Transcription factor Foxo1 is a negative regulator of natural killer cell maturation and function.

Little is known about the role of negative regulators in controlling natural killer (NK) cell development and effector functions. Foxo1 is a multifunctional transcription factor of the forkhead family. Using a mouse model of conditional deletion in NK cells, we found that Foxo1 negatively controlled NK cell differentiation and function. Immature NK cells expressed abundant Foxo1 and little Tbx21 relative to mature NK cells, but these two transcription factors reversed their expression as NK cells proceeded through development. Foxo1 promoted NK cell homing to lymph nodes by upregulating CD62L expression and inhibited late-stage maturation and effector functions by repressing Tbx21 expression. Loss of Foxo1 rescued the defect in late-stage NK cell maturation in heterozygous Tbx21(+/-) mice. Collectively, our data reveal a regulatory pathway by which the negative regulator Foxo1 and the positive regulator Tbx21 play opposing roles in controlling NK cell development and effector functions.

Tumor-suppressive effects of psoriasin (S100A7) are mediated through the ß-catenin/T cell factor 4 protein pathway in estrogen receptor-positive breast cancer cells.

Psoriasin (S100A7) is expressed in several epithelial malignancies including breast cancer. Although S100A7 is associated with the worst prognosis in estrogen receptor α-negative (ERα(-)) invasive breast cancers, its role in ERα-positive (ERα(+)) breast cancers is relatively unknown. We investigated the significance of S100A7 in ERα(+) breast cancer cells and observed that S100A7 overexpression in ERα(+) breast cancer cells, MCF7 and T47D, exhibited decreased migration, proliferation, and wound healing. These results were confirmed in vivo in nude mouse model system. Mice injected with S100A7-overexpressing MCF7 cells showed significant reduction in tumor size compared with mice injected with vector control cells. Further mechanistic studies revealed that S100A7 mediates the tumor-suppressive effects via a coordinated regulation of the ß-catenin/TCF4 pathway and an enhanced interaction of ß-catenin and E-cadherin in S100A7-overexpressing ERα(+) breast cancer cells. We observed down-regulation of ß-catenin, p-GSK3ß, TCF4, cyclin D1, and c-myc in S100A7-overexpressing ERα(+) breast cancer cells. In addition, we observed increased expression of GSK3ß. Treatment with GSK3ß inhibitor CHIR 99021 increased the expression of ß-catenin and its downstream target c-myc in S100A7-overexpressing cells. Tumors derived from mice injected with S100A7-overexpressing MCF7 cells also showed reduced activation of the ß-catenin/TCF4 pathway. Therefore, our studies reveal for the first time that S100A7-overexpressing ERα(+) breast cancer cells exhibit tumor suppressor capabilities through down-modulation of the ß-catenin/TCF4 pathway both in vitro and in vivo. Because S100A7 has been shown to enhance tumorigenicity in ERα(-) cells, our studies suggest that S100A7 may possess differential activities in ERα(+) compared with ERα(-) cells.

3,3'-Diindolylmethane negatively regulates Cdc25A and induces a G2/M arrest by modulation of microRNA 21 in human breast cancer cells.

3,3'-Diindolylmethane (DIM) is a potential chemopreventive phytochemical derived from Brassica vegetables. In this study, we assessed the effects of DIM on cell cycle regulation in both estrogen-dependent MCF-7 and estrogen receptor negative p53 mutant MDA-MB-468 human breast cancer cells. In-vitro culture studies showed that DIM dose dependently inhibited the proliferation of both cells. In addition, in-vivo xenograft model showed that DIM strongly inhibited the development of human breast tumors. Fluorescence activated cell sorter analysis showed a DIM-mediated G2/M cell cycle arrest in MCF-7 and MDA-MB-468 cells. Western blot analysis showed that DIM downregulated the expression of cyclin-dependent kinases 2 and 4 and Cdc25A, which plays an important role in G2/M phase. Furthermore, treatment of MCF-7 cells with DIM, which increased microRNA 21 expression, caused a downregulation of Cdc25A, resulting in an inhibition of breast cancer cell proliferation. Taken together, our data show that DIM is able to stop the cell cycle progression of human breast cancer cells regardless of their estrogen-dependence and p53 status, by differentially modulating cell cycle regulatory pathways. The modulation of microRNA 21 mediates the DIM cell cycle regulator effect in MCF-7 cells.

Hemizygous disruption of Cdc25A inhibits cellular transformation and mammary tumorigenesis in mice.

