c-Myc depletion inhibits proliferation of human tumor cells at various stages of the cell cycle

Wang, H; Mannava, Sudha; Grachtchouk, Vladimir; Zhuang, Dazhong; Soengas, Marı́a S.; Gudkov, Andrei V.; Prochownik, Edward V.; Nikiforov, Mikhail A.

doi:10.1038/sj.onc.1210823

Cited by 145 publications

(135 citation statements)

References 51 publications

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“…18 Here, we confirmed these observations for three melanoma cell lines (SK-Mel-19, SK-Mel-29 and SK-Mel-109) using two lentivirus-delivered C-MYC-specific shRNAs: C-MYCshRNA-1 (M1) and C-MYC-shRNA-2 (M2) (see Materials and Methods), (Suppl. Fig.…”

Section: Resultssupporting

confidence: 64%

“…shRNAs for C-MYC gene were described previously. 18 Lentiviral packaging reactions were performed in the 293-FT cell line in the presence of packaging plasmids VSG and ΔDR using Superfect Transfection Reagent (Qiagen). Viral supernatants were collected 48 hr after transfection, filtered through disposable 0.45 μM cellulose acetate filters and frozen in individual aliquots at -80°C.…”

Section: Methodsmentioning

confidence: 99%

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Direct role of nucleotide metabolism in C-MYC-dependent proliferation of melanoma cells

Mannava¹,

Grachtchouk²,

Wheeler³

et al. 2008

Cell Cycle

195

178

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To identify C-MYC targets rate-limiting for proliferation of malignant melanoma, we stably inhibited C-MYC in several human metastatic melanoma lines via lentivirus-based shRNAs approximately to the levels detected in normal melanocytes. C-MYC depletion did not significantly affect levels of E2F1 protein reported to regulate expression of many S-phase specific genes, but resulted in the repression of several genes encoding enzymes rate-limiting for dNTP metabolism. These included thymidylate synthase (TS), inosine monophosphate dehydrogenase 2 (IMPDH2) and phosphoribosyl pyrophosphate synthetase 2 (PRPS2). C-MYC depletion also resulted in reduction in the amounts of deoxyribonucleoside triphosphates (dNTPs) and inhibition of proliferation. shRNA-mediated suppression of TS, IMPDH2 or PRPS2 resulted in the decrease of dNTP pools and retardation of the cell cycle progression of melanoma cells in a manner similar to that of C-MYC-depletion in those cells. Reciprocally, concurrent overexpression of cDNAs for TS, IMPDH2 and PRPS2 delayed proliferative arrest caused by inhibition of C-MYC in melanoma cells. Overexpression of C-MYC in normal melanocytes enhanced expression of the above enzymes and increased individual dNTP pools. Analysis of in vivo C-MYC interactions with TS, IMPDH2 and PRPS2 genes confirmed that they are direct C-MYC targets. Moreover, all three proteins express at higher levels in cells from several metastatic melanoma lines compared to normal melanocytes. Our data establish a novel functional link between C-MYC and dNTP metabolism and identify its role in proliferation of tumor cells.

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Section: Resultssupporting

confidence: 64%

Section: Methodsmentioning

confidence: 99%

Direct role of nucleotide metabolism in C-MYC-dependent proliferation of melanoma cells

Mannava¹,

Grachtchouk²,

Wheeler³

et al. 2008

Cell Cycle

195

178

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“…c-Myc seems to be essential for sustaining proliferation of human tumor cells (Wang et al, 2007). Vectortreated tumors showed very strong c-Myc nuclear immunostaining.…”

Section: Wif1 Inhibits Cervical Cancer Growth and Invasion I Ramachanmentioning

confidence: 98%

“…The overexpression of c-Myc, cyclin D1 and CD44 has been shown to promote tumor cell proliferation (Abbasi et al, 1993;Wielenga et al, 1999;Fu et al, 2004;Wang et al, 2007;Orian-Rousseau, 2010) and to associate with poor cervical cancer prognosis (Kainz et al, 1995;Speiser et al, 1997;Bae et al, 2001;Vijayalakshmi et al, 2002;Lu et al, 2005;Orian-Rousseau, 2010). Importantly, WIF1-treated tumors demonstrated a marked decrease in the expression of b-catenin, TCF-4, c-Myc, cyclin D1 and CD44, with a consequent significant decrease in the tumor mitotic index.…”

Section: Wif1 Inhibits Cervical Cancer Growth and Invasion I Ramachanmentioning

confidence: 99%

Wnt inhibitory factor 1 induces apoptosis and inhibits cervical cancer growth, invasion and angiogenesis in vivo

