RB in Breast Cancer: The Crossroads of Tumorigenesis and Treatment

Cancer is a highly heterogeneous disease, wherein specific determinants modulate disease severity and therapeutic outcomes. In breast cancer, significant effort has been channeled into defining critical genetic effectors of disease behavior. One key molecular determinant is the retinoblastoma tumor suppressor (RB), which is functionally inactivated in the majority of human cancers, and aberrant in nearly half of breast cancers. Deficiency in RB function compromises cell cycle checkpoints, contributes to aggressive tumor proliferation, and is associated with advanced disease. Recent investigation indicates that RB-deficiency has dramatic and disparate effects on the response to therapeutic modalities utilized in the treatment of breast cancer. Loss of RB function promotes inappropriate cell cycle progression during therapeutic challenge. In the context of cytotoxic therapies, this lack of checkpoint function leads to increased sensitivity to the agent. However, RB-deficiency efficiently bypasses the anti-mitogenic function of hormonal therapies and is associated with early disease recurrence following tamoxifen therapy. Thus, RB-pathway status has powerful effects on both tumorigenic proliferation and therapeutic response, and may represent a critical basis for informing breast cancer therapy.     

Role of the retinoblastoma protein in cell cycle arrest mediated by a novel cell surface proliferation inhibitor

A novel cell regulatory sialoglycopeptide (CeReS-18), purified from the cell surface of bovine cerebral cortex cells has been shown to be a potent and reversible inhibitor of proliferation of a wide array of fibroblasts as well as epithelial-like cells and nontransformed and transformed cells. To investigate the possible mechanisms by which CeReS-18 exerts its inhibitory action, the effect of the inhibitor on the posttranslational regulation of the retinoblastoma susceptibility gene product (RB), a tumor suppressor gene, has been examined. It is shown that CeReS-18 mediated cell cycle arrest of both human diploid fibroblasts (HSBP) and mouse fibroblasts (Swiss 3T3) results in the maintenance of the RB protein in the hypophosphorylated state, consistent with a late G1 arrest site. Although their normal nontransformed counterparts are sensitive to cell cycle arrest mediated by CeReS-18, cell lines lacking a functional RB protein, through either genetic mutation or DNA tumor virus oncoprotein interaction, are less sensitive. The refractory nature of these cells is shown to be independent of specific surface receptors for the inhibitor, and another tumor suppressor gene (p53) does not appear to be involved in the CeReS-18 inhibition of cell proliferation. The requirement for a functional RB protein product, in order for CeReS-18 to mediate cell cycle arrest, is discussed in light of regulatory events associated with density-dependent growth inhibition. © 1994 Wiley-Liss, Inc.     

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.     

Role of the retinoblastoma tumor suppressor in the maintenance of genome integrity

The retinoblastoma tumor suppressor (RB) is functionally inactivated at high frequency in human cancers. Based on the role of RB as a negative regulator of cell cycle this event would be expected to contribute to deregulated proliferation. However, evidence suggests that loss of RB not only mediates aberrant proliferation, but compromises the fidelity of cell cycle transitions leading to a breakdown in genome integrity. This review is focused on the mechanisms underlying this facet of RB function and the contribution of this process to tumorigenesis.     

Differential requirements for ras and the retinoblastoma tumor suppressor protein in the androgen dependence of prostatic adenocarcinoma cells.

Prostate cells are dependent on androgen for proliferation, but during tumor progression prostate cancer cells achieve independence from the androgen requirement. We report that androgen withdrawal fails to inhibit cell cycle progression or influence the expression of cyclin-dependent kinase (CDK)/cyclins in androgen-independent prostate cancer cells, indicating that these cells signal for cell cycle progression in the absence of androgen. However, phosphorylation of the retinoblastoma tumor suppressor protein (RB) is still required for G1-S progression in androgen-independent cells, since the expression of constitutively active RB (PSM-RB) or p16ink4a caused cell cycle arrest and mimicked the effects of androgen withdrawal on downstream targets in androgen-dependent LNCaP cells. Since Ras is known to mediate mitogenic signaling to RB, we hypothesized that active V12Ras would induce androgen-independent cell cycle progression in LNCaP cells. Although V12Ras was able to stimulate ERK phosphorylation and induce cyclin D1 expression in the absence of androgen, it was not sufficient to promote androgen-independent cell cycle progression. Similarly, ectopic expression of CDK4/cyclin D1, which stimulated RB phosphorylation in the presence of androgen, was incapable of inactivating RB or driving cell cycle progression in the absence of androgen. We show that androgen regulates both CDK4/cyclin D1 and CDK2 complexes to inactivate RB and initiate cell cycle progression. Together, these data show that androgen independence is achieved via deregulation of the androgen to RB signal, and that this signal can only be partially initiated by the Ras pathway in androgen-dependent cells.     

