p21CIP1 attenuates Ras- and c-Myc-dependent breast tumor epithelial mesenchymal transition and cancer stem cell-like gene expression in vivo.

p21(CIP1/WAF1) is a downstream effector of tumor suppressors and functions as a cyclin-dependent kinase inhibitor to block cellular proliferation. Breast tumors may derive from self-renewing tumor-initiating cells (BT-ICs), which contribute to tumor progression, recurrence, and therapy resistance. The role of p21(CIP1) in regulating features of tumor stem cells in vivo is unknown. Herein, deletion of p21(CIP1), which enhanced the rate of tumorigenesis induced by mammary-targeted Ha-Ras or c-Myc, enhanced gene expression profiles and immunohistochemical features of epithelial mesenchymal transition (EMT) and putative cancer stem cells in vivo. Silencing of p21(CIP1) enhanced, and expression of p21(CIP1) repressed, features of EMT in transformed immortal human MEC lines. p21(CIP1) attenuated oncogene-induced BT-IC and mammosphere formation. Thus, the in vitro cell culture assays reflect the changes observed in vivo in transgenic mice. These findings establish a link between the loss of p21(CIP1) and the acquisition of breast cancer EMT and stem cell properties in vivo.

Dachshund inhibits oncogene-induced breast cancer cellular migration and invasion through suppression of interleukin-8.

Oncogene-mediated signaling to the host environment induces a subset of cytokines and chemokines. The Drosophila Dac gene promotes migration of the morphogenetic furrow during eye development. Expression of the cell-fate determination factor Dachshund (DACH1) was lost in poor prognosis invasive breast cancer. Mouse embryo fibroblasts derived from Dach1(-/-) mice demonstrated endogenous Dach1 constitutively represses cellular migration. DACH1 inhibited cellular migration and invasion of oncogene (Ras, Myc, ErbB2, c-Raf)-transformed human breast epithelial cells. An unbiased proteomic analysis identified and immunoneutralizing antibody and reconstitution experiments demonstrated IL-8 is a critical target of DACH1 mediating breast cancer cellular migration and metastasis in vivo. DACH1 bound the endogenous IL-8 promoter in ChIP assays and repressed the IL-8 promoter through the AP-1 and NF-kappaB binding sites. Collectively, our data identify a pathway by which an endogenous cell-fate determination factor blocks oncogene-dependent tumor metastasis via a key heterotypic mediator.

Deeply Encoding Stable Patterns From Contaminated Data for Scenery Image Recognition.

Effectively recognizing different sceneries with complex backgrounds and varied lighting conditions plays an important role in modern AI systems. Competitive performance has recently been achieved by the deep scene categorization models. However, these models implicitly hypothesize that the image-level labels are 100% correct, which is too restrictive. Practically, the image-level labels for massive-scale scenery sets are usually calculated by external predictors such as ImageNet-CN. These labels can easily become contaminated because no predictors are completely accurate. This article proposes a new deep architecture that calculates scene categories by hierarchically deriving stable templates, which are discovered using a generative model. Specifically, we first construct a semantic space by incorporating image-level labels using subspace embedding. Afterward, it is noticeable that in the semantic space, the superpixel distributions from identically labeled images remain unchanged, regardless of the image-level label noises. On the basis of this observation, a probabilistic generative model learns the stable templates for each scene category. To deeply represent each scenery category, a novel aggregation network is developed to statistically concatenate the CNN features learned from scene annotations predicted by HSA. Finally, the learned deep representations are integrated into an image kernel, which is subsequently incorporated into a multiclass SVM for distinguishing scene categories. Thorough experiments have shown the performance of our method. As a byproduct, an empirical study of 33 SIFT-flow categories shows that the learned stable templates remain almost unchanged under a nearly 36% image label contamination rate.

Nuclear factor-kappaB enhances ErbB2-induced mammary tumorigenesis and neoangiogenesis in vivo.

