Rb Function Research Articles

P2-02-10: Differential Targeting of the RB-Pathway To Expand the Therapeutic Index in the Treatment of Triple Negative Breast Cancer.

Abstract Background: The retinoblastoma tumor suppressor (RB) is inactivated in a significant fraction of triple negative breast cancers (TNBC). RB loss is believed to contribute to tumor initiation and progression, and provocative data suggest that RB loss is associated with improved response to cytotoxic chemotherapy. Unfortunately, such therapies have significant side effects that impinge on patient quality of life. Here we explored the ability to specifically target RB loss in TNBC to expand the therapeutic index by protecting against undesired side-effects of chemotherapy. Experimental Design: A combination of cell culture and xenograft models of triple negative breast cancer were used to evaluate how RB-status impinges on sensitivity to chemotherapy. These models were complemented by the analyses of clinical specimens from patients treated with chemotherapy. Parallel use of agents that activate RB function with high specificity were interrogated as potential tissue protectors to ameliorate side effect of chemotherapy. Results: RB loss in preclinical models is associated with increased sensitivity to conventional cytotoxic chemotherapies that are frequently employed in the treatment of TNBC. The findings were recapitulated through the analyses of clinical specimens, and suggest that the loss of RB could be particularly useful in defining TNBC patients with improved response to chemotherapy. Combination studies of pharmacological activation of the RB pathway and cytotoxic chemotherapies commonly used to treat TNBC demonstrated that activation of RB can modulate the efficacy of cytotoxic compounds in the treatment of TNBC. Importantly, functional studies indicated that RB pathway activation limits cardiotoxicity associated with anthracycline based treatments and correspondingly improve cardiac function in animal studies. Conclusions: Combined, these studies indicate that RB pathway status could be utilized in the clinic to direct combination treatments that would exploit the sensitivity of RB-deficient tumors to cytotoxic chemotherapies while simultaneously providing protection for normal tissues. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-02-10.

Abstract B233: Identification and characterization of LY2835219: A potent oral inhibitor of the cyclin-dependent kinases 4 and 6 (CDK4/6) with broad in vivo antitumor activity.

Abstract Dysregulation of the cell cycle, which normally regulates cell proliferation in response to mitogenic signaling and other extracellular stimuli, is a hallmark of cancer. The G1 restriction point is a primary mechanism controlling cell cycle progression and is controlled by the CDK4/6 pathway (CDK4/6-cyclin D1-Rb-CDKN2). The importance of this pathway is highlighted by inactivation of restriction point control in a majority of human tumors. Transition through the restriction point requires phosphorylation of Rb by CDK4/6, and these kinases are considered highly validated cancer drug targets. We have identified and characterized a potent and selective dual CDK4/6 inhibitor, LY2835219. Preclinical characterization was performed with the monomesylate salt (LY2835219-MsOH), which inhibits these kinases with a IC50 of 2 and 10 nM for CDK4 and CDK6, respectively. In vitro, LY2835219-MsOH is a potent inhibitor of Rb phosphorylation resulting in a G1 arrest, and its activity is specific for tumors that have functional Rb protein. In a multiplexed in vivo target inhibition assay (IVTI), LY2835219-MsOH is a potent inhibitor of Rb phosphorylation and induces complete cell cycle arrest 24 hrs after a single dose. In tumor-bearing mice, oral administration of LY2835219-MsOH inhibits tumor growth in a dose-dependent manner in colon (colo-205), glioblastoma (U87MG), acute myeloid leukemia (MV4–11), mantle cell lymphoma (Jeko-1), and lung (H460) xenografts. LY2835219-MsOH may be administered up to 56 days without adverse events or tumor outgrowth. LY2835219-MsOH enhances the in vivo activity of cytotoxic drugs, suggesting that this novel CDK4/6 inhibitor can be used in combination with these anti-neoplastic agents. In summary, we have identified an oral small molecule inhibitor of CDK4/6 that may provide therapeutic benefit to cancer patients with tumors that have functional Rb protein. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B233.

