Statins in Cancer Prevention and Therapy.

Statins, a class of HMG-CoA reductase inhibitors best known for their cholesterol-reducing and cardiovascular protective activity, have also demonstrated promise in cancer prevention and treatment. This review focuses on their potential applications in head and neck cancer (HNC), a common malignancy for which established treatment often fails despite incurring debilitating adverse effects. Preclinical and clinical studies have suggested that statins may enhance HNC sensitivity to radiation and other conventional therapies while protecting normal tissue, but the underlying mechanisms remain poorly defined, likely involving both cholesterol-dependent and -independent effects on diverse cancer-related pathways. This review brings together recent discoveries concerning the anticancer activity of statins relevant to HNC, highlighting their anti-inflammatory activity and impacts on DNA-damage response. We also explore molecular targets and mechanisms and discuss the potential to integrate statins into conventional HNC treatment regimens to improve patient outcomes.

CDK-mediated Yku80 Phosphorylation Regulates the Balance Between Non-homologous End Joining (NHEJ) and Homologous Directed Recombination (HDR).

There are two major pathways for repairing DNA double-strand breaks (DSBs): homologous directed recombination (HDR) and non-homologous end-joining (NHEJ). While NHEJ functions throughout the cell cycle, HDR is only possible during S/G2 phases, suggesting that there are cell cycle-specific mechanisms regulating the balance between the two repair systems. The regulation exerted by CDKs on HDR has been extensively demonstrated, and here we present evidence that the CDK Pho85, in association with the G1 cyclin Pcl1, phosphorylates Yku80 on Ser 623 to regulate NHEJ activity. Cells bearing a non-phosphorylatable version of Yku80 show increased NHEJ and reduced HDR activity. Accordingly, yku80S623A cells present diminished viability upon treatment with the DSB-producer bleomycin, specifically in the G2 phase of the cell cycle. Interestingly, the mutation of the equivalent residue in human Ku80 increases sensitivity to bleomycin in several cancer cell lines, suggesting that this mechanism is conserved in humans. Altogether, our results reveal a new mechanism whereby G1-CDKs mediate the choice between HDR and NHEJ repair pathways, putting the error prone NHEJ on a leash and enabling error free HDR in G2 when homologous sequences are available.

Mevalonate pathway activity as a determinant of radiation sensitivity in head and neck cancer.

Radioresistance is a major hurdle in the treatment of head and neck squamous cell carcinoma (HNSCC). Here, we report that concomitant treatment of HNSCCs with radiotherapy and mevalonate pathway inhibitors (statins) may overcome resistance. Proteomic profiling and comparison of radioresistant to radiosensitive HNSCCs revealed differential regulation of the mevalonate biosynthetic pathway. Consistent with this finding, inhibition of the mevalonate pathway by pitavastatin sensitized radioresistant SQ20B cells to ionizing radiation and reduced their clonogenic potential. Overall, this study reinforces the view that the mevalonate pathway is a promising therapeutic target in radioresistant HNSCCs.

Comprehensive and quantitative analysis of G1 cyclins. A tool for studying the cell cycle.

In eukaryotes, the cell cycle is driven by the actions of several cyclin dependent kinases (CDKs) and an array of regulatory proteins called cyclins, due to the cyclical expression patterns of the latter. In yeast, the accepted pattern of cyclin waves is based on qualitative studies performed by different laboratories using different strain backgrounds, different growing conditions and media, and different kinds of genetic manipulation. Additionally, only the subset of cyclins regulating Cdc28 was included, while the Pho85 cyclins were excluded. We describe a comprehensive, quantitative and accurate blueprint of G1 cyclins in the yeast Saccharomyces cerevisiae that, in addition to validating previous conclusions, yields new findings and establishes an accurate G1 cyclin blueprint. For the purposes of this research, we produced a collection of strains with all G1 cyclins identically tagged using the same and most respectful procedure possible. We report the contribution of each G1 cyclin for a broad array of growing and stress conditions, describe an unknown role for Pcl2 in heat-stress conditions and demonstrate the importance of maintaining the 3'UTR sequence of cyclins untouched during the tagging process.

O-GlcNAcylation Enhances Double-Strand Break Repair, Promotes Cancer Cell Proliferation, and Prevents Therapy-Induced Senescence in Irradiated Tumors.

