Tumor Suppressors RB1 and CDKN2a Cooperatively Regulate Cell-Cycle Progression and Differentiation During Cardiomyocyte Development and Repair.

RATIONALE: Although rare cardiomyogenesis is reported in the adult mammalian heart, whether this results from differentiation or proliferation of cardiomyogenic cells remains controversial. The tumor suppressor genes RB1 (retinoblastoma) and CDKN2a (cyclin-dependent kinase inhibitor 2a) are critical cell-cycle regulators, but their roles in human cardiomyogenesis remains unclear. OBJECTIVE: We hypothesized that developmental activation of RB1 and CDKN2a cooperatively cause permanent cell-cycle withdrawal of human cardiac precursors (CPCs) driving terminal differentiation into mature cardiomyocytes, and that dual inactivation of these tumor suppressor genes promotes myocyte cell-cycle reentry. METHODS AND RESULTS: Directed differentiation of human pluripotent stem cells (hPSCs) into cardiomyocytes revealed that RB1 and CDKN2a are upregulated at the onset of cardiac precursor specification, simultaneously with GATA4 (GATA-binding protein 4) homeobox genes PBX1 (pre-B-cell leukemia transcription factor 1) and MEIS1 (myeloid ecotropic viral integration site 1 homolog), and remain so until terminal cardiomyocyte differentiation. In both GATA4+ hPSC cardiac precursors and postmitotic hPSC-cardiomyocytes, RB1 is hyperphosphorylated and inactivated. Transient, stage-specific, depletion of RB1 during hPSC differentiation enhances cardiomyogenesis at the cardiac precursors stage, but not in terminally differentiated hPSC-cardiomyocytes, by transiently upregulating GATA4 expression through a cell-cycle regulatory pathway involving CDKN2a. Importantly, cytokinesis in postmitotic hPSC-cardiomyocytes can be induced with transient, dual RB1, and CDKN2a silencing. The relevance of this pathway in vivo was suggested by findings in a porcine model of cardiac cell therapy post-MI, whereby dual RB1 and CDKN2a inactivation in adult GATA4+ cells correlates with the degree of scar size reduction and endogenous cardiomyocyte mitosis, particularly in response to combined transendocardial injection of adult human hMSCs (bone marrow-derived mesenchymal stromal cells) and cKit+ cardiac cells. CONCLUSIONS: Together these findings reveal an important and coordinated role for RB1 and CDKN2a in regulating cell-cycle progression and differentiation during human cardiomyogenesis. Moreover, transient, dual inactivation of RB1 and CDKN2a in endogenous adult GATA4+ cells and cardiomyocytes mediates, at least in part, the beneficial effects of cell-based therapy in a post-MI large mammalian model, a finding with potential clinical implications.

Concomitant inactivation of Rb and E2f8 in hematopoietic stem cells synergizes to induce severe anemia.

The retinoblastoma (Rb) tumor suppressor plays important roles in regulating hematopoiesis, particularly erythropoiesis. In an effort to understand whether Rb function can be mediated by E2F transcription factors in a BM-derived hematopoietic system in mice, we uncovered a functional synergy between Rb and E2F8 to promote erythropoiesis and to prevent anemia. Specifically, whereas Mx1-Cre-mediated inactivation of Rb or E2f8 in hematopoietic stem cells only led to mild erythropoietic defects, concomitant inactivation of both genes resulted in marked ineffective erythropoiesis and mild hemolysis, leading to severe anemia despite the presence of enhanced extramedullary erythropoiesis. Interestingly, although ineffective erythropoiesis was already present in the RbΔ/Δ mice and exacerbated in the RbΔ/Δ;E2f8Δ/Δ mice, hemolysis was exclusively manifested in the double-knockout mice. Using an adoptive transfer system and an erythroid-specific knockout system, we have shown that the synergy of Rb and E2f8 deficiency in triggering severe anemia is intrinsic to the erythroid lineage. Surprisingly, concomitant inactivation of Rb and E2f7, a close family member of E2f8, did not substantially worsen the erythropoietic defect resulted from Rb deficiency. The results of the present study reveal the specificity of E2F8 in mediating Rb function in erythropoiesis and suggest critical and overlapping roles of Rb and E2f8 in maintaining normal erythropoiesis and in preventing hemolysis.

