PDGFRß promotes oncogenic progression via STAT3/STAT5 hyperactivation in anaplastic large cell lymphoma.

BACKGROUND: Anaplastic large cell lymphoma (ALCL) is an aggressive non-Hodgkin T cell lymphoma commonly driven by NPM-ALK. AP-1 transcription factors, cJUN and JUNb, act as downstream effectors of NPM-ALK and transcriptionally regulate PDGFRß. Blocking PDGFRß kinase activity with imatinib effectively reduces tumor burden and prolongs survival, although the downstream molecular mechanisms remain elusive. METHODS AND RESULTS: In a transgenic mouse model that mimics PDGFRß-driven human ALCL in vivo, we identify PDGFRß as a driver of aggressive tumor growth. Mechanistically, PDGFRß induces the pro-survival factor Bcl-xL and the growth-enhancing cytokine IL-10 via STAT5 activation. CRISPR/Cas9 deletion of both STAT5 gene products, STAT5A and STAT5B, results in the significant impairment of cell viability compared to deletion of STAT5A, STAT5B or STAT3 alone. Moreover, combined blockade of STAT3/5 activity with a selective SH2 domain inhibitor, AC-4-130, effectively obstructs tumor development in vivo. CONCLUSIONS: We therefore propose PDGFRß as a novel biomarker and introduce PDGFRß-STAT3/5 signaling as an important axis in aggressive ALCL. Furthermore, we suggest that inhibition of PDGFRß or STAT3/5 improve existing therapies for both previously untreated and relapsed/refractory ALK+ ALCL patients.

Activation of c-Myc uncouples DNA replication from activation of G1-cyclin-dependent kinases.

Proto-oncogenes like c-myc are thought to control exit from the cell cycle rather than progression through the cell cycle itself. We now present a different view of Myc function. Exponentially growing Rat1-MycER fibroblasts were size-fractionated by centrifugal elutriation. In these cells, activation of cyclin E- and cyclin A-dependent kinases, degradation of p27, hyperphosphorylation of retinoblastoma protein and activation of E2F occur sequentially at specific cell sizes. Upon activation of Myc, however, these transitions all occur simultaneously in small cells immediately after exit from mitosis. In contrast, Myc has no discernible effect on the cell size at which DNA replication is initiated. These data show first that Myc controls the activity of G1 cyclin-dependent kinases independently from the transition between quiescence and proliferation and from any effect on cell growth in size. These data also provide evidence of at least one dominant mechanism besides activation of E2F and of cyclin E/cdk2 kinase, which prevents DNA replication unless a critical cell size has been reached.

Deregulated expression of E2F-1 induces cyclin A- and E-associated kinase activities independently from cell cycle position.

The transcription factor E2F activates genes required for S phase, such as cyclin E and cyclin A. We show that, contrary to long term effects of E2F-1 overexpression, short ectopic overexpression of this transcription factor in logarithmically growing cells does neither affect the cell cycle distribution nor the cell size, but heavily induces cyclin E and A expression as well as cyclin E- and A-dependent kinase activities. We further separated logarithmically growing E2F-1-overexpressing cells according to their different cell cycle phases by centrifugal elutriation. These experiments revealed that deregulated E2F-1 expression triggers high levels of cyclin E and A expression and kinase activities in small early G1 cells, normally not exhibiting these activities. These effects on the regulation of cyclin E- and A-associated kinases are not accompanied by any detectable alteration in the rate of progression through the cell cycle, suggesting that these changes are independent of any mitogenic properties of E2F-1.

The role of p16 in the E2F-dependent thymidine kinase regulation.

The role of alterations of the MTS1 tumor suppressor gene on chromosome 9p21, which encodes p16, the inhibitor of cyclin-dependent-kinase-4 and 6, in tumorigenesis is not yet clear. Phosphorylation of the retinoblastoma protein by cyclin-dependent kinases 4 and 6 prevents its interaction with the transcription factor E2F, which subsequently promotes the expression of S phase regulated genes, such as thymidine kinase. Although a role of p16 in this regulation has been presumed, there is no proof so far that loss of this tumor suppressor gene really affects E2F-mediated regulations. We investigated the regulation of thymidine kinase in phytohemagglutinin-stimulated normal human lymphocytes and in the p16-negative human acute lymphoblastic leukemia cell lines, MOLT-4 and CEM. Compared to normal lymphocytes, MOLT-4 and CEM cells exhibited an altered cell cycle regulation of thymidine kinase, a much higher intracellular activity of this enzyme, and higher thymidine kinase mRNA expression. Transient expression of p16 in normal human lymphocytes caused arrest in G1, but was without effect on the cell growth of MOLT-4 and CEM cells, although all of them express functional retinoblastoma protein. Nevertheless, in the two leukemia cell lines transient overexpression of p16 reestablished the normal regulation of thymidine kinase, paralleled by an increase of the underphosphorylated form of retinoblastoma protein and decrease of free E2F bound to its motif in the thymidine kinase promoter. We demonstrate that loss of p16 causes upregulation of this DNA precursor pathway enzyme via activation of E2F by a mechanism involving retinoblastoma protein.