视网膜母细胞瘤基因1(RB1)研究进展

视网膜母细胞瘤基因1(Retinoblastoma1,RB1)是人类第一个分离克隆的抑癌基因。RB1基因是细胞周期的负调控因子,通过与转录因子结合调节细胞增殖和分化所需基因的表达,从而维持细胞生长发育的平衡。因此,该基因的功能与细胞周期、细胞衰老、细胞凋亡、细胞分化和生长抑制等有关。文章对RB1基因的结构、表达及其功能的研究进展进行了综述。     

MicroRNA-192 targeting retinoblastoma 1 inhibits cell proliferation and induces cell apoptosis in lung cancer cells

microRNAs play an important roles in cell growth, differentiation, proliferation and apoptosis. They can function either as tumor suppressors or oncogenes. We found that the overexpression of miR-192 inhibited cell proliferation in A549, H460 and 95D cells, and inhibited tumorigenesis in a nude mouse model. Both caspase-7 and the PARP protein were activated by the overexpression of miR-192, thus suggesting that miR-192 induces cell apoptosis through the caspase pathway. Further studies showed that retinoblastoma 1 (RB1) is a direct target of miR-192. Over-expression of miR-192 decreased RB1 mRNA and protein levels and repressed RB1-3'-UTR reporter activity. Knockdown of RB1 using siRNA resulted in a similar cell morphology as that observed for overexpression of miR-192. Additionally, RB1-siRNA treatment inhibited cell proliferation and induced cell apoptosis in lung cancer cells. Analysis of miRNA expression in clinical samples showed that miR-192 is significantly downregulated in lung cancer tissues compared to adjacent non-cancerous lung tissues. In conclusion, our results demonstrate that miR-192 is a tumor suppressor that can target the RB1 gene to inhibit cell proliferation and induce cell apoptosis in lung cancer cells. Furthermore, miR-192 was expressed at low levels in lung cancer samples, indicating that it might be a promising therapeutic target for lung cancer treatment.     

RB-dependent S-phase response to DNA damage

The retinoblastoma tumor suppressor protein (RB) is a potent inhibitor of cell proliferation. RB is expressed throughout the cell cycle, but its antiproliferative activity is neutralized by phosphorylation during the G(1)/S transition. RB plays an essential role in the G(1) arrest induced by a variety of growth inhibitory signals. In this report, RB is shown to also be required for an intra-S-phase response to DNA damage. Treatment with cisplatin, etoposide, or mitomycin C inhibited S-phase progression in Rb(+/+) but not in Rb(-/-) mouse embryo fibroblasts. Dephosphorylation of RB in S-phase cells temporally preceded the inhibition of DNA synthesis. This S-phase dephosphorylation of RB and subsequent inhibition of DNA replication was observed in p21(Cip1)-deficient cells. The induction of the RB-dependent intra-S-phase arrest persisted for days and correlated with a protection against DNA damage-induced cell death. These results demonstrate that RB plays a protective role in response to genotoxic stress by inhibiting cell cycle progression in G(1) and in S phase.     

Regulated expression of the RB "tumor suppressor gene" in normal lymphocyte mitogenesis: elevated expression in transformed leukocytes and role as a "status quo" gene.

Expression of the RB retinoblastoma tumor suppressor gene product is regulated early during the stimulation of normal human peripheral blood lymphocytes, suggesting a regulatory role for the amount of this protein in mitogenesis of normal cells. When normal human peripheral blood lymphocytes were mitogenically stimulated with pokeweed mitogen, bivariate flow cytometric measurements of cellular DNA and RB protein content showed an early decrease in the amount of RB protein per cell, anteceding onset of S phase. A subsequent increase in the amount of RB protein per cell occurred with cell proliferation. Thus the amount of RB protein relative to the total cell mass underwent a biphasic response with mitogenesis. The resulting proliferating cells had a slightly elevated level of RB protein per cell compared to the unstimulated cells. Comparison of other proliferating leukocytes to normal lymphocytes showed that both EBV virally transformed lymphocytes and human promyelocytic leukemia cells (HL-60) had elevated levels of RB protein per cell compared to normal peripheral blood lymphocytes. Mitogenic stimulation or transformation by other means thus is associated with regulation of the amount of RB protein per cell, suggesting a regulatory role for the RB protein in normal cell growth control.     