A negative feedback signaling network underlies oncogene-induced senescence.

Oncogene-induced senescence functions to limit tumor development. However, a complete understanding of the signals that trigger this type of senescence is currently lacking. We found that mutations affecting NF1, Raf, and Ras induce a global negative feedback response that potently suppresses Ras and/or its effectors. Moreover, these signals promote senescence by inhibiting the Ras/PI3K pathway, which can impact the senescence machinery through HDM2 and FOXO. This negative feedback program is regulated in part by RasGEFs, Sprouty proteins, RasGAPs, and MKPs. Moreover, these signals function in vivo in benign human tumors. Thus, the ultimate response to the aberrant activation of the Ras pathway is a multifaceted negative feedback signaling network that terminates the oncogenic signal and participates in the senescence response.

Adenovirus-mediated retinoblastoma gene therapy suppresses spontaneous pituitary melanotroph tumors in Rb+/- mice.

The retinoblastoma gene (RB) is the prototypic tumor suppressor. Studies to date have demonstrated cancer suppression with tumor cells reconstituted with RB ex vivo and implanted into immunodeficient mice, as well as with germline transmission of a human RB transgene into tumor-prone Rb +/- mice. To mimic the therapy of cancer more closely, spontaneous pituitary melanotroph tumors arising in immunocompetent Rb +/- mice were treated with a recombinant adenovirus carrying RB cDNA. Intratumoral RB gene transfer decreased tumor cell proliferation, reestablished innervation by growth-regulatory dopaminergic neurons, inhibited the growth of tumors, and prolonged the life spans of treated animals.

Retinoblastoma tumor suppressor gene expression determines the response to sequential flavopiridol and doxorubicin treatment in small-cell lung carcinoma.

PURPOSE: Small-cell lung cancers (SCLC) are defective in many regulatory mechanisms that control cell cycle progression, i.e., functional retinoblastoma protein (pRb). Flavopiridol inhibits proliferation and induces apoptosis in SCLC cell lines. We hypothesized that the sequence flavopiridol followed by doxorubicin would be synergistic in pRb-deficient SCLC cells. EXPERIMENTAL DESIGN: A H69 pRb-deficient SCLC cell line, H865, with functional pRb and H865 pRb small interfering RNA (siRNA) knockdown cells were used for in vitro and in vivo experiments. The in vivo efficiencies of various sequential combinations were tested using nude/nude athymic mice and human SCLC xenograft models. RESULTS: Flavopiridol then doxorubicin sequential treatment was synergistic in the pRB-negative H69 cell line. By knocking down pRb with specific siRNA, H865 clones with complete pRb knockdown became sensitive to flavopiridol and doxorubicin combinations. pRb-deficient SCLC cell lines were highly sensitive to flavopiridol-induced apoptosis. pRb-positive H865 cells arrested in G0-G1 with flavopiridol exposure, whereas doxorubicin and all flavopiridol/doxorubicin combinations caused a G2-M block. In contrast, pRb-negative SCLC cells did not arrest in G0-G1 with flavopiridol exposure. Flavopiridol treatment alone did not have an in vivo antitumor effect, but sequential flavopiridol followed by doxorubicin treatment provided tumor growth control and a survival advantage in Rb-negative xenograft models, compared with the other sequential treatments. CONCLUSIONS: Flavopiridol and doxorubicin sequential treatment induces potent in vitro and in vivo synergism in pRb-negative SCLC cells and should be clinically tested in tumors lacking functional pRB.

The retinoblastoma protein and the regulation of cell cycling.

Increasing attention has been focused on how the retinoblastoma (RB) protein regulates cell growth. Recent evidence indicates that it is a substrate for phosphorylation by cyclin-dependent kinase-cyclin complexes and suggests that this phosphorylation modulates the ability of this protein to regulate transit through the cell cycle, perhaps in its G1 phase.

New roles for the RB tumor suppressor protein.