Expression of the cyclin-dependent kinase inhibitor p16 during the ongoing cell cycle.

It has been demonstrated that protein expression of p16, the inhibitor of cyclin-dependent kinase 4 and 6, increases 4 fold at the G1/S transition when serum-arrested cells are restimulated to logarithmic growth. We examined the cell cycle regulation of this cyclin-dependent kinase inhibitor in cells separated according to their cell cycle phases by centrifugal elutriation. Neither p16 mRNA nor its protein expression are regulated during the cell cycle of normal phytohemagglutinin-stimulated lymphocytes, retinoblastoma protein-negative cells, papilloma virus-transformed cells, and acute promyelocytic leukemia cells. p16 mRNA is constitutively expressed in cells in which we detected the normal E2F-dependent S-phase specific expression of thymidine kinase mRNA. We further observed a G1-phase specific expression of cyclin D1 mRNA in the same cells separated by centrifugal elutriation.

Specific transformation abolishes cyclin D1 fluctuation throughout the cell cycle.

We analysed cyclin D1 mRNA and protein expression in several different cell types after separating these cells according to their different cell cycle phases by centrifugal elutriation. In normal human and rat fibroblasts cyclin D1 expression is high in early to mid G1 and decreases about 6-7 fold before onset of replication. It has been demonstrated that specific transforming events, such as loss of functional retinoblastoma protein, overexpression of c-myc, and transfection with the human papillomavirus oncoproteins E6 and E7 cause transcriptional downregulation of cyclin D1 expression in logarithmically growing cells. We found that such transformed cells exhibit loss of the cell cycle-dependent cyclin D1 fluctuation accompanied with reduced upregulation of cyclin D1 in G1 phase. The data presented here provide the experimental support for a recently suggested model involving the function of the retinoblastoma protein in cyclin D1 cell cycle regulation.

Deregulated expression of CDK2- or CDK3-associated kinase activities enhances c-Myc-induced apoptosis.

Activation of high ectopic levels of c-Myc in serum-deprived Rat1-MycER cells by 4-hydroxytamoxifen induces both proliferation and apoptosis. To further elucidate the role of G1 cyclin-dependent kinases (CDKs) in the process of Myc-induced apoptosis, we generated Rat1-MycER cells stably overexpressing CDK2 or CDK3. Ectopic expression of these CDKs in Myc-overexpressing cells was accompanied by upregulation of the specific kinase activities. Whereas neither high ectopic CDK2 nor CDK3 alone induced apoptosis in serum-deprived Rat1 cells, both CDKs markedly elevated the incidence of Myc-induced apoptosis. It was shown earlier that in Rat1-MycER cells, which are resistant to tumor necrosis factor-alpha (TNF) when grown in high serum concentrations, the addition of TNF with the concomitant activation of Myc resulted in apoptotic cell death. Here, we show that neither CDK2 nor CDK3 induces susceptibility to the cytotoxic action of TNF in Rat1 cells. However, both molecules heavily elevated the incidence of apoptosis induced by TNF together with Myc. It has earlier been reported that Myc-induced apoptosis in serum-deprived Rat1 fibroblasts is inhibited by specific cytokines, such as platelet-derived growth factor (PDGF). Here, we demonstrate that PDGF-mediated protection from Myc-induced apoptosis is almost lost in Rat1 cells overexpressing CDK2 or CDK3. These apoptotic effects of CDK2 or CDK3 are not accompanied by alterations of proliferation parameters, such as DNA distribution, time the cells spend in each phase of the cell cycle, thymidine incorporation into DNA, or cell size analyzed during Myc-induced apoptosis. However, we found CDK3 to deregulate E2F-dependent transcription. In this report, we provide evidence for a not yet described property of CDK2 or CDK3 besides their activity in promoting proliferation: these G1-CDKs can promote apoptosis by interfering with the cell's response to survival factors.

Cellular targets for activation by c-Myc include the DNA metabolism enzyme thymidine kinase.

Although a remarkable number of genes has been identified that are either activated or repressed via c-Myc, only few of them obviously contribute to Myc's biological effect--the induction of proliferation. We found that in logarithmically growing cells overexpression of Myc specifically induces thymidine kinase (TK) mRNA expression and enzyme activity, whereas loss of one allele of Myc causes downregulation of this enzyme. We show that activation of Myc triggers high levels of this normally strictly S-phase-regulated DNA metabolism enzyme in serum arrested G0 cells and causes high and constant levels of TK expression throughout the entire ongoing cell cycle. Induction of TK by Myc requires an intact transcriptional activation domain. Myc-induced deregulation of this enzyme is paralleled by alterations of protein binding at the E2F-site of the TK promoter. We further show that cell growth arrest by the cyclin-dependent kinase inhibitor p16 is abrogated by overexpression of Myc and that co-overexpression of p16 cannot inhibit the Myc-induced up-regulation of TK expression. Our data demonstrate TK to be a cellular target of Myc independently of the status of cell proliferation and provide evidence that the transcription factor E2F might be involved in this process.