Unique Impact of RB Loss on Hepatic Proliferation: TUMORIGENIC STRESSES UNCOVER DISTINCT PATHWAYS OF CELL CYCLE CONTROL

The retinoblastoma (RB) tumor suppressor pathway is disrupted at high frequency in hepatocellular carcinoma. However, the mechanisms through which RB modulates physiological responses in the liver remain poorly defined. Despite the well established role of RB in cell cycle control, the deletion of RB had no impact on the kinetics of cell cycle entry or the restoration of quiescence during the course of liver regeneration. Although these findings indicated compensatory effects from the RB-related proteins p107 and p130, even the dual deletion of RB with p107 or p130 failed to deregulate hepatic proliferation. Furthermore, although these findings suggested a modest role for the RB-pathway in the context of proliferative control, RB loss had striking effects on response to the genotoxic hepatocarcinogen diethylnitrosamine. With diethylnitrosamine, RB deletion resulted in inappropriate cell cycle entry that facilitated secondary genetic damage and further uncoupling of DNA replication with mitotic entry. Analysis of the mechanism underlying the differential impact of RB status on liver biology revealed that, while liver regeneration is associated with the conventional induction of cyclin D1 expression, the RB-dependent cell cycle entry, occurring with diethylnitrosamine treatment, was independent of cyclin D1 levels and associated with the specific induction of E2F1. Combined, these studies demonstrate that RB loss has disparate effects on the response to unique tumorigenic stresses, which is reflective of distinct mechanisms of cell cycle entry.     

RB status governs differential sensitivity to cytotoxic and molecularly-targeted therapeutic agents

The retinoblastoma tumor suppressor (RB) is frequently inactivated in human cancers and has been shown to modulate the anti-proliferative effects of DNA-damaging therapies. However, the impact of RB loss on response to disparately functioning cytotoxic agents as well as targeted therapies is poorly understood. Here 3T3-immortalized and Ras-transformed mouse adult fibroblasts (MAFs) containing conditional RB alleles were utilized to investigate the consequence of RB loss on cellular response to cytotoxic agents and therapies targeting the MEK and PI3K pathways. Using these models, we demonstrate that RB deficiency is associated with bypass of therapy-induced checkpoints in response to both cytotoxic and targeted treatments. Interestingly, while checkpoint bypass following treatment with cytotoxic therapy results in an agent specific increase in drug sensitivity, checkpoint bypass following treatment targeting MEK and PI3K function results in increased cellular proliferation. These results indicate that RB status differentially impacts therapeutic response and should be considered when evaluating the efficacy of molecularly targeted therapeutics.     

p53 Moves to Mitochondria: A Turn on the Path to Apoptosis

It has been said that no matter which direction cancer research turns, the p53 tumor suppressor protein comes into view. The widespread role of p53 as a suppressor of tumor development is believed to rely on its ability to induce programmed cell death in response to stress, either the replicative stress associated with uncontrolled cellular proliferation, or the environmental stresses that accompany tumor development, such as hypoxia. For some time it has been believed that the role of p53 in inducing apoptosis in response to such stress was as a master regulator coordinating the expression of other molecules whose ultimate role was the execution of the cell. New data, however, suggest that p53 itself also has a direct role in accomplishing cell death, at the mitochondria.     

Differential Impact of Tumor Suppressor Pathways on DNA Damage Response and Therapy-Induced Transformation in a Mouse Primary Cell Model

