Sir Peter MacCallum Department of Oncology - Research Publications
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ItemNo Preview AvailablemTOR-Dependent regulation of ribosomal gene transcription requires S6K1 and is mediated by phosphorylation of the carboxy-terminal activation domain of the nucleolar transcription factor UBF(AMER SOC MICROBIOLOGY, 2003-12)Mammalian target of rapamycin (mTOR) is a key regulator of cell growth acting via two independent targets, ribosomal protein S6 kinase 1 (S6K1) and 4EBP1. While each is known to regulate translational efficiency, the mechanism by which they control cell growth remains unclear. In addition to increased initiation of translation, the accelerated synthesis and accumulation of ribosomes are fundamental for efficient cell growth and proliferation. Using the mTOR inhibitor rapamycin, we show that mTOR is required for the rapid and sustained serum-induced activation of 45S ribosomal gene transcription (rDNA transcription), a major rate-limiting step in ribosome biogenesis and cellular growth. Expression of a constitutively active, rapamycin-insensitive mutant of S6K1 stimulated rDNA transcription in the absence of serum and rescued rapamycin repression of rDNA transcription. Moreover, overexpression of a dominant-negative S6K1 mutant repressed transcription in exponentially growing NIH 3T3 cells. Rapamycin treatment led to a rapid dephosphorylation of the carboxy-terminal activation domain of the rDNA transcription factor, UBF, which significantly reduced its ability to associate with the basal rDNA transcription factor SL-1. Rapamycin-mediated repression of rDNA transcription was rescued by purified recombinant phosphorylated UBF and endogenous UBF from exponentially growing NIH 3T3 cells but not by hypophosphorylated UBF from cells treated with rapamycin or dephosphorylated recombinant UBF. Thus, mTOR plays a critical role in the regulation of ribosome biogenesis via a mechanism that requires S6K1 activation and phosphorylation of UBF.
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ItemPalbociclib synergizes with BRAF and MEK inhibitors in treatment naive melanoma but not after the development of BRAF inhibitor resistance(WILEY, 2018-05-15)Increased CDK4 activity occurs in the majority of melanomas and CDK4/6 inhibitors in combination with BRAF and MEK inhibitors are currently in clinical trials for the treatment of melanoma. We hypothesize that the timing of the addition of CDK4/6 inhibitors to the current BRAF and MEK inhibitor regime will impact on the efficacy of this triplet drug combination. The efficacy of BRAF, MEK and CDK4/6 inhibitors as single agents and in combination was assessed in human BRAF mutant cell lines that were treatment naïve, BRAF inhibitor tolerant or had acquired resistance to BRAF inhibitors. Xenograft studies were then performed to test the in vivo efficacy of the BRAF and CDK4/6 inhibitor combination. Melanoma cells that had developed early reversible tolerance or acquired resistance to BRAF inhibition remained sensitive to palbociclib. In drug-tolerant cells, the efficacy of the combination of palbociclib with BRAF and/or MEK inhibitors was equivalent to single agent palbociclib. Similarly, acquired BRAF inhibitor resistance cells lost efficacy to the palbociclib and BRAF combination. In contrast, upfront treatment of melanoma cells with palbociclib in combination with BRAF and/or MEK inhibitors induced either cell death or senescence and was superior to a BRAF plus MEK inhibitor combination. In vivo palbociclib plus BRAF inhibitor induced rapid and sustained tumor regression without the development of therapy resistance. In summary, upfront dual targeting of CDK4/6 and mutant BRAF signaling enables tumor cells to evade resistance to monotherapy and is required for robust and sustained tumor regression. Melanoma patients whose tumors have acquired resistance to BRAF inhibition are less likely to have favorable responses to subsequent treatment with the triplet combination of BRAF, MEK and CDK4/6 inhibitors.
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ItemNo Preview AvailableAKT-independent PI3-K signaling in cancer - emerging role for SGK3.(Informa UK Limited, 2013)The phosphoinositide 3-kinase (PI3-K) signaling pathway plays an important role in a wide variety of fundamental cellular processes, largely mediated via protein kinase B/v-akt murine thymoma viral oncogene homolog (PKB/AKT) signaling. Given the crucial role of PI3-K/AKT signaling in regulating processes such as cell growth, proliferation, and survival, it is not surprising that components of this pathway are frequently dysregulated in cancer, making the AKT kinase family members important therapeutic targets. The large number of clinical trials currently evaluating PI3-K pathway inhibitors as a therapeutic strategy further emphasizes this. The serum- and glucocorticoid-inducible protein kinase (SGK) family is made up of three isoforms, SGK1, 2, and 3, that are PI3-K-dependent, serine/threonine kinases, with similar substrate specificity to AKT. Consequently, the SGK family also regulates similar cell processes to the AKT kinases, including cell proliferation and survival. Importantly, there is emerging evidence demonstrating that SGK3 plays a critical role in AKT-independent oncogenic signaling. This review will focus on the role of SGK3 as a key effector of AKT-independent PI3-K oncogenic signaling.