The (HER2/Neu) ErbB2 oncogene is commonly overexpressed in human breast cancer and is sufficient for mammary tumorigenesis in transgenic mice. Nuclear factor (NF)-kappaB activity is increased in both human and murine breast tumors. The immune response to mammary tumorigenesis may regulate tumor progression. The role of endogenous mammary epithelial cell NF-kappaB had not previously been determined in immune-competent animals. Furthermore, the role of the NF-kappaB components, p50 and p65, in tumor growth was not known. Herein, the expression of a stabilized form of the NF-kappaB-inhibiting IkappaBalpha protein (IkappaBalphaSR) in breast tumor cell lines that express oncogenic ErbB2 inhibited DNA synthesis and growth in both two- and three-dimensional cultures. Either NF-kappaB inhibition or selective silencing of p50 or p65 led to a loss of contact-independent tumor growth in vitro. IkappaBalphaSR reversed the features of the oncogene-induced phenotype under three-dimensional growth conditions. The NF-kappaB blockade inhibited ErbB2-induced mammary tumor growth in both immune-competent and immune-deficient mice. These findings were associated with both reduced tumor microvascular density and a reduction in the amount of vascular endothelial growth factor. The expression of IkappaBalphaSR in breast cancer tumors inhibited angiogenesis. Thus, mammary epithelial cell NF-kappaB activity enhances ErbB2-mediated mammary tumorigenesis in vivo by promoting both growth and survival signaling via the promotion of tumor vasculogenesis.

Cyclin D1 regulates cellular migration through the inhibition of thrombospondin 1 and ROCK signaling.

Cyclin D1 is overexpressed in human tumors, correlating with cellular metastasis, and is induced by activating Rho GTPases. Herein, cyclin D1-deficient mouse embryo fibroblasts (MEFs) exhibited increased adhesion and decreased motility compared with wild-type MEFs. Retroviral transduction of cyclin D1 reversed these phenotypes. Mutational analysis of cyclin D1 demonstrated that its effects on cellular adhesion and migration were independent of the pRb and p160 coactivator binding domains. Genomewide expression arrays identified a subset of genes regulated by cyclin D1, including Rho-activated kinase II (ROCKII) and thrombospondin 1 (TSP-1). cyclin D1(-/-) cells showed increased Rho GTP and ROCKII activity and signaling, with increased phosphorylation of LIM kinase, cofilin (Ser3), and myosin light chain 2 (Thr18/Ser19). Cyclin D1 repressed ROCKII and TSP-1 expression, and the migratory defect of cyclin D1(-/-) cells was reversed by ROCK inhibition or TSP-1 immunoneutralizing antibodies. cyclin E knockin to the cyclin D1(-/-) MEFs rescued the DNA synthesis defect of cyclin D1(-/-) MEFs but did not rescue either the migration defect or the abundance of ROCKII. Cyclin D1 promotes cellular motility through inhibiting ROCK signaling and repressing the metastasis suppressor TSP-1.

Cyclin D1 determines mitochondrial function in vivo.

The cyclin D1 gene encodes a regulatory subunit of the holoenzyme that phosphorylates and inactivates the pRb tumor suppressor to promote nuclear DNA synthesis. cyclin D1 is overexpressed in human breast cancers and is sufficient for the development of murine mammary tumors. Herein, cyclin D1 is shown to perform a novel function, inhibiting mitochondrial function and size. Mitochondrial activity was enhanced by genetic deletion or antisense or small interfering RNA to cyclin D1. Global gene expression profiling and functional analysis of mammary epithelial cell-targeted cyclin D1 antisense transgenics demonstrated that cyclin D1 inhibits mitochondrial activity and aerobic glycolysis in vivo. Reciprocal regulation of these genes was observed in cyclin D1-induced mammary tumors. Cyclin D1 thus integrates nuclear DNA synthesis and mitochondrial function.

Cyclin D1 integrates G9a-mediated histone methylation.

Lysine methylation of histones and non-histone substrates by the SET domain containing protein lysine methyltransferase (KMT) G9a/EHMT2 governs transcription contributing to apoptosis, aberrant cell growth, and pluripotency. The positioning of chromosomes within the nuclear three-dimensional space involves interactions between nuclear lamina (NL) and the lamina-associated domains (LAD). Contact of individual LADs with the NL are dependent upon H3K9me2 introduced by G9a. The mechanisms governing the recruitment of G9a to distinct subcellular sites, into chromatin or to LAD, is not known. The cyclin D1 gene product encodes the regulatory subunit of the holoenzyme that phosphorylates pRB and NRF1 thereby governing cell-cycle progression and mitochondrial metabolism. Herein, we show that cyclin D1 enhanced H3K9 dimethylation though direct association with G9a. Endogenous cyclin D1 was required for the recruitment of G9a to target genes in chromatin, for G9a-induced H3K9me2 of histones, and for NL-LAD interaction. The finding that cyclin D1 is required for recruitment of G9a to target genes in chromatin and for H3K9 dimethylation, identifies a novel mechanism coordinating protein methylation.