The metabolic reprogramming associated with characteristic increases in glucose and glutamine metabolism in advanced cancer is often ascribed to answering a higher demand for metabolic intermediates required for rapid tumor cell growth. Instead, recent discoveries have pointed to an alternative role for glucose and glutamine metabolites as cofactors for chromatin modifiers and other protein posttranslational modification enzymes in cancer cells. Beyond epigenetic mechanisms regulating gene expression, many chromatin modifiers also modulate DNA repair, raising the question whether cancer metabolic reprogramming may mediate resistance to genotoxic therapy and genomic instability. Our prior work had implicated N-acetyl-glucosamine (GlcNAc) formation by the hexosamine biosynthetic pathway (HBP) and resulting protein O-GlcNAcylation as a common means by which increased glucose and glutamine metabolism can drive double-strand break (DSB) repair and resistance to therapy-induced senescence in cancer cells. We have examined the effects of modulating O-GlcNAcylation on the DNA damage response (DDR) in MCF7 human mammary carcinoma in vitro and in xenograft tumors. Proteomic profiling revealed deregulated DDR pathways in cells with altered O-GlcNAcylation. Promoting protein O-GlcNAc modification by targeting O-GlcNAcase or simply treating animals with GlcNAc protected tumor xenografts against radiation. In turn, suppressing protein O-GlcNAcylation by blocking O-GlcNAc transferase activity led to delayed DSB repair, reduced cell proliferation, and increased cell senescence in vivo. Taken together, these findings confirm critical connections between cancer metabolic reprogramming, DDR, and senescence and provide a rationale to evaluate agents targeting O-GlcNAcylation in patients as a means to restore tumor sensitivity to radiotherapy. IMPLICATIONS: The finding that the HBP, via its impact on protein O-GlcNAcylation, is a key determinant of the DDR in cancer provides a mechanistic link between metabolic reprogramming, genomic instability, and therapeutic response and suggests novel therapeutic approaches for tumor radiosensitization.

Intertwined control of the cell cycle and nucleocytoplasmic transport by the cyclin-dependent kinase Pho85 and RanGTPase Gsp1 in Saccharomyces cerevisiae.

Deciphering the molecular mechanisms that connect cell cycle progression and nucleocytoplasmic transport is of particular interest: this intertwined relationship, once understood, may provide useful insight on the diseases resulting from the malfunction of these processes. In the present study we report on findings that indicate a biochemical connection between the cell cycle regulator CDK Pho85 and Ran-GTPase Gsp1, an essential nucleocytoplasmic transport component. When Gsp1 cannot be phosphorylated by Pho85, the cell cycle progression is impaired. Accordingly, a nonphosphorylatable version of Gsp1 abnormally localizes to the nucleus, which impairs the nuclear transport of molecules, including key components of cell cycle progression. Furthermore, our results suggest that the physical interaction of Gsp1 and the Kap95 karyopherin, essential to the release of nuclear cargoes, is altered. Altogether, the present findings point to the involvement of a biochemical mechanism in the interlocked regulation of the cell cycle and nuclear transport.

HMG-CoA Reductase Inhibition Delays DNA Repair and Promotes Senescence After Tumor Irradiation.

Despite significant advances in combinations of radiotherapy and chemotherapy, altered fractionation schedules and image-guided radiotherapy, many cancer patients fail to benefit from radiation. A prevailing hypothesis is that targeting repair of DNA double strand breaks (DSB) can enhance radiation effects in the tumor and overcome therapeutic resistance without incurring off-target toxicities. Unrepaired DSBs can block cancer cell proliferation, promote cancer cell death, and induce cellular senescence. Given the slow progress to date translating novel DSB repair inhibitors as radiosensitizers, we have explored drug repurposing, a proven route to improving speed, costs, and success rates of drug development. In a prior screen where we tracked resolution of ionizing radiation-induced foci (IRIF) as a proxy for DSB repair, we had identified pitavastatin (Livalo), an HMG-CoA reductase inhibitor commonly used for lipid lowering, as a candidate radiosensitizer. Here, we report that pitavastatin and other lipophilic statins are potent inhibitors of DSB repair in breast and melanoma models both in vitro and in vivo When combined with ionizing radiation, pitavastatin increased persistent DSBs, induced senescence, and enhanced acute effects of radiation on radioresistant melanoma tumors. shRNA knockdown implicated HMG-CoA reductase, farnesyl diphosphate synthase, and protein farnesyl transferase in IRIF resolution, DSB repair, and senescence. These data confirm on-target activity of statins, although via inhibition of protein prenylation rather than cholesterol biosynthesis. In light of prior studies demonstrating enhanced efficacy of radiotherapy in patients taking statins, this work argues for clinical evaluation of lipophilic statins as nontoxic radiosensitizers to enhance the benefits of image-guided radiotherapy. Mol Cancer Ther; 17(2); 407-18. ©2017 AACRSee all articles in this MCT Focus section, "Developmental Therapeutics in Radiation Oncology."

The quantitative changes in the yeast Hsp70 and Hsp90 interactomes upon DNA damage.