Conserved RB functions in development and tumor suppression

The variety of human cancers in which the retinoblastoma protein pRb is inactivated reflects both its broad importance for tumor suppression and its multitude of cellular functions. Accumulating evidence indicates that pRb contributes to a diversity of cellular functions, including cell proliferation, differentiation, cell death, and genome stability. pRb performs these diverse functions through the formation of large complexes that include E2F transcription factors and chromatin regulators. In this review we will discuss some of the recent advances made in understanding the structure and function of pRb as they relate to tumor suppression, and highlight research using Drosophila melanogaster that reveals important, evolutionarily conserved functions of the RB family.

RB Restricts DNA Damage-Initiated Tumorigenesis through an LXCXE-Dependent Mechanism of Transcriptional Control

The LXCXE peptide motif facilitates interaction between the RB tumor suppressor and a large number of cellular proteins that are expected to impinge on diverse biological processes. Invitro and invivo analyses demonstrated that LXCXE binding function is dispensable for RB promoter association and control of basal gene expression. Dependence on this function of RB is unmasked after DNA damage, wherein LXCXE binding is essential for exerting control over E2F3 and suppressing cell-cycle progression in the presence of genotoxic stress. Gene expression profiling revealed that the transcriptional program coordinated by this specific aspect of RB is associated with progression of human hepatocellular carcinoma and poor disease outcome. Consistent with these findings, biological challenge revealeda requirement for LXCXE binding in suppression of genotoxin-initiated hepatocellular carcinoma invivo. Together, these studies establish an essential role ofthe LXCXE binding motif for RB-mediated transcriptional control, response to genotoxic insult, and tumor suppression.

Gene expression profiling of choroid plexus in Alzheimer's disease reveals important implications of CSF dynamics

(entorhinal and temporal cortices, and hippocampus) assembled in tissuemicroarray (TMA), immunohistochemistry was performed to examine the expression patterns of Rb in the following groups: 1) asymptomatic AD elderly individuals with AD-related pathology, but no clinical evidence of dementia (Braak IV-VI, CERAD B or C, CDR 0); 2) symptomatic AD elderly individuals with AD-related pathology and dementia (Braak IV-VI, CERAD B or C, CDR 1⁄4 2); and 3) healthy normal individuals (controls) (Braak I-III, CERAD 0 or A, CDR 0). Results: The nuclear staining was low in all groups, although in the group of normal individuals the number of positive nucleus was higher than in the Alzheimer’s groups. However, there also was cytoplasmatic staining with a stronger immunoreactivity in the normal group and a progressive decrease of reactivity in the asymptomatic and symptomatic AD groups, respectively. Conclusions: Our results show a progressive lost of Rb expression in AD patient’s brain, mainly in the cytoplasm. This shift at the subcellular localization of several makers implicated with cell cycle has been shown in AD, but the real biological significance of this change is still unclear. Considering that Rb function as a cell cycle suppressor, this expression lost could facilitate the cell cycle re-entry and neuronal death. In asymptomatic AD individuals, the Rb expression level higher than in symptomatic AD subjects could be helping, together with other mechanisms, the keeping of their normal cognitive function.

Functional Interactions between Retinoblastoma and c-MYC in a Mouse Model of Hepatocellular Carcinoma

Inactivation of the RB tumor suppressor and activation of the MYC family of oncogenes are frequent events in a large spectrum of human cancers. Loss of RB function and MYC activation are thought to control both overlapping and distinct cellular processes during cell cycle progression. However, how these two major cancer genes functionally interact during tumorigenesis is still unclear. Here, we sought to test whether loss of RB function would affect cancer development in a mouse model of c-MYC-induced hepatocellular carcinoma (HCC), a deadly cancer type in which RB is frequently inactivated and c-MYC often activated. We found that RB inactivation has minimal effects on the cell cycle, cell death, and differentiation features of liver tumors driven by increased levels of c-MYC. However, combined loss of RB and activation of c-MYC led to an increase in polyploidy in mature hepatocytes before the development of tumors. There was a trend for decreased survival in double mutant animals compared to mice developing c-MYC-induced tumors. Thus, loss of RB function does not provide a proliferative advantage to c-MYC-expressing HCC cells but the RB and c-MYC pathways may cooperate to control the polyploidy of mature hepatocytes.