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ItemAutophagy Induction Is a Tor- and Tp53-Independent Cell Survival Response in a Zebrafish Model of Disrupted Ribosome Biogenesis(PUBLIC LIBRARY SCIENCE, 2013-02)Ribosome biogenesis underpins cell growth and division. Disruptions in ribosome biogenesis and translation initiation are deleterious to development and underlie a spectrum of diseases known collectively as ribosomopathies. Here, we describe a novel zebrafish mutant, titania (tti(s450)), which harbours a recessive lethal mutation in pwp2h, a gene encoding a protein component of the small subunit processome. The biochemical impacts of this lesion are decreased production of mature 18S rRNA molecules, activation of Tp53, and impaired ribosome biogenesis. In tti(s450), the growth of the endodermal organs, eyes, brain, and craniofacial structures is severely arrested and autophagy is up-regulated, allowing intestinal epithelial cells to evade cell death. Inhibiting autophagy in tti(s450) larvae markedly reduces their lifespan. Somewhat surprisingly, autophagy induction in tti(s450) larvae is independent of the state of the Tor pathway and proceeds unabated in Tp53-mutant larvae. These data demonstrate that autophagy is a survival mechanism invoked in response to ribosomal stress. This response may be of relevance to therapeutic strategies aimed at killing cancer cells by targeting ribosome biogenesis. In certain contexts, these treatments may promote autophagy and contribute to cancer cells evading cell death.
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ItemAutophosphorylation of serine 608 in the p85 regulatory subunit of wild type or cancer-associated mutants of phosphoinositide 3-kinase does not affect its lipid kinase activity(BMC, 2012-12-27)BACKGROUND: The α-isoform of the Type 1A Phosphoinositide 3-kinases (PI3Kα) has protein kinase activity as well as phosphoinositide lipid kinase activity. The best described substrate for its protein kinase activity is its regulatory subunit, p85α, which becomes phosphorylated on Serine 608. Phosphorylation of Serine 608 has been reported to down-regulate its lipid kinase activity. RESULTS: We have assessed whether oncogenic mutants of PI3Kα, which have up-regulated lipid kinase activity, have altered levels of Serine 608 phosphorylation compared to wild type PI3Kα, and whether differential phosphorylation of Serine 608 contributes to increased activity of oncogenic forms of PI3Kα with point mutations in the helical or the kinase domains. Despite markedly increased lipid kinase activity, protein kinase activity was not altered in oncogenic compared to wild type forms of PI3Kα. By manipulating levels of phosphorylation of Serine 608 in vitro, we found no evidence that the protein kinase activity of PI3Kα affects its phosphoinositide lipid kinase activity in either wild-type or oncogenic mutants of PI3Kα. CONCLUSIONS: Phosphorylation of p85α S608 is not a significant regulator of wild-type or oncogenic PI3Kα lipid kinase activity.
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ItemDeregulation of MYCN, LIN28B and LET7 in a Molecular Subtype of Aggressive High-Grade Serous Ovarian Cancers(PUBLIC LIBRARY SCIENCE, 2011-04-13)Molecular subtypes of serous ovarian cancer have been recently described. Using data from independent datasets including over 900 primary tumour samples, we show that deregulation of the Let-7 pathway is specifically associated with the C5 molecular subtype of serous ovarian cancer. DNA copy number and gene expression of HMGA2, alleles of Let-7, LIN28, LIN28B, MYC, MYCN, DICER1, and RNASEN were measured using microarray and quantitative reverse transcriptase PCR. Immunohistochemistry was performed on 127 samples using tissue microarrays and anti-HMGA2 antibodies. Fluorescence in situ hybridisation of bacterial artificial chromosomes hybridized to 239 ovarian tumours was used to measure translocation at the LIN28B locus. Short interfering RNA knockdown in ovarian cell lines was used to test the functionality of associations observed. Four molecular subtypes (C1, C2, C4, C5) of high-grade serous ovarian cancers were robustly represented in each dataset and showed similar pattern of patient survival. We found highly specific activation of a pathway involving MYCN, LIN28B, Let-7 and HMGA2 in the C5 molecular subtype defined by MYCN amplification and over-expression, over-expression of MYCN targets including the Let-7 repressor LIN28B, loss of Let-7 expression and HMGA2 amplification and over-expression. DICER1, a known Let-7 target, and RNASEN were over-expressed in C5 tumours. We saw no evidence of translocation at the LIN28B locus in C5 tumours. The reported interaction between LIN28B and Let-7 was recapitulated by siRNA knockdown in ovarian cancer cell lines. Our results associate deregulation of MYCN and downstream targets, including Let-7 and oncofetal genes, with serous ovarian cancer. We define for the first time how elements of an oncogenic pathway, involving multiple genes that contribute to stem cell renewal, is specifically altered in a molecular subtype of serous ovarian cancer. By defining the drivers of a molecular subtype of serous ovarian cancers we provide a novel strategy for targeted therapeutic intervention.