Cyclin D1 induction of cellular migration requires p27(KIP1).

The cyclin D1 gene is amplified and overexpressed in human breast cancer, functioning as a collaborative oncogene. As the regulatory subunit of a holoenzyme phosphorylating Rb, cyclin D1 promotes cell cycle progression and a noncatalytic function has been described to sequester the cyclin-dependent kinase inhibitor protein p27. Cyclin D1 overexpression correlates with tumor metastasis and cyclin D1-deficient fibroblasts are defective in migration. The genetic mechanism by which cyclin D1 promotes migration and movement is poorly understood. Herein, cyclin D1 promoted cellular migration and cytokinesis of mammary epithelial cells. Cyclin D1 enhanced cellular migratory velocity. The induction of migration by cyclin D1 was abolished by mutation of K112 or deletion of NH(2)-terminal residues 46 to 90. These mutations of cyclin D1 abrogated physical interaction with p27(KIP1). Cyclin D1(-/-) cells were p27(KIP1) deficient and the defect in migration was rescued by p27(KIP1) reintroduction. Conversely, the cyclin D1 rescue of cyclin D1(-/-) cellular migration was reversed by p27(KIP1) small interfering RNA. Cyclin D1 regulated p27(KIP1) abundance at the posttranslational level, inhibiting the Skp2 promoter, Skp2 abundance, and induced p27(KIP1) phosphorylation at Ser(10). Together, these studies show cyclin D1 promotes mammary epithelial cell migration. p27(KIP1) is required for cyclin D1-mediated cellular migration.

Cyclin D1-mediated microRNA expression signature predicts breast cancer outcome.

Background: Genetic classification of breast cancer based on the coding mRNA suggests the evolution of distinct subtypes. Whether the non-coding genome is altered concordantly with the coding genome and the mechanism by which the cell cycle directly controls the non-coding genome is poorly understood. Methods: Herein, the miRNA signature maintained by endogenous cyclin D1 in human breast cancer cells was defined. In order to determine the clinical significance of the cyclin D1-mediated miRNA signature, we defined a miRNA expression superset from 459 breast cancer samples. We compared the coding and non-coding genome of breast cancer subtypes. Results: Hierarchical clustering of human breast cancers defined four distinct miRNA clusters (G1-G4) associated with distinguishable relapse-free survival by Kaplan-Meier analysis. The cyclin D1-regulated miRNA signature included several oncomirs, was conserved in multiple breast cancer cell lines, was associated with the G2 tumor miRNA cluster, ERα+ status, better outcome and activation of the Wnt pathway. The coding and non-coding genome were discordant within breast cancer subtypes. Seed elements for cyclin D1-regulated miRNA were identified in 63 genes of the Wnt signaling pathway including DKK. Cyclin D1 restrained DKK1 via the 3'UTR. In vivo studies using inducible transgenics confirmed cyclin D1 induces Wnt-dependent gene expression. Conclusion: The non-coding genome defines breast cancer subtypes that are discordant with their coding genome subtype suggesting distinct evolutionary drivers within the tumors. Cyclin D1 orchestrates expression of a miRNA signature that induces Wnt/ß-catenin signaling, therefore cyclin D1 serves both upstream and downstream of Wnt/ß-catenin signaling.

Stromal cyclin D1 promotes heterotypic immune signaling and breast cancer growth.