For a gene whose existence was first postulated in 1971, was cloned in 1986 and whose functions have been extensively characterized ever since, you might be inclined to think there was not much new to report regarding the retinoblastoma tumor suppressor gene (RB)--but you would be wrong to make such an assumption. RB is still piquing our interest with several activities defined over the past year that reveal new and exciting roles for this key tumor suppressor gene. These functions include regulation of senescence through specific gene silencing mechanisms, control of developmental processes in extra-embryonic tissues, maintaining tissue homeostasis and determining survival responses to chemotherapy.

Molecular genetics of RB1--the retinoblastoma gene.

Thirty-seven years ago Alfred Knudson proposed his "two-hit" theory of the molecular etiology of retinoblastoma, establishing an elegant conceptual paradigm for tumorigenesis in general. A great body of work has subsequently elucidated the structure and function of the RB1 gene and the biology of its protein product, pRB. Multiple categories of mutations have been characterized and correlated with phenotypic expression, including translocations, deletions, insertions, and point mutations. The purpose of this review is to provide a concise overview of the molecular genetics and genotype-phenotype correlations in retinoblastoma.

HBeAg-induced miR-106b promotes cell growth by targeting the retinoblastoma gene.

Chronic HBV infection is a major cause of hepatocellular carcinoma (HCC). The association between hepatitis B "e" antigen (HBeAg) and HCC is well-established by epidemiological studies. Nonetheless, the biological role of HBeAg in HCC remains enigmatic. We investigate the role of HBeAg in HBV-related HCC. Our findings suggest that HBeAg enhances cell proliferation and accelerates progression from G0/G1 phase to the S phase of the cell cycle in Huh7 cells. Examination of host gene expression and miRNA expression profiles reveals a total of 21 host genes and 12 host miRNAs that were differentially regulated in cells expressing HBeAg. Importantly, HBeAg induced the expression of miR-106b, an oncogenic miRNA. Interestingly, HBeAg-expression results in a significant reduction in the expression of retinoblastoma (Rb) gene, an experimentally validated target of miR-106b. Inhibition of miR-106b significantly increased the expression of the Rb gene, resulting in reduced cell proliferation and slowing of cell cycle progression from the G0/G1 phase to S phase. These observations suggest that the up-regulation of miR-106b by HBeAg contributes to the pathogenesis of HBV-related HCC by down-regulating the Rb gene. Our results highlight a role for HBeAg in HCC and provide a novel perspective on the molecular mechanisms underlying HBV-related HCC.

The retinoblastoma gene regulates somatic growth during mouse development.

Overexpression of the retinoblastoma gene (Rb) in mice leads to the dwarf phenotype. To explore the potential mechanism of Rb effects on the somatic growth, bitransgenic mice with tetracycline-regulated Rb expression were generated, and their phenotypes were compared with those of previously established Rb mouse models. By gestational day 12.5, embryos lacking Rb and those expressing twice the regular amount of Rb are 15% larger and 10-30% smaller, respectively, compared with their wild-type littermates. The dwarf phenotype is associated with increased plasma levels of insulin-like growth factor-I (IGF-I) but not with growth hormone and glucose concentrations. Down-regulation of the Rb transgene expression results in a reduction of the IGF-I plasma concentrations to normalcy and an increase of somatic growth prenatally and postnatally. Consistent with the in vivo results, cells overexpressing Rb require higher thresholds of IGF-I to stimulate proliferation. Thus, Rb plays an integral role for mouse somatic growth and maintenance during ontogenesis, and IGF-I pathway is likely to be a target for such regulation.

Transforming growth factor beta 1 suppresses genomic instability independent of a G1 arrest, p53, and Rb.

Alterations in expression of or responsiveness to transforming growth factor beta (TGF-beta) are frequently found in human and animal epithelial cancers and are though to be important for loss of growth control in the neoplastic cell. We show here that keratinocyte cell lines from mice with a targeted deletion of the TGF-beta 1 gene have significantly increased frequencies of gene amplification in response to the drug N-phosphonoacetyl-L-aspartate (PALA) compared to TGF-beta 1-expressing control keratinocyte cell lines. In contrast to the control lines, the PALA-mediated G1 arrest did not occur in the TGF-beta 1 null keratinocytes despite the presence of wild-type p53 in both genotypes. Exogenous TGF-beta 1 suppresses gene amplification in the null keratinocytes at concentrations that do not cause a G1 growth arrest and in human tumor cell lines that are insensitive to TGF-beta 1-mediated growth inhibition. The pathway of TGF-beta 1 suppression is independent of the p53 and Rb genes, but requires an intact TGF-beta type II receptor. These studies reveal a novel TGF-beta-mediated pathway regulating genomic stability and suggest that defects in TGF-beta signaling may have profound effects on tumor progression independent of cell proliferation.