CDC25A phosphatase activates multiple cyclin-dependent kinases (CDK) during cell cycle progression. Inactivation of CDC25A by ubiquitin-mediated degradation is a major mechanism of DNA damage-induced S-G(2) checkpoint. Although increased CDC25A expression has been reported in various human cancer tissues, it remains unclear whether CDC25A activation is a critical rate-limiting step of carcinogenesis. To assess the role for CDC25A in cell cycle control and carcinogenesis, we used a Cdc25A-null mouse strain we recently generated. Whereas Cdc25A(-/-) mice exhibit early embryonic lethality, Cdc25A(+/-) mice show no appreciable developmental defect. Cdc25A(+/-) mouse embryonic fibroblasts (MEF) exhibit normal kinetics of cell cycle progression at early passages, modestly enhanced G(2) checkpoint response to DNA damage, and shortened proliferative life span, compared with wild-type MEFs. Importantly, Cdc25A(+/-) MEFs are significantly resistant to malignant transformation induced by coexpression of H-ras(V12) and a dominant negative p53 mutant. The rate-limiting role for CDC25A in transformation is further supported by decreased transformation efficiency in MCF-10A human mammary epithelial cells stably expressing CDC25A small interfering RNA. Consistently, Cdc25A(+/-) mice show substantially prolonged latency in mammary tumorigenesis induced by MMTV-H-ras or MMTV-neu transgene, whereas MMTV-myc-induced tumorigenesis is not significantly affected by Cdc25A heterozygosity. Mammary tissues of Cdc25A(+/-);MMTV-neu mice before tumor development display less proliferative response to the oncogene with increased tyrosine phosphorylation of CDK1/2, but show no significant change in apoptosis. These results suggest that Cdc25A plays a rate-limiting role in transformation and tumor initiation mediated by ras activation.

Transforming growth factor beta facilitates beta-TrCP-mediated degradation of Cdc25A in a Smad3-dependent manner.

Ubiquitin-dependent degradation of Cdc25A is a major mechanism for damage-induced S-phase checkpoint. Two ubiquitin ligases, the Skp1-cullin-beta-TrCP (SCFbeta-TrCP) complex and the anaphase-promoting complex (APCCdh1), are involved in Cdc25A degradation. Here we demonstrate that the transforming growth factor beta (TGF-beta)-Smad3 pathway promotes SCF(beta-TrCP)-mediated Cdc25A ubiquitination. Cells treated with TGF-beta, as well as cells transfected with Smad3 or a constitutively active type I TGF-beta receptor, exhibit increased ubiquitination and markedly shortened half-lives of Cdc25A. Furthermore, Cdc25A is stabilized in cells transfected with Smad3 small interfering RNA (siRNA) and cells from Smad3-null mice. TGF-beta-induced ubiquitination is associated with Cdc25A phosphorylation at the beta-TrCP docking site (DS82G motif) and physical association of Cdc25A with Smad3 and beta-TrCP. Cdc25A mutant proteins deficient in DS82G phosphorylation are resistant to TGF-beta-Smad3-induced degradation, whereas a Cdc25A mutant protein defective in APCCdh1 recognition undergoes efficient degradation. Smad3 siRNA inhibits beta-TrCP-Cdc25A interaction and Cdc25A degradation in response to TGF-beta. beta-TrCP2 siRNA also inhibits Smad3-induced Cdc25A degradation. In contrast, Cdh1 siRNA had no effect on Cdc25A down-regulation by Smad3. These data suggest that Smad3 plays a key role in the regulation of Cdc25A ubiquitination by SCFbeta-TrCP and that Cdc25A stabilization observed in various cancers could be associated with defects in the TGF-beta-Smad3 pathway.

CDK4 deficiency promotes genomic instability and enhances Myc-driven lymphomagenesis.

The G1 kinase CDK4 is amplified or overexpressed in some human tumors and promotes tumorigenesis by inhibiting known tumor suppressors. Here, we report that CDK4 deficiency markedly accelerated lymphoma development in the Eµ-Myc transgenic mouse model of B lymphoma and that silencing or loss of CDK4 augmented the tumorigenic potential of Myc-driven mouse and human B cell lymphoma in transplant models. Accelerated disease in CDK4-deficient Eµ-Myc transgenic mice was associated with rampant genomic instability that was provoked by dysregulation of a FOXO1/RAG1/RAG2 pathway. Specifically, CDK4 phosphorylated and inactivated FOXO1, which prevented FOXO1-dependent induction of Rag1 and Rag2 transcription. CDK4-deficient Eµ-Myc B cells had high levels of the active form of FOXO1 and elevated RAG1 and RAG2. Furthermore, overexpression of RAG1 and RAG2 accelerated lymphoma development in a transplant model, with RAG1/2-expressing tumors exhibiting hallmarks of genomic instability. Evaluation of human tumor samples revealed that CDK4 expression was markedly suppressed, while FOXO1 expression was elevated, in several subtypes of human non-Hodgkin B cell lymphoma. Collectively, these findings establish a context-specific tumor suppressor function for CDK4 that prevents genomic instability, which contributes to B cell lymphoma. Furthermore, our data suggest that targeting CDK4 may increase the risk for the development and/or progression of lymphoma.

