Sudha Mannava

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

A major role for c-Myc in the proliferation of normal cells is attributed to its ability to promote progression through G1 and into S phase of the cell cycle. The absolute requirement of c-Myc for cell cycle progression in human tumor cells has not been comprehensively addressed. In the present work, we used a lentiviral-based short hairpin RNA (shRNA) expression vector to stably reduce c-Myc expression in a large number of human tumor cell lines and in three different types of normal human cells. In all cases, cell proliferation was severely inhibited, with normal cells ultimately undergoing G0/G1 growth arrest. In contrast, tumor cells demonstrated a much more variable cell cycle response with cells from several lines accumulating in S or G2/M phases. Moreover, in some tumor lines, the phase of cell cycle arrest caused by inhibition of c-Myc could be altered by depleting tumor suppressor protein p53 or its transcriptional target p21CIP/WAF. Our data suggest that, as in the case of normal cells, c-Myc is essential for sustaining proliferation of human tumor cells. However its rate-limiting role in cell cycle control is variable and is reliant upon the status of other cell cycle regulators.

Nrf2 Amplifies Oxidative Stress via Induction of Klf9

Reactive oxygen species (ROS) activate NF-E2-related transcription factor 2 (Nrf2), a key transcriptional regulator driving antioxidant gene expression and protection from oxidant injury. Here, we report that in response to elevation of intracellular ROS above a critical threshold, Nrf2 stimulates expression of transcription Kruppel-like factor 9 (Klf9), resulting in further Klf9-dependent increases in ROS and subsequent cell death. We demonstrated that Klf9 independently causes increased ROS levels in various types of cultured cells and in mouse tissues and is required for pathogenesis of bleomycin-induced pulmonary fibrosis in mice. Mechanistically, Klf9 binds to the promoters and alters the expression of several genes involved in the metabolism of ROS, including suppression of thioredoxin reductase 2, an enzyme participating in ROS clearance. Our data reveal an Nrf2-dependent feedforward regulation of ROS and identify Klf9 as a ubiquitous regulator of oxidative stress and lung injury.

Depletion of Deoxyribonucleotide Pools Is an Endogenous Source of DNA Damage in Cells Undergoing Oncogene-Induced Senescence

In normal human cells, oncogene-induced senescence (OIS) depends on induction of DNA damage response. Oxidative stress and hyperreplication of genomic DNA have been proposed as major causes of DNA damage in OIS cells. Here, we report that down-regulation of deoxyribonucleoside pools is another endogenous source of DNA damage in normal human fibroblasts (NHFs) undergoing HRAS(G12V)-induced senescence. NHF-HRAS(G12V) cells underexpressed thymidylate synthase (TS) and ribonucleotide reductase (RR), two enzymes required for the entire de novo deoxyribonucleotide biosynthesis, and possessed low dNTP levels. Chromatin at the promoters of the genes encoding TS and RR was enriched with retinoblastoma tumor suppressor protein and histone H3 tri-methylated at lysine 9. Importantly, ectopic coexpression of TS and RR or addition of deoxyribonucleosides substantially suppressed DNA damage, senescence-associated phenotypes, and proliferation arrest in two types of NHF-expressing HRAS(G12V). Reciprocally, short hairpin RNA-mediated suppression of TS and RR caused DNA damage and senescence in NHFs, although less efficiently than HRAS(G12V). However, overexpression of TS and RR in quiescent NHFs did not overcome proliferation arrest, suggesting that unlike quiescence, OIS requires depletion of dNTP pools and activated DNA replication. Our data identify a previously unknown role of deoxyribonucleotides in regulation of OIS.

KLF9 is a novel transcriptional regulator of bortezomib- and LBH589-induced apoptosis in multiple myeloma cells

Bortezomib, a therapeutic agent for multiple myeloma (MM) and mantle cell lymphoma, suppresses proteosomal degradation leading to substantial changes in cellular transcriptional programs and ultimately resulting in apoptosis. Transcriptional regulators required for bortezomib-induced apoptosis in MM cells are largely unknown. Using gene expression profiling, we identified 36 transcription factors that displayed altered expression in MM cells treated with bortezomib. Analysis of a publically available database identified Kruppel-like family factor 9 (KLF9) as the only transcription factor with significantly higher basal expression in MM cells from patients who responded to bortezomib compared with nonresponders. We demonstrated that KLF9 in cultured MM cells was up-regulated by bortezomib; however, it was not through the induction of endoplasmic reticulum stress. Instead, KLF9 levels correlated with bortezomib-dependent inhibition of histone deacetylases (HDAC) and were increased by the HDAC inhibitor LBH589 (panobinostat). Furthermore, bortezomib induced binding of endogenous KLF9 to the promoter of the proapoptotic gene NOXA. Importantly, KLF9 knockdown impaired NOXA up-regulation and apoptosis caused by bortezomib, LBH589, or a combination of theses drugs, whereas KLF9 overexpression induced apoptosis that was partially NOXA-dependent. Our data identify KLF9 as a novel and potentially clinically relevant transcriptional regulator of drug-induced apoptosis in MM cells.

