Role of Ku70 in deubiquitination of Mcl-1 and suppression of apoptosis

Mcl-1 is a unique antiapoptotic Bcl2 family member with a short half-life due to its rapid turnover through ubiquitination. We discovered that Ku70, a DNA double-strand break repair protein, functions as a deubiquitinase to stabilize Mcl-1. Ku70 knockout in mouse embryonic fibroblast (MEF) cells or depletion from human lung cancer H1299 cells leads to the accumulation of polyubiquitinated Mcl-1 and a reduction in its half-life and protein expression. Conversely, expression of exogenous Ku70 in Ku70^−/− MEF cells restores Mcl-1 expression. Subcellular fractionation indicates that Ku70 extensively colocalizes with Mcl-1 in mitochondria, endoplasmic reticulum and nucleus in H1299 cells. Ku70 directly interacts with Mcl-1 via its C terminus (that is, aa 536–609), which is required and sufficient for deubiquitination and stabilization of Mcl-1, leading to suppression of apoptosis. Purified Ku70 protein directly deubiquitinates Mcl-1 by removing K48-linked polyubiquitin chains. Ku70 knockdown not only promotes Mcl-1 turnover but also enhances antitumor efficacy of the BH3-mimetic ABT-737 in human lung cancer xenografts. These findings identify Ku70 as a novel Mcl-1 deubiquitinase that could be a potential target for cancer therapy by manipulating Mcl-1 deubiquitination.Mcl-1 is an antiapoptotic molecule that is overexpressed in various types of cancers, including lung cancer,¹ leukemia,² lymphoma,³ hepatocellular carcinoma⁴ and so on. In addition to its antiapoptotic function, Mcl-1 is also an oncoprotein that promotes the development of cancer.⁵ In contrast to other Bcl2 family members such as Bcl2 and Bcl-XL, Mcl-1 is unique in its short half-life (30 min–3 h) and short-term prosurvival function, which probably relates to the presence of a long proline-, glutamic acid-, serine- and threonine-rich (PEST) region upstream of the Bcl2 homology (BH) domain.¹ The mechanism(s) that stabilizes the Mcl-1 protein are critical for its long-term survival function. Mcl-1 protein can be phosphorylated at multiple sites that distinctly regulate Mcl-1 protein turnover. For example, extracellular signal-regulated kinase 1/2-mediated T163 site phosphorylation enhances the half-life and antiapoptotic function of Mcl-1.^{1, 6} In contrast, S159 phosphorylation by GSK-3β facilitates Mcl-1 ubiquitination and degradation to reduce its survival activity.⁷Ubiquitination and deubiquitination are two reversible processes that can control protein stability. E3 ligases and deubiquitinases (deubiquitinating enzymes (DUBs)) are two groups of regulatory enzymes that orchestrate the ubiquitination levels of target proteins in eukaryotic cells.⁸ Recently, Mule and FBW7 have been identified as Mcl-1 ubiquitin E3 ligases that can directly induce polyubiquitination and degradation of Mcl-1.^{9, 10} Inversely, USP9X has been demonstrated as the Mcl-1 deubiquitinase that removes the Lys 48-linked polyubiquitin chains that normally mark Mcl-1 for proteasomal degradation, leading to stabilization of Mcl-1.³ Therefore, the stability of Mcl-1 in cells is tightly regulated by its E3 ligases and deubiquitinase, which is dependent on Mcl-1 phosphorylation status.^{3, 11}Ku70 is a protein that binds to DNA double-strand break (DSB) ends and is required for the non-homologous end-joining pathway of DSB repair.^{12, 13, 14, 15} The Ku70 protein consists of three structural domains, including the N-terminal, central (that is, DNA binding) and C-terminal domains.^{16, 17} Ku70 usually heterodimerizes with Ku86, which forms a functional complex for DSB repair. By forming a bridge between the broken DNA ends, the Ku70/Ku86 heterodimer acts to structurally support and align the DNA ends, to protect them from degradation and to prevent promiscuous binding to unbroken DNA. Ku70/Ku86 effectively aligns the DNA, while still allowing access of polymerases, nucleases and ligases to the broken DNA ends to promote end joining.¹⁸ In some cases, a fourth domain is present at the C terminus of Ku86, which binds to the DNA-dependent protein kinase catalytic subunit.¹⁹ Importantly, Ku70 also regulates apoptosis independent of its DSB repair activity. For example, a recent report revealed that Ku70 regulates the proapoptotic function of Bax by sequestering Bax from the mitochondria and mediating Bax deubiquitylation.²⁰ Here we discovered that Ku70 functions as a novel Mcl-1 deubiquitinase that directly removes polyubiquitin chains from Mcl-1 protein, leading to reduced Mcl-1 ubiquitination/degradation, enhanced stability and suppression of apoptosis.     