Changes in growth and tumorigenicity following reconstitution of retinoblastoma gene function in various human cancer cell types by microcell transfer of chromosome 13.

Functional loss of the retinoblastoma (RB) gene has been implicated in the initiation or progression of several human tumor types including cancer of the eye, bone, bladder, and prostate. To examine the consequence of adding one RB allele containing its normal regulatory elements back into representative examples of each of these cancer types, as well as to compare the results to those previously reported using various RB complementary DNA constructs, a neomycin resistant marked 13 chromosome was transferred by microcell fusion. Several attempts to obtain RB positive osteosarcoma cells failed. In addition, only one RB positive retinoblastoma clone was isolated. This clone contained many large cells, could not be maintained in long-term culture, and produced only RB negative tumors. Three RB positive bladder cancer cell clones were obtained, all of which grew slower in culture than their RB negative parental counterpart and did not form colonies in soft agar. Tumorigenicity was markedly suppressed in these clones. One clone yielded no tumors, and the other 2 clones produced only one small tumor each, both of which were RB negative. In contrast, the 2 RB positive prostate cancer cell clones isolated had no differences in their cell culture growth properties, including growth in soft agar compared to the parental cells. One of the clones was nontumorigenic, while the other clone produced 4 small tumors, all of which were RB positive. These results indicate that the transfer of one RB allele by microcell transfer produces different levels of growth inhibition as well as tumor suppression, depending on the cell type examined. In the case of prostate cancer, the function of the RB gene in tumor suppression appears to be independent from its growth regulatory function, since no growth inhibition in cell culture was noted in these cells, although tumor suppression was significant.

Inactivation of the retinoblastoma tumor suppressor induces apoptosis protease-activating factor-1 dependent and independent apoptotic pathways during embryogenesis.

Inactivation of the retinoblastoma (Rb) tumor suppressor in the mouse induces mid-gestational death accompanied by massive apoptosis in certain tissues. Herein, we analyzed the role of the apoptosis protease-activating factor Apaf-1, an essential component of the apoptosome, in mediating apoptosis in Rb-deficient mice. Analysis of compound mutant embryos lacking Rb and Apaf-1 revealed that Apaf-1 was absolutely required for apoptosis in the central nervous system and lens. In contrast, apoptosis in the peripheral nervous system and skeletal muscles only partly depended on Apaf-1 function. The dependency on Apaf-1 coincided with the requirement documented previously for E2F1 and p53 in the respective tissues. Loss of Apaf-1 specifically suppressed apoptosis but not the proliferation and differentiation defects in Rb-mutant embryos. We also show that the Apaf1+ but not the Rb+ allele is retained in pituitary tumors arising in Rb+/-:Apaf1+/- double heterozygous mice. Our results indicate that Apaf-1 plays a critical role in apoptosis in a subset of tissues and that both E2F1:p53:Apaf-1-dependent and -independent apoptotic pathways operate downstream of Rb.

Sex hormone-induced carcinogenesis in Rb-deficient prostate tissue.