The cyclin D1 gene encodes the regulatory subunit of a holoenzyme that drives cell autonomous cell cycle progression and proliferation. Herein we show cyclin D1 abundance is increased >30-fold in the stromal fibroblasts of patients with invasive breast cancer, associated with poor outcome. Cyclin D1 transformed hTERT human fibroblast to a cancer-associated fibroblast phenotype. Stromal fibroblast expression of cyclin D1 (cyclin D1Stroma) in vivo, enhanced breast epithelial cancer tumor growth, restrained apoptosis, and increased autophagy. Cyclin D1Stroma had profound effects on the breast tumor microenvironment increasing the recruitment of F4/80+ and CD11b+ macrophages and increasing angiogenesis. Cyclin D1Stroma induced secretion of factors that promoted expansion of stem cells (breast stem-like cells, embryonic stem cells and bone marrow derived stem cells). Cyclin D1Stroma resulted in increased secretion of proinflammatory cytokines (CCL2, CCL7, CCL11, CXCL1, CXCL5, CXCL9, CXCL12), CSF (CSF1, GM-CSF1) and osteopontin (OPN) (30-fold). OPN was induced by cyclin D1 in fibroblasts, breast epithelial cells and in the murine transgenic mammary gland and OPN was sufficient to induce stem cell expansion. These results demonstrate that cyclin D1Stroma drives tumor microenvironment heterocellular signaling, promoting several key hallmarks of cancer.

Cyclin D1 Promotes Androgen-Dependent DNA Damage Repair in Prostate Cancer Cells.

Therapy resistance and poor outcome in prostate cancer is associated with increased expression of cyclin D1. Androgens promote DNA double-strand break repair to reduce DNA damage, and cyclin D1 was also shown to enhance DNA damage repair (DDR). In this study, we investigated the significance of cyclin D1 in androgen-induced DDR using established prostate cancer cells and prostate tissues from cyclin D1 knockout mice. We demonstrate that endogenous cyclin D1 further diminished the dihydrotestosterone (DHT)-dependent reduction of γH2AX foci in vitro. We also show that cyclin D1 was required for the androgen-dependent DNA damage response both in vitro and in vivo. Furthermore, cyclin D1 was required for androgen-enhanced DDR and radioresistance of prostate cancer cells. Moreover, microarray analysis of primary prostate epithelial cells from cyclin D1-deficient and wild-type mice demonstrated that most of the DHT-dependent gene expression changes are also cyclin D1 dependent. Collectively, our findings suggest that the hormone-mediated recruitment of cyclin D1 to sites of DDR may facilitate the resistance of prostate cancer cells to DNA damage therapies and highlight the need to explore other therapeutic approaches in prostate cancer to prevent or overcome drug resistance.

Cyclin D1 silencing suppresses tumorigenicity, impairs DNA double strand break repair and thus radiosensitizes androgen-independent prostate cancer cells to DNA damage.

Patients with hormone-resistant prostate cancer (PCa) have higher biochemical failure rates following radiation therapy (RT). Cyclin D1 deregulated expression in PCa is associated with a more aggressive disease: however its role in radioresistance has not been determined. Cyclin D1 levels in the androgen-independent PC3 and 22Rv1 PCa cells were stably inhibited by infecting with cyclin D1-shRNA. Tumorigenicity and radiosensitivity were investigated using in vitro and in vivo experimental assays. Cyclin D1 silencing interfered with PCa oncogenic phenotype by inducing growth arrest in the G1 phase of cell cycle and reducing soft agar colony formation, migration, invasion in vitro and tumor formation and neo-angiogenesis in vivo. Depletion of cyclin D1 significantly radiosensitizes PCa cells by increasing the RT-induced DNA damages by affecting the NHEJ and HR pathways responsible of the DNA double-strand break repair. Following treatment of cells with RT the abundance of a biomarker of DNA damage, γ-H2AX, was dramatically increased in sh-cyclin D1 treated cells compared to shRNA control. Concordant with these observations DNA-PKcs-activation and RAD51-accumulation, part of the DNA double-strand break repair machinery, were reduced in shRNA-cyclin D1 treated cells compared to shRNA control. We further demonstrate the physical interaction between CCND1 with activated-ATM, -DNA-PKcs and RAD51 is enhanced by RT. Finally, siRNA-mediated silencing experiments indicated DNA-PKcs and RAD51 are downstream targets of CCND1-mediated PCa cells radioresistance. In summary, these observations suggest that CCND1 is a key mediator of PCa radioresistance and could represent a potential target for radioresistant hormone-resistant PCa.

v-Src Oncogene Induces Trop2 Proteolytic Activation via Cyclin D1.