The molecular chaperones Hsp70 and Hsp90 participate in many important cellular processes, including how cells respond to DNA damage. Here we show the results of applied quantitative affinity-purification mass spectrometry (AP-MS) proteomics to understand the protein network through which Hsp70 and Hsp90 exert their effects on the DNA damage response (DDR). We characterized the interactomes of the yeast Hsp70 isoform Ssa1 and Hsp90 isoform Hsp82 before and after exposure to methyl methanesulfonate. We identified 256 chaperone interactors, 146 of which are novel. Although the majority of chaperone interaction remained constant under DNA damage, 5 proteins (Coq5, Ast1, Cys3, Ydr210c and Rnr4) increased in interaction with Ssa1 and/or Hsp82. This data presented here are related to [1] (Truman et al., in press). The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE partner repository (Vizcaino et al. (2013) [2]) with the dataset identifier PXD001284.

Quantitative proteomics of the yeast Hsp70/Hsp90 interactomes during DNA damage reveal chaperone-dependent regulation of ribonucleotide reductase.

The highly conserved molecular chaperones Hsp90 and Hsp70 are indispensible for folding and maturation of a significant fraction of the proteome, including many proteins involved in signal transduction and stress response. To examine the dynamics of chaperone-client interactions after DNA damage, we applied quantitative affinity-purification mass spectrometry (AP-MS) proteomics to characterize interactomes of the yeast Hsp70 isoform Ssa1 and Hsp90 isoform Hsp82 before and after exposure to methyl methanesulfonate. Of 256 proteins identified and quantified via (16)O(/18)O labeling and LC-MS/MS, 142 are novel Hsp70/90 interactors. Nearly all interactions remained unchanged or decreased after DNA damage, but 5 proteins increased interactions with Ssa1 and/or Hsp82, including the ribonucleotide reductase (RNR) subunit Rnr4. Inhibiting Hsp70 or 90 chaperone activity destabilized Rnr4 in yeast and its vertebrate homolog hRMM2 in breast cancer cells. In turn, pre-treatment of cancer cells with chaperone inhibitors sensitized cells to the RNR inhibitor gemcitabine, suggesting a novel chemotherapy strategy. All MS data have been deposited in the ProteomeXchange with identifier PXD001284. BIOLOGICAL SIGNIFICANCE: This study provides the dynamic interactome of the yeast Hsp70 and Hsp90 under DNA damage which suggest key roles for the chaperones in a variety of signaling cascades. Importantly, the cancer drug target ribonucleotide reductase was shown to be a client of Hsp70 and Hsp90 in both yeast and breast cancer cells. As such, this study highlights the potential of a novel cancer therapeutic strategy that exploits the synergy of chaperone and ribonucleotide reductase inhibitors.

Redundancy or specificity? The role of the CDK Pho85 in cell cycle control.

It is generally accepted that progression through the eukaryotic cell cycle is driven by cyclin-dependent kinases (CDKs), which are regulated by interaction with oscillatory expressed proteins called cyclins. CDKs may be separated into 2 categories: essential and non-essential. Understandably, more attention has been focused on essential CDKs because they are shown to control cell cycle progression to a greater degree. After clearly determining the basic and "core" mechanisms of essential CDKs, several questions arise. What role do non-essential CDKs play? Are these CDKs functionally redundant and do they serve as a mere backup? Or might they be responsible for some accessory tasks in cell cycle progression or control? In the present review we will try to answer these questions based on recent findings on the involvement of non-essential CDKs in cell cycle progression. We will analyse the most recent information with regard to these questions in the yeast Saccharomyces cerevisiae, a well-established eukaryotic model, and in its unique non-essential CDK involved in the cell cycle, Pho85. We will also briefly extend our discussion to higher eukaryotic systems.

Defective in mitotic arrest 1 (Dma1) ubiquitin ligase controls G1 cyclin degradation.

Progression through the G(1) phase of the cell cycle is controlled by diverse cyclin-dependent kinases (CDKs) that might be associated to numerous cyclin isoforms. Given such complexity, regulation of cyclin degradation should be crucial for coordinating progression through the cell cycle. In Saccharomyces cerevisiae, SCF is the only E3 ligase known to date to be involved in G(1) cyclin degradation. Here, we report the design of a genetic screening that uncovered Dma1 as another E3 ligase that targets G(1) cyclins in yeast. We show that the cyclin Pcl1 is ubiquitinated in vitro and in vivo by Dma1, and accordingly, is stabilized in dma1 mutants. We demonstrate that Pcl1 must be phosphorylated by its own CDK to efficiently interact with Dma1 and undergo degradation. A nonphosphorylatable version of Pcl1 accumulates throughout the cell cycle, demonstrating the physiological relevance of the proposed mechanism. Finally, we present evidence that the levels of Pcl1 and Cln2 are independently controlled in response to nutrient availability. This new previously unknown mechanism for G(1) cyclin degradation that we report here could help elucidate the specific roles of the redundant CDK-cyclin complexes in G(1).