The RB and p53 tumor suppressors are mediators of DNA damage response, and compound inactivation of RB and p53 is a common occurrence in human cancers. Surprisingly, their cooperation in DNA damage signaling in relation to tumorigenesis and therapeutic response remains enigmatic. In the context of individuals with heritable retinoblastoma, there is a predilection for secondary tumor development, which has been associated with the use of radiation-therapy to treat the primary tumor. Furthermore, while germline mutations of the p53 gene are critical drivers for cancer predisposition syndromes, it is postulated that extrinsic stresses play a major role in promoting varying tumor spectrums and disease severities. In light of these studies, we examined the tumor suppressor functions of these proteins when challenged by exposure to therapeutic stress. To examine the cooperation of RB and p53 in tumorigenesis, and in response to therapy-induced DNA damage, a combination of genetic deletion and dominant negative strategies was employed. Results indicate that loss/inactivation of RB and p53 is not sufficient for cellular transformation. However, these proteins played distinct roles in response to therapy-induced DNA damage and subsequent tumorigenesis. Specifically, RB status was critical for cellular response to damage and senescence, irrespective of p53 function. Loss of RB resulted in a dramatic evolution of gene expression as a result of alterations in epigenetic programming. Critically, the observed changes in gene expression have been specifically associated with tumorigenesis, and RB-deficient, recurred cells displayed oncogenic characteristics, as well as increased resistance to subsequent challenge with discrete therapeutic agents. Taken together, these findings indicate that tumor suppressor functions of RB and p53 are particularly manifest when challenged by cellular stress. In the face of such challenge, RB is a critical suppressor of tumorigenesis beyond p53, and RB-deficiency could promote significant cellular evolution, ultimately contributing to a more aggressive disease.     

Multiple pathways counteract cell death induced by RB1 loss: Implications for cancer

Inactivation of the tumor suppressor RB1 leads to cell proliferation, cell death and abortive differentiation in certain tissues and physiological contexts. Anti-apoptotic signals are thought to be the most important mechanism by which RB1-mutant cells escape cell death. Indeed, in the course of neoplastic transformation RB1 is often inactivated in conjunction with a mutation in the pro-apoptotic tumor suppressor p53. We have previously devised a biological framework to identify factors that maintain survival of differentiating Rb-deficient muscle fibers. We showed that differentiating Rb-deficient myoblasts fuse to form short myotubes that degenerate in a process associated with enhanced autophagy, and that degeneration was rescued by antagonists of apoptosis or autophagy, induction of mitochondrial-biogenesis or hypoxia-induced glycolytic shift, leading to long, twitching myotubes. Here, we also show that lithium slows the collapse of Rb-deficient myotubes and surprisingly, this is independent of autophagy, cyclin D3 and β-catenin. Thus, several distinct processes can suppress cell death induced by RB1 loss. We discuss these pathways and how they may cooperate with RB1 inactivation in the course of cancer initiation.     

Radiation dose-dependent maintenance of G(2) arrest requires retinoblastoma protein

In response to ionizing radiation (IR), cell cycle checkpoints are activated to provide time for DNA repair. Several different checkpoint mechanisms have been elucidated. However, mechanisms that regulate the duration of cell cycle arrest are not understood. Previous studies have shown that the retinoblastoma tumor suppressor protein (RB) is required for radiation-induced G1 arrest. Working with primary fibroblasts derived from Rb+/+ and Rb-/- mouse embryos, we show that RB also regulates the duration of G2 arrest. The initial G2 checkpoint response is enhanced in Rb-/- cells due to a defect in G1 arrest. However, the permanent arrest in G2 induced by higher doses of IR does not occur in Rb-/- cells. Rb-/- cells either resumed proliferation or underwent apoptosis at IR doses that caused the majority of Rb+/+ cells to arrest permanently in G2. The prolongation of G2 arrest in Rb+/+ cells correlated with a gradual accumulation of hypophosphorylated RB. Thus, regulation of the RB function may be an important aspect in the maintenance of cell cycle checkpoints in DNA damage response.     

The 100-kDa Proteolytic Fragment of RB Is Retained Predominantly within the Nuclear Compartment of Apoptotic Cells

The retinoblastoma tumor suppressor protein (RB) has been shown to play a role in regulating the eukaryotic cell cycle, promoting cellular differentiation, and modulating programmed cell death. Although regulation of RB tumor suppressor activity is mediated by reversible phosphorylation, an additional posttranslational modification involves the cleavage of 42 residues from the carboxy terminus of RB during the onset of drug-induced or receptor-mediated apoptosis. We now demonstrate that a recombinant p100cl RB species localizes to the nucleus where it may retain wildtype “pocket” protein binding activity. In addition, using immunocytochemistry, we show that cleavage of the endogenous RB protein occurs in vivo in human cells and that p100cl is predominantly retained within the nuclear compartment of cells during early apoptosis. We also show that the carboxy-terminal cleavage of RB is detected immediately following caspase-3 and PARP cleavage during FAS-mediated apoptosis of MCF10 cells. These findings suggest that this cleavage event may be a component of a downstream cascade during programmed cell death.