S100A7 enhances mammary tumorigenesis through upregulation of inflammatory pathways.

S100A7/psoriasin, a member of the epidermal differentiation complex, is widely overexpressed in invasive estrogen receptor (ER)α-negative breast cancers. However, it has not been established whether S100A7 contributes to breast cancer growth or metastasis. Here, we report the consequences of its expression on inflammatory pathways that impact breast cancer growth. Overexpression of human S100A7 or its murine homologue mS100a7a15 enhanced cell proliferation and upregulated various proinflammatory molecules in ERα-negative breast cancer cells. To examine in vivo effects, we generated mice with an inducible form of mS100a7a15 (MMTV-mS100a7a15 mice). Orthotopic implantation of MVT-1 breast tumor cells into the mammary glands of these mice enhanced tumor growth and metastasis. Compared with uninduced transgenic control mice, the mammary glands of mice where mS100a7a15 was induced exhibited increased ductal hyperplasia and expression of molecules involved in proliferation, signaling, tissue remodeling, and macrophage recruitment. Furthermore, tumors and lung tissues obtained from these mice showed further increases in prometastatic gene expression and recruitment of tumor-associated macrophages (TAM). Notably, in vivo depletion of TAM inhibited the effects of mS100a7a15 induction on tumor growth and angiogenesis. Furthermore, introduction of soluble hS100A7 or mS100a7a15 enhanced chemotaxis of macrophages via activation of RAGE receptors. In summary, our work used a powerful new model system to show that S100A7 enhances breast tumor growth and metastasis by activating proinflammatory and metastatic pathways.

Cdc25A regulates matrix metalloprotease 1 through Foxo1 and mediates metastasis of breast cancer cells.

Cdc25A is a cell cycle-activating phosphatase, and its overexpression in breast cancers has been shown to correlate with poor prognosis. Most recent studies related to Cdc25A and tumor progression have focused on its role in regulating cell cycle progression. However, less is known about how Cdc25A modulates the metastasis of breast cancer cells. In this study, we revealed that Cdc25A enhances Foxo1 stability by dephosphorylating Cdk2, and Foxo1 was shown to directly regulate transcription of the metastatic factor MMP1. Further studies have shown that overexpression of Cdc25A in breast cancer cells enhances metastasis, whereas its downmodulation inhibits metastasis in mouse models, and the effects of Cdc25A on breast cancer cell metastasis are independent of cell proliferation and apoptosis. Furthermore, we have demonstrated that aberrant Cdc25A in breast cancer patient samples directly correlates with the metastatic phenotype. Further insights into this critical role of Cdc25A in the metastasis of breast cancer cells and the trial of an anti-Cdc25A strategy in mouse models may reveal its therapeutic potential in prevention and treatment of breast cancer cell dissemination.

Crosstalk between chemokine receptor CXCR4 and cannabinoid receptor CB2 in modulating breast cancer growth and invasion.