The retinoblastoma (Rb) gene product is a prototypic tumor suppressor. Mice lacking the Rb gene are not viable and die in utero at approximately 13 days of gestation. In this study, we have rescued Rb-/- prostates by grafting pelvic organ rudiments from Rb-/- mouse embryos under the renal capsule of adult male nude mouse hosts. Grafts of embryonic pelvic organs developed into functional prostatic tissue. Some of the prostatic tissue generated was further used to construct chimeric prostatic tissue recombinants by combining wild-type rat urogenital mesenchyme (rUGM) with Rb-/- and Rb+/+ prostatic epithelium (PRE). The tissue recombinants were grown as subcapsular renal grafts and treated from the time of grafting with Silastic capsules containing 25 mg of testosterone plus 2.5 mg of estradiol. During 5-8 weeks of hormone treatment, rUGM+Rb+/+PRE tissue recombinants developed prostatic hyperplasia, whereas PRE in rUGM+Rb-/-PRE tissue recombinants developed hyperplasia, atypical hyperplasia, and carcinoma. During carcinogenesis in rUGM+Rb-/-PRE tissue recombinants, prostatic epithelial cells of the basal lineage disappeared, whereas the luminal cells underwent carcinogenesis. Epithelial E-cadherin almost totally disappeared. In all cases, epithelial PCNA labeling was elevated in tissue recombinants containing Rb-/- versus Rb+/+ epithelium. These epithelial changes were associated with almost total loss of smooth muscle cells in the stroma. In contrast, in untreated hosts rUGM+Rb+/+PRE tissue recombinants developed normally, and rUGM+Rb-/-PRE tissue recombinants developed mild epithelial hyperplasia. The results of this study demonstrate that Rb-/- prostatic tissue can be rescued from embryonic lethal mice and used to test its susceptibility to hormonal carcinogenesis. Deletion of the Rb gene predisposes prostatic epithelium to hyperplasia and increases proliferative activity Susceptibility to hormonal carcinogenesis in response to exogenous testosterone + estradiol is manifested in the progression from atypica hyperplasia to carcinoma. Thus, these findings demonstrate that the absence of the Rb tumor suppressor gene may predispose prostatic epithelial cells to carcinogenesis. Rescue of organs from Rb-/- embryos not only provides an opportunity to analyze the Rb gene pathway in the development and progression of prostate cancer but also provides an opportunity for specifically evaluating the role of the Rb pathway in development and carcinogenesis in other organs, such as the mammary gland and colon. Because rUGM greatly stimulates prostatic epithelial proliferation, the tissue recombinant model is a particularly useful tool for assessing the functional role of other genes in prostatic carcinogenesis through use of the appropriate transgenic or gene knockout mice.

Abrogation of p53 function by transfection of HPV16 E6 gene enhances the resistance of human diploid fibroblasts to ionizing radiation.

In order to examine the role of p53 expression on the sensitivity of cells to radiation-induced reproductive failure, we examined the radiosensitivity of a human diploid fibroblast cell strain (AG1521) before and after transfection with the E6 or E6/E7 genes of human papillomavirus 16 (HPV16)3. HPV E6 binds to p53, promoting its degradation and abrogating wild-type p53 function. AG1521 cells transfected with either E6 or E6/E7 showed no radiation induction of either p53 or WAF1/Cip1. The radioresistance of these cells were significantly increased; the D0 of the survival curves rose from 130 +/- 4 cGy for wild type (neo-transfected) cells to 178-192 cGy for three subclones transfected with E6 alone, and 151-218 cGy for eight subclones transfected with E6/E7. The change in radiosensitivity took place before the process of cellular immortalization and transformation produced by transfection with these genes. Thus, the effect on radiosensitivity appears to be an early effect of the loss of p53 function in non-transformed cells, perhaps related to the loss of the G1 checkpoint and of the capacity for programmed death amongst radiation damaged cells.

Compensatory apoptosis in response to SV40 large T antigen expression in the liver.

Transgenesis allows the in vivo determination of the effects of oncogene expression in normal tissues. In an attempt to understand the mechanism underlying liver transformation, we have previously created transgenic mice carrying the SV40 early gene sequences, which developed hepatocarcinoma in a reproducible way. In the present study, we show that constant expression of the transgene was directly correlated to an abnormally increased hepatocyte proliferation, even at the adult stage. We further demonstrate in this model that the preneoplastic stage of hepatocarcinoma is characterized by marked ploidy alterations as early as 1 month, including the emergence of aneuploid and hyperpolyploid cells, and the persistence of an important diploid cell population. We show that this elevated proliferation is early and transiently counterbalanced by a mechanism of apoptosis, which maintains liver homeostasis. The disappearance of this programmed cell death response effective during preneoplasia might signal the commitment of the liver to neoplasia.

The retinoblastoma protein modulates expression of genes coding for diverse classes of proteins including components of the extracellular matrix.