PP2A-B56α controls oncogene-induced senescence in normal and tumor human melanocytic cells

Oncoprotein C-MYC is overexpressed in human metastatic melanomas and melanoma-derived cells where it is required for the suppression of oncogene-induced senescence (OIS). The genetic events that maintain high levels of C-MYC in melanoma cells and their role in OIS are unknown. Here we report that C-MYC in cells from several randomly chosen melanoma lines was upregulated at the protein level, and largely because of the increased protein stability. Of all known regulators of C-MYC stability, levels of B56α subunit of the PP2A tumor suppressor complex were substantially suppressed in all human melanoma cells compared with normal melanocytes. Accordingly, immunohistochemical analysis revealed that the lowest and the highest amounts of PP2A-B56α were predominantly detected in metastatic melanoma tissues and in primary melanomas from patients with good clinical outcome, respectively. Importantly, PP2A-B56α overexpression suppressed C-MYC in melanoma cells and induced OIS, whereas depletion of PP2A-B56α in normal human melanocytes upregulated C-MYC protein levels and suppressed BRAFV600E- and, less efficiently, NRASQ61R-induced senescence. Our data reveal a mechanism of C-MYC overexpression in melanoma cells and identify a functional role for PP2A-B56α in OIS of melanocytic cells.

MLH1 Deficiency Enhances Radiosensitization with 5-Fluorodeoxyuridine by Increasing DNA Mismatches

The antitumor drug 5-fluoro-2′-deoxyuridine (FdUrd) also sensitizes tumor cells to ionizing radiation in vitro and in vivo. Although radiosensitization with FdUrd requires dTTP depletion and S-phase arrest, the exact mechanism by which these events produce radiosensitization remains unknown. We hypothesized that the depletion of dTTP produces DNA mismatches that, if not repaired before irradiation, would result in radiosensitization. We evaluated this hypothesis in mismatch repair (MMR)-deficient HCT116 0-1 cells that lack the expression of the required MMR protein MLH1 (inactive MLH1), and in MMR-proficient (wild-type MLH1) HCT116 1-2 cells. Although HCT116 0-1 cells were less sensitive to FdUrd (IC50 = 3.5 μM) versus HCT116 1-2 cells (IC50 = 0.75 μM), when irradiation followed FdUrd (IC50) the MLH1-inactivated cells exhibited greater radiosensitization compared with MMR-wild-type cells [radiation enhancement ratio (RER) = 1.8 ± 0.28 versus 1.1 ± 0.1, respectively] and an increase (≥8-fold) in nucleotide misincorporations. In SW620 cells and HCT116 1-2 MLH1-wild-type cells, FdUrd (IC50) did not produce radiosensitization nor did it increase the mutation frequency, but after short hairpin RNA-directed suppression of MLH1 this concentration produced excellent radiosensitization (RER = 1.6 ± 0.10 and 1.5 ± 0.06, respectively) and an increase in nucleotide misincorporations (8-fold and 6-fold, respectively). Incubation with higher concentrations of FdUrd (IC90) after suppression of MLH1 produced a further increase in ionizing radiation sensitivity in both SW620 and HCT116 1-2 cells (RER = 1.8 ± 0.03 and 1.7 ± 0.13, respectively) and nucleotide misincorporations (>10-fold in both cell lines). These results demonstrate an important role for MLH1 and implicate mismatches in radiosensitization by FdUrd.

Abstract 847: Suppression of p53R2 but not R2 radiosensitizes mutant p53 tumor cells