Abstract 267: E2F1 regulates human TRAIL promoter and mediates Interferon alpha 2a response

Abstract The tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL/APO2L) is a member of the TNF gene superfamily that induces apoptosis upon engagement of cognate death receptors. While TRAIL is relatively non-toxic to normal cells, it selectively induces apoptosis in many transformed cells and has been explored as an anti-cancer agent. Earlier studies have shown the upstream region of human TRAIL gene, contain NFAT, AP-1, and Sp1 binding sites which have been shown to regulate expression of multiple cytokines, but the transcriptional regulation of TRAIL has not yet been elucidated in detail. An examination of the human TRAIL promoter showed the presence of five binding sites for the E2F transcription factors. E2F1 is a transcription factor that plays a well-documented role in cell cycle regulation, differentiation and senescence. In addition to its proliferative effects, E2F1 has been shown to have potent apoptotic properties as well, under appropriate conditions. E2F1 can induce apoptosis via several p53-dependent or -independent mechanisms, either through direct transactivation of proapoptotic genes such as p73, apaf-1 (apoptosis protease-activating factor 1) and caspases or transcriptional repression. Studies presented here show that the human TRAIL promoter is E2F responsive in multiple lung cancer cell lines. Chromatin immunoprecipitation assays showed the recruitment of E2F1 as well as Rb to the TRAIL promoter and transient transfection of E2F1 could induce the expression of a TRAIL-luciferase reporter construct. TRAIL could be repressed by Rb, but this repression could be overcome by the transient transfection of ASK-1, Raf-1 or p38 kinases as well as cycling D or E, suggesting that this promoter is responsive to extracellular signals that can regulate Rb function. It has been shown that stimulation of natural killer cells by Interferon alpha-2a leads to upregulated TRAIL expression. We find that Interferon alpha 2a (IFN α 2a) stimulated lung cancer cell lines exhibited high TRAIL luciferase activity and depletion of E2F1 by siRNA techniques prevented the induction of TRAIL. These results suggest that E2F1 as a critical regulator of TRAIL expression and might facilitate death-receptor-mediated apoptosis. These observations raise the possibility that E2F-mediated regulation of TRAIL might have effects on the immune response of lung cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 267. doi:10.1158/1538-7445.AM2011-267

Viscous fingering of a miscible reactive A+B→C interface for an infinitely fast chemical reaction: Nonlinear simulations

Nonlinear dynamics of miscible viscous fingering is analyzed numerically for a reactive system when a solution containing a reactant A is displacing another miscible solution containing another reactant B. A simple A+B→C reaction takes place upon contact of the solutions. The viscosity of the fluid depends on the concentration of the various chemicals. The nonlinear fingering dynamics is studied numerically for an infinite Damköhler number Da, i.e., for an infinitely fast reaction as a function of the log-mobility ratios Rb and Rc quantifying the viscosity ratios of the solutions of B and C, respectively, versus that of the solution of A. If Rb>0, i.e., if the system is genuinely viscously unstable because the displacing solution of A is less viscous than the displaced solution of B, we analyze the changes to classical nonreactive viscous fingering induced by the reaction. If on the contrary Rb<0, which corresponds to a hydrodynamically stable case in absence of reactions, we study chemically driven viscous fingering occurring when the chemical reaction triggers a nonmonotonic viscosity profile. Comparison between the present simulation results and corresponding linear stability analysis and experiments is also conducted.