BACKGROUND: Cannabinoids bind to cannabinoid receptors CB(1) and CB(2) and have been reported to possess anti-tumorigenic activity in various cancers. However, the mechanisms through which cannabinoids modulate tumor growth are not well known. In this study, we report that a synthetic non-psychoactive cannabinoid that specifically binds to cannabinoid receptor CB(2) may modulate breast tumor growth and metastasis by inhibiting signaling of the chemokine receptor CXCR4 and its ligand CXCL12. This signaling pathway has been shown to play an important role in regulating breast cancer progression and metastasis. METHODOLOGY/PRINCIPAL FINDINGS: We observed high expression of both CB(2) and CXCR4 receptors in breast cancer patient tissues by immunohistochemical analysis. We further found that CB(2)-specific agonist JWH-015 inhibits the CXCL12-induced chemotaxis and wound healing of MCF7 overexpressing CXCR4 (MCF7/CXCR4), highly metastatic clone of MDA-MB-231 (SCP2) and NT 2.5 cells (derived from MMTV-neu) by using chemotactic and wound healing assays. Elucidation of the molecular mechanisms using various biochemical techniques and confocal microscopy revealed that JWH-015 treatment inhibited CXCL12-induced P44/P42 ERK activation, cytoskeletal focal adhesion and stress fiber formation, which play a critical role in breast cancer invasion and metastasis. In addition, we have shown that JWH-015 significantly inhibits orthotopic tumor growth in syngenic mice in vivo using NT 2.5 cells. Furthermore, our studies have revealed that JWH-015 significantly inhibits phosphorylation of CXCR4 and its downstream signaling in vivo in orthotopic and spontaneous breast cancer MMTV-PyMT mouse model systems. CONCLUSIONS/SIGNIFICANCE: This study provides novel insights into the crosstalk between CB(2) and CXCR4/CXCL12-signaling pathways in the modulation of breast tumor growth and metastasis. Furthermore, these studies indicate that CB(2) receptors could be used for developing innovative therapeutic strategies against breast cancer.

Cannabinoid receptors, CB1 and CB2, as novel targets for inhibition of non-small cell lung cancer growth and metastasis.

Non-small cell lung cancer (NSCLC) is the leading cause of cancer deaths worldwide; however, only limited therapeutic treatments are available. Hence, we investigated the role of cannabinoid receptors, CB1 and CB2, as novel therapeutic targets against NSCLC. We observed expression of CB1 (24%) and CB2 (55%) in NSCLC patients. Furthermore, we have shown that the treatment of NSCLC cell lines (A549 and SW-1573) with CB1/CB2- and CB2-specific agonists Win55,212-2 and JWH-015, respectively, significantly attenuated random as well as growth factor-directed in vitro chemotaxis and chemoinvasion in these cells. We also observed significant reduction in focal adhesion complex, which plays an important role in migration, upon treatment with both JWH-015 and Win55,212-2. In addition, pretreatment with CB1/CB2 selective antagonists, AM251 and AM630, prior to JWH-015 and Win55,212-2 treatments, attenuated the agonist-mediated inhibition of in vitro chemotaxis and chemoinvasion. In addition, both CB1 and CB2 agonists Win55,212-2 and JWH-133, respectively, significantly inhibited in vivo tumor growth and lung metastasis (∼50%). These effects were receptor mediated, as pretreatment with CB1/CB2 antagonists abrogated CB1/CB2 agonist-mediated effects on tumor growth and metastasis. Reduced proliferation and vascularization, along with increased apoptosis, were observed in tumors obtained from animals treated with JWH-133 and Win55,212-2. Upon further elucidation into the molecular mechanism, we observed that both CB1 and CB2 agonists inhibited phosphorylation of AKT, a key signaling molecule controlling cell survival, migration, and apoptosis, and reduced matrix metalloproteinase 9 expression and activity. These results suggest that CB1 and CB2 could be used as novel therapeutic targets against NSCLC.

A novel mechanism of indole-3-carbinol effects on breast carcinogenesis involves induction of Cdc25A degradation.

The natural compound indole-3-carbinol (I3C; found in vegetables of the genus Brassica) is a promising cancer prevention or therapy agent. The cell division cycle 25A (Cdc25A) phosphatase is overexpressed in a variety of human cancers and other diseases. In the present study, I3C induced degradation of Cdc25A, arrest of the G(1) cell cycle, and inhibition of the growth of breast cancer cells. We also showed that the Ser124 site of Cdc25A, which is related to cyclin-dependent kinase 2, is required for I3C-induced degradation of Cdc25A in breast cancer cells, and that interruption of the ATM-Chk2 pathway suppressed I3C-induced destruction of Cdc25A. Our in vivo studies of different mutated forms of Cdc25A found that the mutation Cdc25A(S124A) (Ser124 to Ala124), which confers resistance to I3C-induced degradation of Cdc25A, attenuated I3C inhibition of breast tumorigenesis in a mouse xenograft model. The present in vitro and in vivo studies together show that I3C-induced activation of the ATM-Chk2 pathway and degradation of Cdc25A represent a novel molecular mechanism of I3C in arresting the G(1) cell cycle and inhibiting the growth of breast cancer cells. The finding that I3C induces Cdc25A degradation underscores the potential use of this agent for preventing and treating cancers and other human diseases with Cdc25A overexpression.