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Gemcitabine (2’,2’-difluroro-2’-deoxycytidine;dFdCyd) is a potent radiosensitizer in tumor cells in vitro and in vivo. dFdCyd elicits cytotoxicity primarily via incorporation of its triphosphate, dFdCTP, into DNA, whereas inhibition of ribonucleotide reductase (RR) by dFdCDP produces a profound depletion of dATP which correlates to radiosensitization. We have demonstrated that dNTP imbalances generated by dFdCyd produce mismatches in DNA, which augment sensitivity to subsequent ionizing radiation (IR) but are not required to elicit cytotoxicity. We have proposed that RR suppression would be as effective as dFdCyd for radiosensitization. RR is a heterodimeric tetramer composed of the regulatory and active site subunit R1 paired with either R2 or its p53-inducible homolog, p53R2, as the catalytic and rate-limiting subunit. We used a RNAi approach to suppress either R2 or p53R2 (≥ 90% suppression of either R2 or p53R2, with little effect (≤20%) on expression of its homolog) in two p53 wild type cell lines, MCF7 breast carcinoma and A549 lung carcinoma. This approach produced equivalent radiosensitization, dATP depletion, cytotoxicity and increase in DNA mismatches compared to inactivation of RR by dFdCyd (IC50). These results reinforce our prior finding that radiosensitization with dFdCyd is the result of inhibition of RR and not incorporation into DNA or cytotoxicity. We then proposed that R2 but not p53R2 suppression would radiosensitize mutant p53 tumor cells. Interestingly, R2 shRNA suppression did not radiosensitize mutant p53 MCF7/ADR cells (radiation enhancement ratio (RER) = 1.07 ± 0.06). Despite the mutant p53 status of MCF7/ADR cells, p53R2 was elevated (≥ 50%) following suppression of R2 compared to untreated cells (no shRNA). We hypothesized that this increase in p53R2 permits RR to continue producing dNTPs for DNA replication and repair in the absence of R2, thus preventing radiosensitization. Indeed, simultaneous suppression of R2 and p53R2 produced excellent radiosensitization (RER = 1.68 ± 0.04 (shRNAs) vs. 1.6 ± 0.01 (dFdCyd (IC50)), and similar cytotoxicity (surviving fraction (SF) = 76.5 ± 8.3% (shRNA) vs. 71.3 ± 6.7% (dFdCyd)). Impressively, suppression of p53R2 alone produced excellent radiosensitization (RER= 1.76 ± 0.30) with similar cytotoxicity (SF = 84 ± 20%) compared to the double knockdown or dFdCyd. Considering that most solid tumors express mutant p53, suppression of p53R2 instead of R2 may be a more effective method for radiosensitization. These studies suggest that deoxynucleotide biosynthesis is regulated differently in p53 wild type compared to mutant p53 tumor cells, and may be used to maintain DNA replication and repair and prevent anticancer efficacy after damage induced by IR or other DNA damaging agents. The mechanism by which mutant p53 tumor cells upregulate p53R2 warrants further investigation. Note: This abstract was not presented at the meeting. Citation Format: Sheryl A. Flanagan, Jeffrey J. Ackroyd, Sudha Mannava, Mikhail A. Nikiforov, Donna S. Shewach. Suppression of p53R2 but not R2 radiosensitizes mutant p53 tumor cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 847. doi:10.1158/1538-7445.AM2014-847

Abstract 445: shRNA mediated suppression of either of the small subunits of ribonucleotide reductase, R2 and p53R2, elicits a robust increase in sensitivity to ionizing radiation.

Gemcitabine (2’,2’-difluroro-2’-deoxycytidine;dFdCyd) is a potent radiosensitizer in tumor cells in vitro and in vivo. dFdCyd elicits cytotoxicity primarily via incorporation of its triphosphate, dFdCTP, into DNA, whereas inhibition of ribonucleotide reductase (RR) by dFdCDP produces a profound depletion of dATP which correlates to radiosensitization. We have demonstrated that dNTP imbalances generated by dFdCyd produce mismatches in DNA, which, if not repaired, augment sensitivity to subsequent ionizing radiation (IR). DNA mismatches were not required to elicit cytotoxicity but were necessary for radiosensitization to occur. We therefore hypothesized that RNAi mediated suppression of RR activity would also radiosensitize cells. RR is a heterodimeric tetramer composed of the regulatory and active site subunit R1 paired with either R2 or its p53-inducible homolog, p53R2, as the catalytic and rate-limiting subunit. To determine whether the suppression of either R2 or p53R2 produced a similar extent of radiosensitization compared to dFdCyd, two different shRNAs were used to suppress each subunit in wild-type p53 MCF7 breast carcinoma and A549 non-small cell lung carcinoma cells, producing a profound (≥ 90%) suppression of the target protein, R2 or p53R2, with little effect (≤20%) on expression of its homolog. shRNAs for both R2 and p53R2 produced effects similar to those observed with dFdCyd on dATP depletion (% control value) (MCF7: 66 ± 6% (shRNA) vs. 85 ± 10 % (dFdCyd); A549: 75 ± 20% (shRNA) vs. 80 ± 5% (dFdCyd), and cytotoxicity (MCF7: 35-60% (shRNA) vs. 40-50% (FdCyd); A549: 40-65% (shRNA) vs. 40-60% (dFdCyd)). When shRNA suppression was followed by IR, radiosensitivity was similar with the shRNAs (R2 and p53R2) vs. dFdCyd (MCF7: radiation enhancement ratio (RER)) = 1.45 ± 0.08 (shRNA) vs. 1.7 ± 0.14 (dFdCyd); A549: RER= 1.51 ± 0.09 (shRNA) vs. 1.55 ± 0.14(dFdCyd)), and a similar increase in DNA mismatches compared to untreated wild type cells (MCF7: ≥ 7-fold (dFdCyd) vs. 5-7 fold (shRNA); A549: ≥ 3 fold (dFdCyd) vs. 3-5 fold (shRNA)) was observed. Although p53R2 is thought to be induced primarily in response to DNA damage, its effects on dATP depletion and DNA mismatches prior to IR suggest that it may play a previously unidentified role in normal maintenance of DNA replication. These results reinforce our prior finding that the mechanism of radiosensitization with dFdCyd is the decrease in dATP and not its incorporation into DNA or its cytotoxicity. Excellent radiosensitization with suppression of either R2 or p53R2, compared to dFdCyd, suggests that this targeted approach merits in vivo evaluation. Elevated expression of R2 is known to enhance the invasiveness of cancer cells, and increase resistance to dFdCyd, thus its suppression would further enhance anticancer activity in addition to its effect on radiosensitization. Citation Format: Sheryl A. Flanagan, Kristin Cooper, Sudha Mannava, Mikhail Nikiforov, Donna S. Shewach. shRNA mediated suppression of either of the small subunits of ribonucleotide reductase, R2 and p53R2, elicits a robust increase in sensitivity to ionizing radiation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 445. doi:10.1158/1538-7445.AM2013-445