A non-functional retinoblastoma tumor suppressor (RB) pathway in premenopausal breast cancer is associated with resistance to tamoxifen

The retinoblastoma tumor suppressor (RB) is important for retaining cell cycle control and loss of RB function is commonly observed in various malignancies. Experimental and animal studies have shown that RB knockdown in ER+ (estrogen receptor) cell lines and xenografts leads to resistance to tamoxifen, indicating that RB-inactivation could be linked to impaired response to specific cancer treatments. To address this issue, we utilized a unique randomized trial including 500 premenopausal breast cancer patients receiving either two years of adjuvant tamoxifen treatment or no treatment after surgery, and defined the tamoxifen response in RB-subgroups. Non-functional RB tumors were defined by lack of concordance between RB-phosphorylation and proliferation, in comparison to RB-functional tumors displaying comparable RB-phosphorylation and proliferation. In the ER+ tumors harboring a functional RB pathway (N=204), patients benefited from adjuvant tamoxifen with fewer breast cancer recurrences (HR=0.53, 95% CI 0.34-0.81, P=0.003). In the small subgroup of ER+ and RB non-functional tumors there was no benefit of tamoxifen (HR=2.28, 95% CI 0.51-10.3, P=0.28). In a multivariate analysis, the interaction between status of the RB pathway and treatment was significant (P=0.010), validating that despite being a small subgroup of ER+ breast cancer, RB functional status appears to be linked to response to tamoxifen treatment. These findings are in line with earlier experimental data altogether suggesting that analyses of RB status in breast cancer have the potential to be one among other future predictive factors that needs to be analyzed in order to successfully identify patients that will benefit from tamoxifen treatment.

"Molecular-Targeting Prevention" of Cancer -The Theory and Its Possibilities-

In previous prevention studies, molecular targets were not intended. We then proposed the concept termed "molecular-targeting prevention" and applied it to cancer prevention. In most malignant tumors, tumor-suppressor genes, the retinoblastoma gene (RB) and/or the p53 gene are considered to be inactivated. We therefore hypothesized that RB and/or p53 might be good candidates for the molecular-targeting prevention of cancer. Interestingly, many cancer-preventive food factors were found to reactivate the lost functions of RB and/or p53 by a "gene-regulating chemoprevention" strategy. We next proposed the concept termed "combination-oriented molecular-targeting prevention", in which only the preventive effects are synergistically enhanced. We then investigated the TNF-related apoptosis-inducing ligand (TRAIL)-death receptor 5 (DR5) pathway as a candidate of the target, and found that many cancer-preventive food factors could enhance the pathway resulting in the synergistic apoptosis of various cancer cells. We hope that these strategies will contribute to the prevention of cancer.

Preimplantation-embryo-specific Cell-cycle Regulation is Attributable to a Low Expression of Retinoblastoma Protein Rather Than Its Phosphorylation

Mammalian preimplantation embryos enter the S phase immediately after the end of the M phase; their cell cycle lacks a substantial G1 phase. Previously, we suggested that the absence of the G1 phase was attributable to a loss of retinoblastoma protein (RB) function, which is required for suppression of S phase entrance and that this loss of RB function in turn was attributable to the low RB expression level during preimplantation development in mouse embryos. The present study aimed to examine whether or not RB inhibition by CDK4/6-cyclin D-dependent phosphorylation is involved in the loss of RB function in preimplantation mouse embryos by the expression of p16(INK4a), a potent endogenous inhibitor of CDK4/6-cyclin D. First, the decrease in RB expression between the four-cell and morula stages was confirmed in in vivo-derived mouse embryos. We then examined the efficiency of the p16(INK4a) expression vector in inhibiting RB phosphorylation and cell cycle progression using NIH-3T3 cells and obtained gradual RB dephosphorylation and a significantly lower proliferation rate in p16(INK4a)-transfected cells than in control cells. This indicated the successful p16(INK4a) effects on cell-cycle progression by the vector used. On the other hand, the development rate of mouse embryos injected with the p16(INK4a) expression vector was the same as that of the control embryos, although p16(INK4a) expression was detected at mRNA and protein levels in the former group but not in the control group. These results suggest that RB phosphorylation is not involved in RB dysfunction or in the lack of a G1 phase in mouse embryos and that the decrease in RB expression is important for preimplantation-embryo-specific cell-cycle regulation. Moreover, the present study indicates the similarity between preimplantation embryos and cancer cells, which p16(INK4a) expression does not arrest at the G1 phase.