Mir-660 is downregulated in lung cancer patients and its replacement inhibits lung tumorigenesis by targeting MDM2-p53 interaction

Lung cancer represents the leading cause of cancer-related death in developed countries. Despite the advances in diagnostic and therapeutic techniques, the 5-year survival rate remains low. The research for novel therapies directed to biological targets has modified the therapeutic approach, but the frequent engagement of resistance mechanisms and the substantial costs, limit the ability to reduce lung cancer mortality. MicroRNAs (miRNAs) are small noncoding RNAs with known regulatory functions in cancer initiation and progression. In this study we found that mir-660 expression is downregulated in lung tumors compared with adjacent normal tissues and in plasma samples of lung cancer patients with poor prognosis, suggesting a potential functional role of this miRNA in lung tumorigenesis. Transient and stable overexpression of mir-660 using miRNA mimics reduced migration, invasion, and proliferation properties and increased apoptosis in p53 wild-type lung cancer cells (NCI-H460, LT73, and A549). Furthermore, stable overexpression using lentiviral vectors in NCI-H460 and A549 cells inhibited tumor xenograft growth in immunodeficient mice (95 and 50% reduction compared with control, respectively), whereas the effects of mir-660 overexpression were absent in H1299, a lung cancer cell line lacking p53 locus, both in in vitro and in vivo assays. We identified and validated mouse double minute 2 (MDM2) gene, a key regulator of the expression and function of p53, as a new direct target of mir-660. In addition, mir-660 expression reduced both mRNA and protein expression of MDM2 in all cell lines and stabilized p53 protein levels resulting in an upregulation of p21^WAF1/CIP1 in p53 wild-type cells. Our finding supports that mir-660 acts as a tumor suppressor miRNA and we suggest the replacement of mir-660 as a new therapeutic approach for p53 wild-type lung cancer treatment.Lung cancer is the leading cause of cancer death worldwide, resulting in >1.4 million deaths/year.¹ Lung tumors are often discovered as locally advanced or metastatic disease, and despite improvements in molecular diagnosis and targeted therapies, the overall 5-year survival rate remains in the 10–20% range. Indeed, nonsmall cell lung cancer (NSCLC) is poorly chemosensitive to most of the available agents with response rates ranging from 10 to 25%.² The discovery of recurrent mutations in the epidermal growth factor receptor (EGFR) kinase,³ as well as gene fusion products involving the anaplastic lymphoma kinase (ALK),⁴ has led to a marked change in the treatment of patients with lung adenocarcinoma, the most common type of lung cancer.^{5, 6} To date, patients with mutations in the EGFR gene, suitable for targeting by EGFR tyrosine kinase inhibitors, represent roughly 10%, whereas the subgroup of tumors with ALK rearrangements, targeted by ALK inhibitors, is only ~5%.⁷ Thus, the majority of lung tumors lack effective treatment and novel therapeutic strategies are still needed.MicroRNAs (miRNAs) are short noncoding RNAs, 20–24 nucleotides long, that have important roles in almost all biological pathways,^{8, 9, 10, 11} and influence cancer-relevant processes, such as proliferation,¹² cell cycle,¹³ apoptosis,¹⁴ and migration.¹⁵ Many studies have reported the critical role of miRNAs in lung cancer pathogenesis and their potential as biomarkers for lung cancer risk stratification,¹⁶ outcome prediction,¹⁷ and classification of histological subtypes.