prognosis of breast cancer patients after BCT (breast-conserving therapy). METHODS: From January 1999 to December 2007, a total of 699 female patients on BCT were analyzed retrospectively. They were grouped according to the results of immunohistochemistry, they were grouped as Luminal A (ER+/PR+/HER2-, Ki-67 < 14%), Luminal B (ER+/PR+/HER2 ± , Ki-67 > 14%), HER2 (ER-/PR-/HER2+) and triple negative breast cancer (TNBC) (ER-/PR-/HER2-). RESULTS: Among them, the recurrence rate, metastasis rate and survival rate within 5 years after BCT accounted for 6.0%, 6.0% and 91.8% respectively. There were statistical differences in metastasis rate and survival rate among patients with different molecular subtypes (P < 0.001), lymph node status (P < 0.001) and on chemotherapy or not (P < 0.001) on multivariate analysis. Though, on univariate analysis, different molecular subtypes had different risks for local failure (P = 0.022). On multivariate analysis, this difference was not statistically significant (P = 0.081). CONCLUSION: Despite a worse prognosis of TNBC versus other subtypes, BCT is a viable option for selected TNBC patients.

The effects of ECE on the benefits of PMRT for breast cancer patients with positive axillary nodes.

BACKGROUND: The purpose of the present study was to retrospectively evaluate the effects of extracapsular extension (ECE) on the benefits of post-mastectomy radiation therapy (PMRT) for groups of patients with varying numbers of positive axillary nodes (1-3, 4-9 and ≥10 positive axillary nodes). METHODS: A total of 1220 axillary node-positive patients who had received mastectomy were involved in this study. Patients were grouped as 'Radio + /ECE + ', 'Radio-/ECE + ', 'Radio + /ECE-' or 'Radio-/ECE-' according to status of ECE and whether receiving PMRT or not, and were evaluated in terms of local region relapse (LRR) rate. The 5-year and 10-year Kaplan-Meier disease-free survival and overall survival (OS) rates were analyzed. RESULTS: ECE-positive differed from ECE-negative groups with statistical significance for all comparisons in favor of the ECE-negative group: 5-year locoregional failure-free survival (LRFFS) (82.69% vs 91.83%, P < 0.001), 10-year LRFFS (75.39% vs 90.02%, P < 0.001); 5-year OS (52.12% vs 74.46%, P < 0.001), 10-year OS (35.17% vs 67.63%, P < 0.001). There were no significant effects of ECE on the benefits of PMRT for patients with 1-3 (P = 0.5720), ≥10(P = 0.0614) positive axillary nodes. However, for the group of patients with 4-9 positive axillary nodes, ECE status had a significant effect on the benefits of PMRT with respect to 5-year and 10-year LRFFS (P < 0.05). CONCLUSION: In our study, regardless of the ECE status, PMRT didn't significantly improve the LRFFS for patients with 1-3 or ≥10 positive axillary nodes. However, for patients with 4-9 positive axillary nodes, ECE could be an important criterion to consider when deciding whether to receive PMRT.

The prognosis of synchronous and metachronous bilateral breast cancer in Chinese patients.

This study was undertaken to determine the differences in the clinicopathology and survival between synchronous bilateral breast cancer (sBBC) and metachronous bilateral breast cancer (mBBC). Additionally, we analyzed the risk factors for single tumors to develop as sBBC or mBBC. Of the 190 bilateral breast cancer (BBC) cases, 84 cases were sBBC and 106 were mBBC. We defined sBBC as two tumors that developed within 12 months, while mBBC was defined as two tumors that developed over more than 12 months. The peak age of onset of the first mBBC tumors was significantly younger than that of sBBC tumors (p = 0.001). There was a higher concordance rate of ER/ER positivity and PR/PR positivity in the first and second tumors of sBBC than mBBC. The two sBBC breast cancers had relatively similar hormone conditions because of the low rate of ER and PR transformation from positive to negative or vice versa. We determined that patients who presented with extracapsular extension (p = 0.008) and ER positivity (p = 0.001) tend to have synchronous cancers, while patients with 3+ HER2 were more likely to develop metachronous tumors. The prognosis for mBBC was better than that for sBBC when the survival time of mBBC was measured from the initial observation of the first tumors.