Abstract P4-01-04: The Impact of the Retinoblastoma Tumor Suppressor Pathway in Her2 Positive Breast Cancer Pathogenesis and Therapeutic Response

Abstract Extensive studies have shown that breast cancer is associated with molecular aberrations that underlie the distinct subtypes of disease that exhibit differential prognosis and susceptibility to targeted therapeutic. A critical oncogenic event used to classify breast cancers is the amplification or overexpression of the human epidermal growth factor receptor 2 (Her2). Tumors harboring Her2 overexpression present aggressive characteristics related to metastatic spread and poor overall disease outcome. However, such tumors are also susceptible to targeted therapeutic agents which effectively impinge on this oncogene. Strikingly, those events that cooperate with Her2 and modulate susceptibility to targeted therapeutics remain poorly understood. Here the significance of the retinoblastoma tumor suppressor (RB) pathway in the context of Her2 positive breast cancer pathogenesis and therapeutic response was evaluated. In silico and histological analyses suggested that a subset of Her2 positive tumors exhibit compromised RB function. To model interactions between Her2 and the RB-pathway genetic deletion and knockdown strategies were employed in the context of Her2 driven tumors. These studies revealed that RB loss accelerated disease progression of such models and further enhanced the aggressive tumor phenotype. To delineate the functionality of the RB-pathway downstream from therapeutic interventions targeting Her2, two complimentary approaches were used. First, the ability to effectively intercede in Her2-positive tumors using drugs that specifically activate the RB-pathway was interrogated. The data revealed that specific CDK4/6 targeted therapies were highly effective at limiting the growth of Her2-positive tumor cells. Second, the impact of RB status on the response the Her2/EGFR antagonist lapatinib was evaluated. These studies demonstrated that loss of RB compromises the effectiveness of lapatinib in the treatment of Her2 positive breast cancer cell lines. Ongoing studies are evaluating the spontaneous mechanisms of resistance to Her2 targeted therapeutics and the significance of reestablishing RB function as a therapeutic alternative in such cancers. Together, these studies underscore the importance of the interplay between Her2 signaling and cell cycle control in breast cancer, and how this crosstalk influences the response to targeted therapeutics in the treatment of breast cancer. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-01-04.

1α,25‐dihydroxyvitamin D3 inhibits C4‐2 prostate cancer cell growth via a retinoblastoma protein (Rb)‐independent G1 arrest

The active metabolite of vitamin D, 1α,25-dihydroxyvitamin D(3) (1,25D) reduces the growth of several prostate cancer cell lines, most commonly by inducing a cell-cycle arrest in G(1). This is mediated, in part, through down-regulation of c-Myc, a positive regulator of the transcription factor, E2F. There is evidence that prostate cancer cells lacking functional retinoblastoma protein (Rb), a negative regulator of E2F activity, are poorly responsive to 1,25D treatment. Since up to 60% of prostate cancers demonstrate a loss of heterozygosity for Rb, we sought to determine whether Rb is required for the growth inhibitory effects of 1,25D. Using siRNA, Rb was reduced in C4-2 prostate cancer cells, and the response of cells to 1,25D treatment or depletion of c-myc measured by [(3)H]-thymidine incorporation and flow cytometry. The effects of 1,25D treatment on E2F levels and activity, and E2F target gene expression were also measured. 1,25D treatment and c-Myc depletion both cause a G(1) arrest inhibiting C4-2 cell proliferation independently of Rb. 1,25D reduces c-Myc expression and causes a decrease in E2F and E2F target genes. Bcl-2, an E2F target and positive regulator of C4-2 cell growth, also is down-regulated by 1,25D independently of Rb. Redundant growth inhibitory pathways compensate for the loss of Rb, and tumors lacking functional Rb may be responsive to 1,25D.