^{18, 19} miRNAs are actively released by various cell types and can be detected in biological fluids, such as plasma and serum, making them suitable as circulating biomarkers in NSCLC.^{20, 21}There is limited evidence of mir-660 deregulation in cancer and little is known about its role in lung tumorigenesis and its putative target genes. Mir-660 has been reported to be upregulated in chronic lymphocytic leukemia^{22, 23} and in leukemic cells after treatment with 4-hydroxynonenal, a compound that induces differentiation and blocks proliferation of leukemic cells.²⁴ In a previous study we demonstrated that mir-660 was one of the 24 miRNAs deregulated in plasma samples of NSCLC patients identified in a low-dose computed tomography (LDCT) screening trial.²⁰The p53 tumor suppressor protein is a key regulator of cell cycle G0/G1 checkpoint, senescence, and apoptosis in response to cellular stress signals.^{25, 26} Mouse double minute 2 (MDM2), a p53–E3 ubiquitin ligase,²⁷ is the principal negative regulator of the expression level and function of p53.^{28, 29} Several studies have illustrated different mechanisms of p53 regulation by MDM2,^{30, 31} such as binding transactivation region of p53,^{32, 33} promoting nuclear export and cytoplasmic accumulation of p53 by monoubiquitination,^{34, 35} and inducing p53 proteosomal degradation by polyubiquitination.³⁶ In addition, MDM2 gene has been reported to be amplified or overexpressed in a variety of human cancers, such as sarcoma,³⁷ lymphoma,³⁸ breast cancer,³⁹ lung cancer,⁴⁰ and testicular germ cell tumor.⁴¹ Several miRNAs targeting MDM2 have been identified, such as the mir-143/mir-145 cluster that can be induced by p53,⁴² as well as mir-25 and mir-32, known to inhibit tumor glioblastoma growth in mouse brain.⁴³In this study, we report that mir-660 is downregulated in tissue and plasma samples of lung cancer patients and demonstrate that mir-660 replacement impairs the functionality of p53 wild-type (wt) lung cancer cells and inhibits in vitro and in vivo tumor growth. We showed that all the effects observed after mir-660 overexpression were absent in p53 ko cells, identified MDM2 as mir-660 direct target gene and indicate impairment of the MDM2/p53 interaction as the mechanism underlying tumor growth inhibition.     

Restoration of miR-101 suppresses lung tumorigenesis through inhibition of DNMT3a-dependent DNA methylation

The deregulation of miR-101 and DNMT3a has been implicated in the pathogenesis of multiple tumor types, but whether and how miR-101 silencing and DNMT3a overexpression contribute to lung tumorigenesis remain elusive. Here we show that miR-101 downregulation associates with DNMT3a overexpression in lung cancer cell lines and patient tissues. Ectopic miR-101 expression remarkably abrogated the DNMT3a 3′-UTR luciferase activity corresponding to the miR-101 binding site and caused an attenuated expression of endogenous DNMT3a, which led to a reduction of global DNA methylation and the re-expression of tumor suppressor CDH1 via its promoter DNA hypomethylation. Functionally, restoration of miR-101 expression suppressed lung cancer cell clonability and migration, which recapitulated the DNMT3a knockdown effects. Interestingly, miR-101 synergized with decitabine to downregulate DNMT3a and to reduce DNA methylation. Importantly, ectopic miR-101 expression was sufficient to trigger in vivo lung tumor regression and the blockage of metastasis. Consistent with these phenotypes, examination of xenograft tumors disclosed an increase of miR-101, a decrease of DNMT3a and the subsequent DNA demethylation. These findings support that the loss or suppression of miR-101 function accelerates lung tumorigenesis through DNMT3a-dependent DNA methylation, and suggest that miR-101-DNMT3a axis may have therapeutic value in treating refractory lung cancer.Owing to a high propensity for recurrence and a high rate of metastasis at the advanced stages,^{1, 2, 3} lung cancer remains the leading cause of cancer-related mortality. DNA methylation is a major epigenetic rule controlling chromosomal stability and gene expression.