Abstract 1390: shRNA mediated suppression of thymidylate synthase results in an increased sensitivity to ionizing radiation similar to 5-fluoro-2′-deoxyuridine

The fluoropyrimidines (FPs), 5-fluorouracil (5-FU) and 5-fluoro-2’-deoxyuridine (FdUrd), are widely used as radiosensitizers in the treatment of gastrointestinal cancer. The FPs exert their cytotoxic effects through activation to 5-fluoro-2’-deoxyuridine 5’-monophosphate (FdUMP), an inhibitor of thymidylate synthase (TS), resulting in depletion of dTTP and inhibition of DNA synthesis. Incorporation of fluorodeoxyuridine 5’-triphosphate (FdUTP) and dUTP into DNA can also elicit cytotoxicity. Radiosensitization by FdUrd correlates with dTTP depletion and S-phase arrest, but not with cytotoxicity. We hypothesized that depletion of dTTP leads to incorrect nucleotide incorporation into DNA which, if not repaired, augments cell death following irradiation. This hypothesis was supported by our findings that mismatch repair (MMR)-deficient HCT116 were better radiosensitized by FdUrd compared to MMR-proficient HCT 116 cells. Furthermore, FdUrd produced nucleotide misincorporations in DNA, measured directly as pSP189 plasmid mutations in HCT116 and SW620 cells, but only at radiosensitizing concentrations. We wished to determine whether suppression of TS protein would be as effective as FPs as a radiosensitizing strategy. To selectively inhibit TS we used each of two shRNAs, TS1 and TS2, both of which produced a ≥ 90% decrease in TS expression. Suppression of TS expression in HCT116 and HT29 cells elicited an increase in sensitivity to ionizing radiation (IR) that was similar to the increase observed with FdUrd (IC 50 ) (Radiation Enhancement Ratio (RER) = HCT116: 1.4 ± 0.08 (TS1), 1.5 ± 0.04 (TS2) vs. 1.5 ± 0.08 (FdUrd); HT29: 1.4 ± 0.06 (TS1), 1.4 ± 0.04 (TS2) vs. 1.6 ± 0.3 (FdUrd)). Additionally, a similar increase in pSP189 plasmid mutations was observed following suppression of TS, and FdUrd (IC 50 ) in both cell lines. S-phase accumulation following TS suppression was slightly attenuated compared to drug (HCT116: 70% (TS1), 60% (TS2) vs. 75% (FdUrd); HT29: 55% (TS1), 75% (TS2) vs. 85% (FdUrd)). FdUrd and TS suppression produced a similar initial depletion of dTTP (HCT116: 65% (TS1), 70% (TS2) vs. 40% (FdUrd); HT29: 75% (TS1), 50% (TS2) vs. 50% (FdUrd)), however dTTP levels rebounded with FdUrd, but remained low with TS suppression. dATP levels increased profoundly with FdUrd, but decreased slightly with TS suppression. Despite these differences in dNTPs, TS suppression produced less cytotoxicity than FdUrd (IC 50 ), but both produced a similar increase in sensitivity to IR. These results support our hypothesis that the mechanism of radiosensitzation by FdUrd is the decrease in dTTP, and not the incorporation of the drug into DNA or its cytotoxicity. Therefore, clinical treatment with FdUrd and IR could be titrated to maximize DNA mismatches in tumors rather than cytotoxicity, or TS could be targeted directly by shRNA methods to decrease normal tissue toxicity. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1390.