The retinoblastoma tumor suppressor controls androgen signaling and human prostate cancer progression

Retinoblastoma (RB; encoded by RB1) is a tumor suppressor that is frequently disrupted in tumorigenesis and acts in multiple cell types to suppress cell cycle progression. The role of RB in tumor progression, however, is poorly defined. Here, we have identified a critical role for RB in protecting against tumor progression through regulation of targets distinct from cell cycle control. In analyses of human prostate cancer samples, RB loss was infrequently observed in primary disease and was predominantly associated with transition to the incurable, castration-resistant state. Further analyses revealed that loss of the RB1 locus may be a major mechanism of RB disruption and that loss of RB function was associated with poor clinical outcome. Modeling of RB dysfunction in vitro and in vivo revealed that RB controlled nuclear receptor networks critical for tumor progression and that it did so via E2F transcription factor 1-mediated regulation of androgen receptor (AR) expression and output. Through this pathway, RB depletion induced unchecked AR activity that underpinned therapeutic bypass and tumor progression. In agreement with these findings, disruption of the RB/E2F/nuclear receptor axis was frequently observed in the transition to therapy resistance in human disease. Together, these data reveal what we believe to be a new paradigm for RB function in controlling prostate tumor progression and lethal tumor phenotypes.

Knockdown of Inwardly Rectifying Potassium Channel Kir2.2 Suppresses Tumorigenesis by Inducing Reactive Oxygen Species–Mediated Cellular Senescence

Senescence is an important determinant of treatment outcome in cancer therapy. In the present study, we show that knockdown of the inwardly rectifying K(+) channel Kir2.2 induced growth arrest without additional cellular stress in cancer cells lacking functional p53, p16, and/or Rb. Kir2.2 knockdown also induced senescence-associated β-galactosidase activity and upregulated senescence marker proteins in multiple cancer cell lines derived from different tissues, including prostate, stomach, and breast. Interestingly, knockdown of Kir2.2 induced a significant increase in reactive oxygen species (ROS) that was accompanied by cell cycle arrest, characterized by significant upregulation of p27, with concomitant downregulation of cyclinA, cdc2, and E2F1. Kir2.2 knockdown cells displayed increased levels of PML bodies, DNA damage (γH2AX) foci, senescence-associated heterochromatin foci, mitochondrial dysfunction, secretory phenotype, and phosphatase inactivation. Conversely, overexpression of Kir2.2 decreased doxorubicin-induced ROS accumulation and cell growth inhibition. Kir2.2 knockdown-induced cellular senescence was blocked by N-acetylcysteine, indicating that ROS is a critical mediator of this pathway. In vivo tumorigenesis analyses revealed that tumors derived from Kir2.2 knockdown cells were significantly smaller than those derived from control cells (P < 0.0001) and showed a remarkable increase in senescence-associated proteins, including senescence-associated β-galactosidase, p27, and plasminogen activator inhibitor-1. Moreover, the preestablished tumors are reduced in size after the injection of siKir2.2 (P = 0.0095). Therefore, we propose for the first time that Kir2.2 knockdown induces senescence of cancer cells by a mechanism involving ROS accumulation that requires p27, but not Rb, p53, or p16.

RB-pathway disruption in breast cancer

In breast cancer, inactivation of the RB tumor suppressor gene is believed to occur via multiple mechanisms to facilitate tumorigenesis. However, the prognostic and predictive value of RB status in disease-specific clinical outcomes has remained uncertain. We investigated RB pathway deregulation in the context of both ER-positive and ER-negative disease using combined microarray datasets encompassing over 900 breast cancer patient samples. Disease-specific characteristics of RB pathway deregulation were investigated in this dataset by evaluating correlation among pathway genes as well as differential expression across patient tumor populations defined by ER status. Survival analysis among these breast cancer samples demonstrates that the RB-loss signature is associated with poor disease outcome within several independent cohorts. Within the ER-negative subpopulation, the RB-loss signature is associated with improved response to chemotherapy and longer relapse-free survival. Additionally, while individual genes in the RB target signature closely reproduce its prognostic value, they also serve to predict and monitor response to therapeutic compounds, such as the cytostatic agent PD-0332991. These results indicate that the RB-loss signature expression is associated with poor outcome in breast cancer, but predicts improved response to chemotherapy based on data in ER-negative populations. While the RB-loss signature, as a whole, demonstrates prognostic and predictive utility, a small subset of markers could be sufficient to stratify patients based on RB function and inform the selection of appropriate therapeutic regimens.