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ItemUBF levels determine the number of active ribosomal RNA genes in mammals(ROCKEFELLER UNIV PRESS, 2008-12-29)In mammals, the mechanisms regulating the number of active copies of the approximately 200 ribosomal RNA (rRNA) genes transcribed by RNA polymerase I are unclear. We demonstrate that depletion of the transcription factor upstream binding factor (UBF) leads to the stable and reversible methylation-independent silencing of rRNA genes by promoting histone H1-induced assembly of transcriptionally inactive chromatin. Chromatin remodeling is abrogated by the mutation of an extracellular signal-regulated kinase site within the high mobility group box 1 domain of UBF1, which is required for its ability to bend and loop DNA in vitro. Surprisingly, rRNA gene silencing does not reduce net rRNA synthesis as transcription from remaining active genes is increased. We also show that the active rRNA gene pool is not static but decreases during differentiation, correlating with diminished UBF expression. Thus, UBF1 levels regulate active rRNA gene chromatin during growth and differentiation.
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ItemToo much or too little Harnessing senescence to control oncogene-driven cancer(TAYLOR & FRANCIS INC, 2012-09-01)
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ItemDrosophila Ribosomal Protein Mutants Control Tissue Growth Non-Autonomously via Effects on the Prothoracic Gland and Ecdysone(PUBLIC LIBRARY SCIENCE, 2011-12)The ribosome is critical for all aspects of cell growth due to its essential role in protein synthesis. Paradoxically, many Ribosomal proteins (Rps) act as tumour suppressors in Drosophila and vertebrates. To examine how reductions in Rps could lead to tissue overgrowth, we took advantage of the observation that an RpS6 mutant dominantly suppresses the small rough eye phenotype in a cyclin E hypomorphic mutant (cycE(JP)). We demonstrated that the suppression of cycE(JP) by the RpS6 mutant is not a consequence of restoring CycE protein levels or activity in the eye imaginal tissue. Rather, the use of UAS-RpS6 RNAi transgenics revealed that the suppression of cycE(JP) is exerted via a mechanism extrinsic to the eye, whereby reduced Rp levels in the prothoracic gland decreases the activity of ecdysone, the steroid hormone, delaying developmental timing and hence allowing time for tissue and organ overgrowth. These data provide for the first time a rationale to explain the counter-intuitive organ overgrowth phenotypes observed for certain members of the Minute class of Drosophila Rp mutants. They also demonstrate how Rp mutants can affect growth and development cell non-autonomously.
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ItemAKT induces senescence in human cells via mTORC1 and p53 in the absence of DNA damage: implications for targeting mTOR during malignancy(NATURE PUBLISHING GROUP, 2012-04)The phosphatidylinositol 3-kinase (PI3K)/AKT and RAS oncogenic signalling modules are frequently mutated in sporadic human cancer. Although each of these pathways has been shown to play critical roles in driving tumour growth and proliferation, their activation in normal human cells can also promote cell senescence. Although the mechanisms mediating RAS-induced senescence have been well characterised, those controlling PI3K/AKT-induced senescence are poorly understood. Here we show that PI3K/AKT pathway activation in response to phosphatase and tensin homolog (PTEN) knockdown, mutant PI3K, catalytic, α polypeptide (PIK3CA) or activated AKT expression, promotes accumulation of p53 and p21, increases cell size and induces senescence-associated β-galactosidase activity. We demonstrate that AKT-induced senescence is p53-dependent and is characterised by mTORC1-dependent regulation of p53 translation and stabilisation of p53 protein following nucleolar localisation and inactivation of MDM2. The underlying mechanisms of RAS and AKT-induced senescence appear to be distinct, demonstrating that different mediators of senescence may be deregulated during transformation by specific oncogenes. Unlike RAS, AKT promotes rapid proliferative arrest in the absence of a hyperproliferative phase or DNA damage, indicating that inactivation of the senescence response is critical at the early stages of PI3K/AKT-driven tumourigenesis. Furthermore, our data imply that chronic activation of AKT signalling provides selective pressure for the loss of p53 function, consistent with observations that PTEN or PIK3CA mutations are significantly associated with p53 mutation in a number of human tumour types. Importantly, the demonstration that mTORC1 is an essential mediator of AKT-induced senescence raises the possibility that targeting mTORC1 in tumours with activated PI3K/AKT signalling may exert unexpected detrimental effects due to inactivation of a senescence brake on potential cancer-initiating cells.
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