Proteomic analysis of castration-resistant prostate cancer demonstrated the enrichment of Src tyrosine kinase activity in approximately 90% of patients. Src is known to induce cyclin D1, and a cyclin D1-regulated gene expression module predicts poor outcome in human prostate cancer. The tumor-associated calcium signal transducer 2 (TACSTD2/Trop2/M1S1) is enriched in the prostate, promoting prostate stem cell self-renewal upon proteolytic activation via a γ-secretase cleavage complex (PS1, PS2) and TACE (ADAM17), which releases the Trop2 intracellular domain (Trop2 ICD). Herein, v-Src transformation of primary murine prostate epithelial cells increased the proportion of prostate cancer stem cells as characterized by gene expression, epitope characteristics, and prostatosphere formation. Cyclin D1 was induced by v-Src, and Src kinase induction of Trop2 ICD nuclear accumulation required cyclin D1. Cyclin D1 induced abundance of the Trop2 proteolytic cleavage activation components (PS2, TACE) and restrained expression of the inhibitory component of the Trop2 proteolytic complex (Numb). Patients with prostate cancer with increased nuclear Trop2 ICD and cyclin D1, and reduced Numb, had reduced recurrence-free survival probability (HR = 4.35). Cyclin D1, therefore, serves as a transducer of v-Src-mediated induction of Trop2 ICD by enhancing abundance of the Trop2 proteolytic activation complex. Cancer Res; 76(22); 6723-34. ©2016 AACR.

The endogenous cell-fate factor dachshund restrains prostate epithelial cell migration via repression of cytokine secretion via a cxcl signaling module.

Prostate cancer is the second leading form of cancer-related death in men. In a subset of prostate cancer patients, increased chemokine signaling IL8 and IL6 correlates with castrate-resistant prostate cancer (CRPC). IL8 and IL6 are produced by prostate epithelial cells and promote prostate cancer cell invasion; however, the mechanisms restraining prostate epithelial cell cytokine secretion are poorly understood. Herein, the cell-fate determinant factor DACH1 inhibited CRPC tumor growth in mice. Using Dach1(fl/fl)/Probasin-Cre bitransgenic mice, we show IL8 and IL6 secretion was altered by approximately 1,000-fold by endogenous Dach1. Endogenous Dach1 is shown to serve as a key endogenous restraint to prostate epithelial cell growth and restrains migration via CXCL signaling. DACH1 inhibited expression, transcription, and secretion of the CXCL genes (IL8 and IL6) by binding to their promoter regulatory regions in chromatin. DACH1 is thus a newly defined determinant of benign and malignant prostate epithelium cellular growth, migration, and cytokine abundance in vivo.

Relationship between tissue factor expression and hepatic metastasis and prognosis in rectal cancer.

OBJECTIVE: To investigate the correlation between tissue factor (TF) expression and hepatic metastasis and prognosis in rectal cancer. METHODS: TF expression was retrospectively studied by immunohistochemical method in specimens of 40 rectal cancer, 3 hepatic metastasis and 6 benign adenoma with relation to their clinicopathologic data. RESULTS: 1. TF expression was detected in 20 (50%) of the 40 primary rectal cancer specimens and all the 3 hepatic metastatic specimens, but not in the 6 benign adenoma or normal mucosa of rectum, 2. Significant correlation was observed between TF expression and synchronic hepatic metastasis (P = 0.002) and heterochronic hepatic metastasis (P = 0.001) and 3. TF was a risk factor for the prognosis of primary rectal cancer (P = 0.024). CONCLUSION: Tissue factor expression may play a role in the process of developing hepatic metastasis. It may be considered as a new clinical indicator for monitor of hepatic metastasis and prognosis of primary rectal cancer.

[Role of tissue factor in the invasive ability of cultured human colon carcinoma cells].

OBJECTIVE: To analyze the role of tissue factor (TF) in the in vitro invasive ability of human colon carcinoma cells (HT-29). METHODS: The eukaryotic expression vectors pcDNA3.1/Zeo bearing either sense or antisense TFcDNA were transfected into HT-29 cells by the way of lipofactamine 2000. TF proteins in transfected cells were detected by Western blot. In vitro Matrigel invasion assays were used to show the invasive ability of HT-29 cells with sense/antisense TFcDNA transfection. RESULTS: HT-29 cells with sense-TFcDNA transfection showed increased TF expression and invasive ability compared with the cells without transfection, but HT-29 cells with antisense-TFcDNA transfection got the contrary change. CONCLUSION: TF can increase the invasive ability of HT-29 cells in vitro.