Loss of the p16/MTS1 tumor suppressor gene causes E2F-mediated deregulation of essential enzymes of the DNA precursor metabolism.

Homozygous deletions of the tumor suppressor gene p16/MTS1 were reported in a wide variety of tumors and tumor cell lines. Its product inhibits the phosphorylation of the retinoblastoma protein (pRb) by CDK4 and CDK6. Because phosphorylation of pRb is a major regulatory event in the activation of the transcription factor E2F, a role for p16 in the regulation of E2F-dependent transcription was presumed. We investigated the effect of the loss of p16 on E2F-mediated transcription in a tumor progression model consisting of three cell lines originating from a common precursor cell--one p16-positive cell line established from the primary biopsy and two lines derived from more advanced stages of the tumor representing the same cell clone after loss of p16. We observed up- and deregulation of E2F-dependent transcription during the cell cycle of the p16-negative cell clones, which returned to normal after transient expression of p16. This p16-dependent regulation affects a set of enzymes necessary for the activation of all four DNA precursors; it is paralleled by the interconversion of transcriptionally active free E2F and transcriptionally inactive higher molecular complexes of E2F and is dependent on the existence of endogenous pRb. Furthermore, we show that p16-negative cell clones exhibit a growth advantage compared to their p16-positive counterparts. One might speculate that one feature of tumor progression could be deregulation of E2F-dependent transcription caused by loss of p16.

Role of the tuberous sclerosis gene-2 product in cell cycle control. Loss of the tuberous sclerosis gene-2 induces quiescent cells to enter S phase.

Tuberous sclerosis is an autosomal dominant disorder characterized by the development of benign growths in many tissues and organs. Linkage analysis revealed two disease-determining genes on chromosome 9 and chromosome 16. The TSC2 gene on chromosome 16 encodes a 1784-amino acid tumor suppressor protein, tuberin, that functions as a GTPase-activating protein for Rap1, a member of the superfamily of Ras-related proteins. By immunoblot analyses, we found TSC2 expression to be high in G0 as well as in early small G1 cells. Analyses after different cell synchronization procedures revealed that TSC2 mRNA and protein expression do not fluctuate throughout the cell cycle. Using inducible expression systems we further demonstrated that TSC2 expression is not affected by overexpression of the mitogenic transcription factor E2F-1 or c-Myc. Nevertheless, antisense inhibition of tuberin expression in logarithmically growing cells markedly decreased the percentage of cells in G1. Furthermore, we found that cells exposed to TSC2 antisense oligonucleotides did not undergo G0 arrest after serum withdrawal. Antisense inhibition of TSC2 expression also induced quiescent G0-arrested fibroblasts to reenter the cell cycle. Our data show for the first time that the absence of tuberin can both induce cells to pass through the G1/S transition of the eukaryotic cell cycle and prevent them from entering a quiescent state. These results have clear implications for the tumor suppressor function of TSC2. We further found that reentry into the cell cycle upon loss of TSC2 is dependent on the activity of the G1 cyclin-dependent kinases (CDKs), Cdk2 or Cdk4. Taken together with our finding that antisense inhibition of TSC2 causes up-regulation of cyclin D1 expression, these results provide the first evidence for a connection between tuberin/Rap1 and the G1 CDK-dependent regulation of the transition from G0/G1 to S phase.

The gag homologue of retrotransposon Ty1 assembles into spherical particles in Escherichia coli.

Expression of TyA (reading frame A) of the yeast retrotransposon Ty1 in Escherichia coli is possible by using efficient transcriptional and translational initiation signals. When expressed in E. coli, the gag homologue of Ty1 assembles into spherical particles similar, but not identical to virus-like particles in the natural host of Ty1, Saccharomyces cerevisiae. Deletion analysis reveals a domain in the C-terminus of TyA that is essential for the assembly process. These findings indicate that an early step of the retroelement life cycle, assembly of the gag homologue into spherical particles, does not depend on specific host factors. The experiments also demonstrate that Ty1 Gag fusion proteins, potential tools for immunization, can be produced in E. coli, an organism that lacks endogenous retrotransposons.

The Tpv2 family of retrotransposons of Phaseolus vulgaris: structure, integration characteristics, and use for genotype classification.

The Tpv2 family of transposable elements of common bean, Phaseolus vulgaris, belongs to the Ty1/copia group of long terminal repeat (LTR) containing retrotransposons. By reverse transcriptase (RT)-PCR and by analysis of genomic clones, we characterized four of the approximately 40 copies present in the Phaseolus genome, and the genomic environment of their integration sites. Tpv2 integrated preferentially into actively transcribed regions. While none of the isolated elements had all the functional domains necessary for transposition, analysis of bean cultivars suggested that some members of the Tpv2 family transposed in recent breeding history. Probes derived from Tpv2, as well as flanking genomic sequences, may be useful for classifying Phaseolus cultivars.