^{4, 5} It is under control of DNA methyltransferases (DNMTs), whose overexpression in lung cancer cells predicts worse outcomes.^{6, 7} It is postulated that DNMT overexpression induces DNA hypermethylation and silencing of tumor suppressor genes (TSGs), leading to an aggressive lung cancer. Indeed, enforced expression of DNMT1 or DNMT3a increases DNA methylation, while the abolition of DNMT expression by genetic depletion, microRNAs (miRs) or small molecules reduces genome-wide and gene-specific DNA methylation and restores TSG expression.^{8, 9, 10, 11, 12, 13} As TSGs are the master controllers for cell multiplicity and their silencing predicts poor prognosis,^{14, 15} TSG re-expression via promoter DNA hypomethylation inhibits cell proliferation and induces cell differentiation.^{13, 16} Thus, DNMT gene abundance could serve as a target for anticancer therapy, but how DNMT upregulation occurs in lung cancer is incompletely understood.MiRs are small non-coding RNAs that crucially regulate target gene expression. Up to 30% of all protein-coding genes are predicted to be targeted by miRs,^{17, 18} supporting the key roles of miRs in controlling cell fate.^{19, 20, 21, 22} Research is showing that certain miRs are frequently dysregulated in cancers, including lung cancer.^{7, 23, 24} As miR targets can promote or inhibit cancer cell expansion, miRs have huge potential for acting as bona fide oncogenes (i.e., miR-21) or TSGs (i.e., miR-29b).^{7, 25} We and others demonstrated that the levels of DNMT1 or DNMT3a or DNMT3b are regulated by miR-29b, miR-148, miR-152 or miR-30c,^{7, 13, 26, 27} and overexpression of these miRs results in DNA hypomethylation and TSG reactivation with the concurrent blockage of cancer cell proliferation.^{7, 13} These findings underscore the importance of miRs as epigenetic modulators and highlight their therapeutic applications.MiR-101 is frequently silenced in human cancers^{28, 29, 30, 31} and, importantly, exhibits antitumorigenic properties when overexpressed. Mechanistically, miR-101 inactivation by genomic loss causes the overexpression of EZH2, a histone methyltransferase, via 3′-UTR targeting, which is followed by histone hypermethylation and aggressive tumorigenesis.^{29, 30, 32} However, whether and how miR-101 silencing contributes to DNA hypermethylation patterning in lung cancer is unclear. In this study, we explore the role of miR-101 in regulating DNMT3a expression and the impacts of miR-101-DNMT3a nexus on lung cancer pathogenesis. We showed that the expression of miR-101 and DNMT3a was negatively correlated in lung cancer. We presented evidence that ectopic miR-101 expression decreased DNMT3a levels, reduced global DNA methylation and upregulated CDH1 via its promoter DNA demethylation. The biological significance of miR-101-mediated DNA hypomethylation and CDH1 re-expression was evident by its inhibition of lung tumor cell growth in vitro and in vivo. Thus, our findings mechanistically and functionally link miR-101 silencing to DNA hypermethylation in lung cancer cells.     