A mutant allele of BARA/LIN-9 rescues the cdk4-/- phenotype by releasing the repression on E2F-regulated genes.

It has been proposed that C. elegans LIN-9 functions downstream of CDK4 in a pathway that regulates cell proliferation. Here, we report that mammalian BARA/LIN-9 is a predominantly nuclear protein that inhibits cell proliferation. More importantly, we demonstrate that BARA/LIN-9 also acts downstream of cyclin D/CDK4 in mammalian cells since (i) its antiproliferative effect is partially blocked by coexpression of cyclin D1, and (ii) a mutant form that lacks the first 84 amino acids rescues several phenotypic alterations observed in mice null for cdk4. Interestingly, mutation of BARA/LIN-9 restores the expression of E2F target genes in CDK4 null MEFs, indicating that the wild-type protein plays a role in the expression of genes required for the G1/S transition.

Cdk4 is indispensable for postnatal proliferation of the anterior pituitary.

For proper development and tissue homeostasis, cell cycle progression is controlled by multilayered mechanisms. Recent studies using knock-out mice have shown that animals can develop relatively normally with deficiency for each of the G1/S-regulatory proteins, D-type and E-type cyclins, cyclin-dependent kinase 4 (Cdk4), and Cdk2. Although Cdk4-null mice show no embryonic lethality, they exhibit specific endocrine phenotypes, i.e. dwarfism, infertility, and diabetes. Here we have demonstrated that Cdk4 plays an essential non-redundant role in postnatal proliferation of the anterior pituitary. Pituitaries from wild-type and Cdk4-null embryos at embryonic day 17.5 are morphologically indistinguishable with similar numbers of cells expressing a proliferating marker, Ki67, and cells expressing a differentiation marker, growth hormone. In contrast, anterior pituitaries of Cdk4-null mice at postnatal 8 weeks are extremely hypoplastic with markedly decreased numbers of Ki67+ cells, suggesting impaired cell proliferation. Pituitary hyperplasia induced by transgenic expression of human growth hormone-releasing hormone (GHRH) is significantly diminished in the Cdk4+/- genetic background and completely abrogated in the Cdk4-/- background. Small interfering RNA (siRNA)-mediated knockdown of Cdk4 inhibits GHRH-induced proliferation of GH3 somato/lactotroph cells with restored expression of GHRH receptors. Cdk4 siRNA also inhibits estrogen-dependent cell proliferation in GH3 cells and closely related GH4 cells. In contrast, Cdk6 siRNA does not diminish proliferation of these cells. Furthermore, Cdk4 siRNA does not affect GHRH-induced proliferation of mouse embryonic fibroblasts or estrogen-dependent proliferation of mammary carcinoma MCF-7 cells. Taken together, Cdk4 is dispensable for prenatal development of the pituitary or proliferation of other non-endocrine tissues but indispensable specifically for postnatal proliferation of somato/lactotrophs.

Cdk4 disruption renders primary mouse cells resistant to oncogenic transformation, leading to Arf/p53-independent senescence.

A large number of human cancers display alterations in the Ink4a/cyclin D/Cdk4 genetic pathway, suggesting that activation of Cdk4 plays an important role in oncogenesis. Here we report that Cdk4-null mouse embryonic fibroblasts are resistant to transformation in response to Ras activation with dominant-negative (DN) p53 expression or in the Ink4a/Arf-null background, judged by foci formation, anchorage-independent growth, and tumorigenesis in athymic mice. Cdk4-null fibroblasts proliferate at normal rates during early passages. Whereas Cdk4(+/+)Ink4a/Arf(-/-) cells are immortal in culture, Cdk4(-/-)Ink4a/Arf(-/-) cells undergo senescence during continuous culture, as do wild-type cells. Activated Ras also induces premature senescence in Cdk4(-/-)Ink4a/Arf(-/-) cells and Cdk4(-/-) cells with DNp53 expression. Thus, Cdk4 deficiency causes senescence in a unique Arf/p53-independent manner, which accounts for the loss of transformation potential. Cdk4-null cells express high levels of p21(Cip1/Waf1) with increased protein stability. Suppression of p21(Cip1/Waf1) by small interfering RNA (siRNA), as well as expression of HPV-E7 oncoprotein, restores immortalization and Ras-mediated transformation in Cdk4(-/-)Ink4a/Arf(-/-) cells and Cdk4(-/-) cells with DNp53 expression. Therefore, Cdk4 is essential for immortalization, and suppression of Cdk4 could be a prospective strategy to recruit cells with inactive Arf/p53 pathway to senescence.