et al. 2011

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Aberrant activation of Wingless-type (Wnt)/b-catenin signaling is widespread in human cervical cancer. However, the underlying mechanisms of Wnt activation and the therapeutic potential of Wnt inhibition remain largely unknown. Here, we demonstrate that the Wnt inhibitory factor 1 (WIF1), a secreted Wnt antagonist, is downregulated in all human primary cervical tumors and cell lines analyzed. Our data reveal that WIF1 downregulation occurs due to promoter hypermethylation and is an early event in cervical oncogenesis. WIF1 re-expression upon 5-aza-2 0 -deoxycytidine treatment or WIF1 gene transfer induces significant apoptosis and G 2 /M arrest, and inhibits cervical cancer cell proliferation in vitro. Consistent with this, treatment of established mice tumor xenografts with peritumoral WIF1 gene transfer results in a significant inhibition of cancer growth and invasion. WIF1 treatment causes a significant decrease in intracellular WNT1 and TCF-4 proteins revealing novel Wnt-regulatory mechanisms. Thus, WIF1 causes a major cellular re-distribution of b-catenin and a significant inhibition of the Wnt/b-catenin pathway in tumor cells, as documented by a remarkable reversion in the expression of Wnt/b-catenin transcriptional target genes (E-cadherin, c-Myc, cyclin D1, CD44 and VEGF). Consequently, multiple critical events in tumor progression and metastasis such as cell proliferation, angiogenesis and invasion were inhibited by WIF1. In addition, WIF1 modulated the expression of specific anti-apoptotic and apoptotic proteins, thereby inducing significant apoptosis in vivo. Our findings demonstrate for the first time that WIF1 downregulation by epigenetic gene silencing is an important mechanism of Wnt activation in cervical oncogenesis. Of major clinical relevance, we show that peritumoral WIF1 gene transfer reduces not only cancer growth but also invasion in well-established tumors. Therefore, our data provide novel mechanistic insights into the role of WIF1 in cervical cancer progression, and the important preclinical validation of WIF1 as a potent drug target in cervical cancer treatment.

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“…In HT-p21 and HT-p16 cells, p21 and p16 expression, can be turned on or off using isopropyl-thio-galactosidase (IPTG). 13,70 Melanoma MEL10 (formally, SK-MEL-147) were obtained from Dr. Mikhail Nikiforov (RPCI, Buffalo, NY, USA), were described previously 71 and were cultured in DMEM plus 10% FBS. Normal retinal pigment epithelial RPE cell lines were obtained from ATCC and were maintained in MEM plus 10% FBS.…”

Section: Methodsmentioning

confidence: 99%

MEK drives cyclin D1 hyperelevation during geroconversion

2013

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When the cell cycle becomes arrested, MTOR (mechanistic Target of Rapamycin) converts reversible arrest into senescence (geroconversion). Hyperexpression of cyclin D1 is a universal marker of senescence along with hypertrophy, beta-Gal staining and loss of replicative/regenerative potential (RP), namely, the ability to restart proliferation when the cell cycle is released. Inhibition of MTOR decelerates geroconversion, although only partially decreases cyclin D1. Here we show that in p21-and p16-induced senescence, inhibitors of mitogen-activated/extracellular signal-regulated kinase (MEK) (U0126, PD184352 and siRNA) completely prevented cyclin D1 accumulation, making it undetectable. We also used MEL10 cells in which MEK inhibitors do not inhibit MTOR. In such cells, U0126 by itself induced senescence that was remarkably cyclin D1 negative. In contrast, inhibition of cyclin-dependent kinase (CDK) 4/6 by PD0332991 caused cyclin D1-positive senescence in MEL10 cells. Both types of senescence were suppressed by rapamycin, converting it into reversible arrest. We confirmed that the inhibitor of CDK4/6 caused cyclin D1 positive senescence in normal RPE cells, whereas U0126 prevented cyclin D1 expression. Elimination of cyclin D1 by siRNA did not prevent other markers of senescence that are consistent with the lack of its effect on MTOR. Our data confirmed that a mere inhibition of the cell cycle was sufficient to cause senescence, providing MTOR was active, and inhibition of MEK partially inhibited MTOR in a cell-type-dependent manner. Second, hallmarks of senescence may be dissociated, and hyperelevated cyclin D1, a marker of hyperactivation of senescent cells, did not necessarily determine other markers of senescence. Third, inhibition of MEK was sufficient to eliminate cyclin D1, regardless of MTOR. In proliferating cells, MTOR (mechanistic Target of Rapamycin) stimulates cellular mass growth, whereas cyclins D and E initiate S-phase transition.1-4 Withdrawal of growth factors causes reversible cell-cycle arrest (quiescence) with low levels of cyclins and MTOR activity, reversible by re-addition of growth factors. In contrast, cell-cycle arrest in the presence of growth stimuli, which activate MTOR, leads to cellular senescence. 5,6 In other words, when the cell cycle is blocked, while growth-promoting pathways such as MTOR remain active, cells continue to grow in size and undergo senescence. A process of conversion from normal reversible arrest into senescence was named as gerogenic conversion (geroconversion).7 Cellular senescence is characterized by large flat morphology (hypertrophy), beta-Gal staining, very high levels of cyclins D1 and E, senescence-associated secretory phenotype and loss of replicative/regenerative potential (RP).7-12 Loss of RP means that cells cannot proliferate even when cell-cycle block is removed and cells re-enter the cell cycle.13,14 Rapamycin decreases hypertrophy and loss of RP, in a dose-dependent manner proportional to S6 dephosphorylation (a marker of MTORC1 activity). 15Other ...

show abstract

c-Myc depletion inhibits proliferation of human tumor cells at various stages of the cell cycle

Cited by 145 publications

References 51 publications

Direct role of nucleotide metabolism in C-MYC-dependent proliferation of melanoma cells

Direct role of nucleotide metabolism in C-MYC-dependent proliferation of melanoma cells

Wnt inhibitory factor 1 induces apoptosis and inhibits cervical cancer growth, invasion and angiogenesis in vivo

MEK drives cyclin D1 hyperelevation during geroconversion

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