Transforming growth factor-beta signaling network regulates plasticity and lineage commitment of lung cancer cells

Identification of target cells in lung tumorigenesis and characterization of the signals that control their behavior is an important step toward improving early cancer diagnosis and predicting tumor behavior. We identified a population of cells in the adult lung that bear the EpCAM+CD104+CD49f+CD44+CD24loSCA1+ phenotype and can be clonally expanded in culture, consistent with the properties of early progenitor cells. We show that these cells, rather than being restricted to one tumor type, can give rise to several different types of cancer, including adenocarcinoma and squamous cell carcinoma. We further demonstrate that these cells can be converted from one cancer type to the other, and this plasticity is determined by their responsiveness to transforming growth factor (TGF)-beta signaling. Our data establish a mechanistic link between TGF-beta signaling and SOX2 expression, and identify the TGF-beta/SMAD/SOX2 signaling network as a key regulator of lineage commitment and differentiation of lung cancer cells.Lung cancer is the leading cause of cancer-related mortality in both men and women worldwide. Lung cancers are divided into two major categories: non-small-cell lung cancer (NSCLC) and small-cell lung cancer. NSCLC accounts for ∼80% of all lung cancers and is divided further into adenocarcinoma (ADC), squamous cell carcinoma (SCC) and large-cell lung carcinoma. Of the four major types of lung cancer, Kras mutations are present in about 30–50% of ADC, a smaller percentage of SCC (5–7%) and <1% of SCLC.^{1, 2} Mutations of the p53 gene are common in all types of lung cancer and range from ∼30% in ADC to more than 70% in SCC and SCLC.³ Other alterations occur at lower frequencies in NSCLC, including mutations in EGFR (15%), EML4-ALK (4%), ERBB2 (2%), AKT1, BRAF, MAP2K1 and MET.^{2, 4} Previous efforts in comprehensive characterization of lung cancer include copy number and gene expression profiling, targeted sequencing of candidate genes and large-scale genome sequencing of tumor samples.^{5, 6, 7, 8, 9} Significant progress has also been made in developing mouse models of lung carcinogenesis.^{10, 11} The unifying theme underlying these studies is that there exists a permissive cellular context for each specific oncogenic lesion, and that only certain types of cells are capable of cancer initiation.^{12, 13, 14}The lung consists of three anatomically distinct regions such as trachea, bronchioles and alveoli, each maintained by a distinct population of progenitor cells, that is, basal, Clara and alveolar type 2 (AT2) cells, respectively.^{15, 16} Previous work has focused upon AT2 cells, Clara cells (or variant Clara cells with low CC10 expression) and the putative bronchioalveolar stem cells (BASCs) as potential cells of origin for lung ADC.^{12, 14, 17} However, to date, only AT2 cells have been conclusively identified as having the potential to be the cells of origin for lung ADC.^{14, 17} This raises the question of whether Clara cells, their restricted subpopulations or the newly identified candidate stem cells, termed distal airway stem cells,¹⁸ alveolar epithelial progenitor cells (AECs)^{19, 20} and BASCs,¹² also have the capacity to give rise to ADC. Current knowledge on the cellular origins of SCC, the second most common type of lung cancer, lags behind that of ADC, partly owing to the fact that squamous cells are not normally present in the respiratory epithelium, and therefore arise through either metaplasia (conversions between stem cell states) or trans-differentiation (conversions between differentiated cells).^{21, 22} Whether the mechanisms of SCC causation vary by cell type, their responses to various cells signaling cascades (e.g., transforming growth factor (TGF)-beta, WNT, etc.), or other tumor characteristics is unknown at present.To address the questions of cell type of origin and signal cascades that control their behavior, we developed in vitro culture conditions that favor the growth of lung epithelial cells with stem cell-like properties. We describe a population of cells isolated from the adult lung that, rather than being restricted to one tumor type, can give rise to several different types of cancer, including ADC and SCC. We also show that these cells can be converted from one cancer type to the other, and this plasticity is largely, if not solely, determined by TGF-beta signaling.     