[The role of tissue factor expression in the invasive and metastatic ability of colorectal carcinoma].

OBJECTIVE: To investigate the role of tissue factor (TF) expression in the invasive and metastatic ability of colorectal carcinoma and explore the influence of TF on the invasive ability of HT-29 cells. METHODS: TF expression of specimens from 85 colorectal carcinomas and 6 colorectal adenomas was observed by immunohistochemistry. The role of TF expression in prognosis and tumor invasion and metastasis was analyzed. The plasmids pcDNA3.1/Zeo bearing either sense or antisense-TFcDNA were transfected into HT-29 cells by the way of Lipofectamine 2000. TF proteins in transfected and untransfected HT-29cells were detected by Western blot. In vitro Matrigel invasion assays were performed to show the invasive ability of those cells. RESULTS: TF expression was positive in 40 (47.1%) of 85 colorectal carcinoma specimens, but negative in normal mucosa and adenoma specimens. TF expression showed significant correlation with tumor invasive depth (r = 0.895, P < 0.01). TF expression showed significant correlation with synchronous and metachronous hepatic metastasis (r = 0.974, P < 0.01 and r = 0.963, P < 0.01 respectively). TF expression was a significant risk factor for hepatic metastasis (P < 0.01) and prognosis (P < 0.01). TF expression in HT-29 cells with sense/antisense-TFcDNA transfection was more/less than that of the cells without transfection. The invasive ability of HT-29 cells with sense-TFcDNA transfection was increased in vitro compared with the untransfected cells, but HT-29 cells with antisense-TFcDNA transfection got the contrary change. CONCLUSIONS: TF may take part in the invasive and metastatic process of primary colorectal carcinoma, and TF expression may be an indicator of hepatic metastasis and prognosis for colorectal carcinoma patients. TF expression may increase the invasive ability of HT-29 cell in vitro.

Cyclin D1 integrates estrogen-mediated DNA damage repair signaling.

The cyclin D1 gene encodes the regulatory subunit of a holoenyzme that phosphorylates the retinoblastoma protein (pRb) and nuclear respiratory factor (NRF1) proteins. The abundance of cyclin D1 determines estrogen-dependent gene expression in the mammary gland of mice. Using estradiol (E2) and an E2-dendrimer conjugate that is excluded from the nucleus, we demonstrate that E2 delays the DNA damage response (DDR) via an extranuclear mechanism. The E2-induced DDR required extranuclear cyclin D1, which bound ERα at the cytoplasmic membrane and augmented AKT phosphorylation (Ser473) and γH2AX foci formation. In the nucleus, E2 inhibited, whereas cyclin D1 enhanced homology-directed DNA repair. Cyclin D1 was recruited to γH2AX foci by E2 and induced Rad51 expression. Cyclin D1 governs an essential role in the E2-dependent DNA damage response via a novel extranuclear function. The dissociable cytoplasmic function to delay the E2-mediated DDR together with the nuclear enhancement of DNA repair uncovers a novel extranuclear function of cyclin D1 that may contribute to the role of E2 in breast tumorigenesis.

CCR5 receptor antagonists block metastasis to bone of v-Src oncogene-transformed metastatic prostate cancer cell lines.

Src family kinases (SFK) integrate signal transduction for multiple receptors, regulating cellular proliferation, invasion, and metastasis in human cancer. Although Src is rarely mutated in human prostate cancer, SFK activity is increased in the majority of human prostate cancers. To determine the molecular mechanisms governing prostate cancer bone metastasis, FVB murine prostate epithelium was transduced with oncogenic v-Src. The prostate cancer cell lines metastasized in FVB mice to brain and bone. Gene expression profiling of the tumors identified activation of a CCR5 signaling module when the prostate epithelial cell lines were grown in vivo versus tissue cultures. The whole body, bone, and brain metastatic prostate cancer burden was reduced by oral CCR5 antagonist. Clinical trials of CCR5 inhibitors may warrant consideration in patients with CCR5 activation in their tumors.