The product of the retinoblastoma susceptibility gene, pRb, is a negative regulator of cell growth. It functions by regulating the activity of transcription factors. Rb represses some genes by sequestering or inactivating the positive transcription factor E2F and seems to activate some others by interacting with factors like Sp1 or ATF-2. However, there are only a few examples of genes which are positively regulated by pRb. In order to find out if there are common mechanisms for promoter regulation by pRb, we were interested to identify more genes which are either stimulated or repressed by pRb. Using the method of differential display (DDRT-PCR) in combination with nuclear run-on analyses we were able to detect a number of genes which are upregulated by ectopic expression of the Rb gene in Rb-deficient mammary carcinoma cells. We could demonstrate not only stimulation of the endogenous mutant Rb gene but also positive regulation of genes coding for diverse classes of proteins, including the endothelial growth regulator endothelin-1 and the proteoglycans versican and PG40. As a second approach, we investigated gene expression in cell lines established from Rb deficient heterozygous and homozygous knockout mouse embryos and normal mice. We have identified several genes the expression of which correlates positively or negatively with the presence of Rb. These data provide further evidence for pRb being a master regulator of a complex network of gene activities defining the difference between dividing and resting or differentiated cells.

Expression of the retinoblastoma (RB) tumor suppressor gene inhibits tumor cell invasion in vitro.

To determine if replacement of the retinoblastoma (RB) tumor suppressor gene could inhibit invasion of RB-defective tumor cells, the capacity of tumor cells to migrate or invade was quantitated by the Boyden chamber assay. The studies were done in a diverse group of stable RB-reconstituted human tumor cell lines, including those derived from the osteosarcoma and carcinomas of the lung, breast and bladder. The expression of the exogenous wild-type RB protein in these tumor cell lines was driven by either a constitutively active promoter or an inducible promoter. It was found that significantly more tumor cells from the parental RB-defective cell lines and the RB revertants than from the RB-reconstituted RB+ cell lines penetrated through the Matrigel (P<0.001, two-tailed t-test), although both RB+ and RB- cells migrated at approximately the same rate on uncoated polycarbonate filters in the Boyden chambers. Of note, the inhibition of invasiveness of various RB-defective tumor cells by RB replacement was apparently well correlated with suppression of their tumorigenicity in vivo. In contrast, although either functional RB or p53 re-expression effectively suppressed tumor formation in nude mice of the RB-/p53null osteosarcoma cell line, Saos-2, replacement of the wild-type p53 gene had much less impact on their invasiveness as compared to the RB gene. These studies provided an insight into the broader biological basis of the RB-mediated tumor suppression in RB-defective tumor cells.

Discrete signaling pathways participate in RB-dependent responses to chemotherapeutic agents.

The retinoblastoma (RB) tumor suppressor has been proposed to function as a key mediator of cell cycle checkpoints induced by chemotherapeutic agents. However, these prior studies have relied on embryonic fibroblasts harboring chronic loss of RB, a condition under which compensation of RB functions is known to occur. Here we utilized primary adult fibroblasts derived from mice harboring loxP sites flanking exon 3 of the Rb gene to delineate the action of RB in the chemotherapeutic response. In this system we find that targeted disruption of Rb leads to little overt change in cell cycle distribution. However, these cells exhibited deregulation of RB/E2F target genes and became aneuploid following culture in the absence of RB. When challenged with both DNA damaging and antimetabolite chemotherapeutics, RB was required for primary adult cells to undergo DNA damage checkpoint responses and loss of RB resulted in enhanced aneuploidy following challenge. In contrast, following spontaneous immortalization and the loss of functional p53 signaling, the antimetabolite 5-fluorouracil (5-FU) failed to induce arrest despite the presence of RB. In these immortal cultures RB/E2F targets were deregulated in a complex, gene-specific manner and RB was required for the checkpoint response to camptothecin (CPT). Mechanistic analyses of the checkpoint responses in primary cells indicated that loss of RB leads to increased p53 signaling and decreased viability following both CPT and 5-FU treatment. However, the mechanism through which these agents act to facilitate cell cycle inhibition through RB were distinct. These studies underscore the critical role of RB in DNA-damage checkpoint signaling and demonstrate that RB mediates chemotherapeutic-induced cell cycle inhibition in adult fibroblasts by distinct mechanisms.