The RB-E2F1 Pathway Regulates Autophagy

Autophagy is a protective mechanism that renders cells viable in stressful conditions. Emerging evidence suggests that this cellular process is also a tumor suppressor pathway. Previous studies showed that cyclin-dependent kinase inhibitors (CDKI) induce autophagy. Whether retinoblastoma protein (RB), a key tumor suppressor and downstream target of CDKIs, induces autophagy is not clear. Here, we show that RB triggers autophagy and that the RB activators p16INK4a and p27/kip1 induce autophagy in an RB-dependent manner. RB binding to E2 transcription factor (E2F) is required for autophagy induction and E2F1 antagonizes RB-induced autophagy, leading to apoptosis. Downregulation of E2F1 in cells results in high levels of autophagy. Our findings indicate that RB induces autophagy by repressing E2F1 activity. We speculate that this newly discovered aspect of RB function is relevant to cancer development and therapy.

The Neutrophil Elastase Inhibitor Elafin Triggers Rb-Mediated Growth Arrest and Caspase-Dependent Apoptosis in Breast Cancer

Elafin, an endogenous inhibitor of neutrophil elastase, is expressed in human mammary epithelial cells but is transcriptionally downregulated in breast cancer cells. We hypothesized that elafin may exert a tumor-suppressive activity in the context of breast cancer. In this study, we show that the retinoblastoma (Rb) pathway governs the antitumor properties of elafin. In breast cancer cells with functional Rb, the expression of elafin triggered Rb-dependent cell cycle arrest. Elafin also exhibited suppressive activity in breast cancer cell lines lacking Rb, but this was associated with an induction of caspase-3-dependent, p53-independent apoptotic cell death. Normal mammary epithelial cells were not affected by elafin. Collectively, these results argue that elafin mediates tumor-suppressive effects that are cytostatic or cytotoxic depending on the Rb status. Our findings suggest that elafin could be engineered as a therapeutic modality to treat breast cancer without toxicity to normal proliferating cells.

MiR-335 Directly Targets Rb1 (pRb/p105) in a Proximal Connection to p53-Dependent Stress Response

Loss-of-function mutations of retinoblastoma family (Rb) proteins drive tumorigenesis by overcoming barriers to cellular proliferation. Consequently, factors modulating Rb function are of great clinical import. Here, we show that miR-335 is differentially expressed in human cancer cells and that it tightly regulates the expression of Rb1 (pRb/p105) by specifically targeting a conserved sequence motif in its 3' untranslated region. We found that by altering Rb1 (pRb/p105) levels, miR-335 activates the p53 tumor suppressor pathway to limit cell proliferation and neoplastic cell transformation. DNA damage elicited an increase in miR-335 expression in a p53-dependent manner. miR-335 and p53 cooperated in a positive feedback loop to drive cell cycle arrest. Together, these results indicate that miR-335 helps control proliferation by balancing the activities of the Rb and p53 tumor suppressor pathways. Further, they establish that miR-335 activation plays an important role in the induction of p53-dependent cell cycle arrest after DNA damage.

Gadd45b Mediates Fas-induced Apoptosis by Enhancing the Interaction between p38 and Retinoblastoma Tumor Suppressor

Gadd45b has been known as a positive mediator of apoptosis induced by certain cytokines and oncogenes. Here, we identified Gadd45b as an effector of Fas-induced apoptosis and found that p38-mediated Rb hyperphosphorylation is one of the mechanisms of Fas-induced apoptosis in murine hepatocyte AML12 cells. Gadd45b has been shown to activate p38 through its physical interaction with MTK1 and induce apoptosis. However, in this study, we have showed that the function of Gadd45b during Fas-induced apoptosis in AML12 cells is different from that reported in previous studies. Depletion of Gadd45b expression did not inhibit the phosphorylation of p38, but it suppressed p38-mediated Rb phosphorylation and apoptosis in response to Fas stimulation by reducing the interaction between p38 and Rb. Ectopic expression of Gadd45b was sufficient to enhance this interaction. These findings suggest that Gadd45b mediates p38-induced Rb phosphorylation by enhancing the interaction between p38 and Rb during Fas-induced apoptosis in murine hepatocytes.