The Fcp1-Wee1-Cdk1 axis affects spindle assembly checkpoint robustness and sensitivity to antimicrotubule cancer drugs

To grant faithful chromosome segregation, the spindle assembly checkpoint (SAC) delays mitosis exit until mitotic spindle assembly. An exceedingly prolonged mitosis, however, promotes cell death and by this means antimicrotubule cancer drugs (AMCDs), that impair spindle assembly, are believed to kill cancer cells. Despite malformed spindles, cancer cells can, however, slip through SAC, exit mitosis prematurely and resist killing. We show here that the Fcp1 phosphatase and Wee1, the cyclin B-dependent kinase (cdk) 1 inhibitory kinase, play a role for this slippage/resistance mechanism. During AMCD-induced prolonged mitosis, Fcp1-dependent Wee1 reactivation lowered cdk1 activity, weakening SAC-dependent mitotic arrest and leading to mitosis exit and survival. Conversely, genetic or chemical Wee1 inhibition strengthened the SAC, further extended mitosis, reduced antiapoptotic protein Mcl-1 to a minimum and potentiated killing in several, AMCD-treated cancer cell lines and primary human adult lymphoblastic leukemia cells. Thus, the Fcp1-Wee1-Cdk1 (FWC) axis affects SAC robustness and AMCDs sensitivity.The spindle assembly checkpoint (SAC) delays mitosis exit to coordinate anaphase onset with spindle assembly. To this end, SAC inhibits the ubiquitin ligase Anaphase-Promoting Complex/Cyclosome (APC/C) to prevent degradation of the anaphase inhibitor securin and cyclin B, the major mitotic cyclin B-dependent kinase 1 (cdk1) activator, until spindle assembly.¹ However, by yet poorly understood mechanisms, exceedingly prolonging mitosis translates into cell death induction.^{2, 3, 4, 5, 6, 7} Although mechanistic details are still missing on how activation of cell death pathways is linked to mitosis duration, prolongation of mitosis appears crucial for the ability of antimicrotubule cancer drugs (AMCDs) to kill cancer cells.^{2, 3, 4, 5, 6, 7} These drugs, targeting microtubules, impede mitotic spindle assembly and delay mitosis exit by chronically activating the SAC. Use of these drugs is limited, however, by toxicity and resistance. A major mechanism for resistance is believed to reside in the ability of cancer cells to slip through the SAC and exit mitosis prematurely despite malformed spindles, thus resisting killing by limiting mitosis duration.^{2, 3, 4, 5, 6, 7} Under the AMCD treatment, cells either die in mitosis or exit mitosis, slipping through the SAC, without or abnormally dividing.^{2, 3, 4} Cells that exit mitosis either die at later stages or survive and stop dividing or proliferate, giving rise to resistance.^{2, 3, 4} Apart from a role for p53, what dictates cell fate is still unknown; however, it appears that the longer mitosis is protracted, the higher the chances for cell death pathway activation are.^{2, 3, 4, 5, 6, 7}Although SAC is not required per se for killing,⁶ preventing SAC adaptation should improve the efficacy of AMCD by increasing mitosis duration.^{2, 3, 4, 5, 6, 7} Therefore, further understanding of the mechanisms by which cells override SAC may help to improve the current AMCD therapy. Several kinases are known to activate and sustain SAC, and cdk1 itself appears to be of primary relevance.^{1, 8, 9} By studying mitosis exit and SAC resolution, we recently reported a role for the Fcp1 phosphatase to bring about cdk1 inactivation.^{10, 11} Among Fcp1 targets, we identified cyclin degradation pathway components, such as Cdc20, an APC/C co-activator, USP44, a deubiquitinating enzyme, and Wee1.^{10, 11} Wee1 is a crucial kinase that controls the G2 phase by performing inhibitory phosphorylation of cdk1 at tyr-15 (Y15-cdk1). Wee1 is also in a feedback relationship with cdk1 itself that, in turn, can phosphorylate and inhibit Wee1 in an autoamplification loop to promote the G2-to-M phase transition.¹² At mitosis exit, Fcp1 dephosphorylated Wee1 at threonine 239, a cdk1-dependent inhibitory phosphorylation, to dampen down the cdk1 autoamplification loop, and Cdc20 and USP44, to promote APC/C-dependent cyclin B degradation.^{10, 11, 12} In this study we analysed the Fcp1 relevance in SAC adaptation and AMCD sensitivity.     

Tyr1068-phosphorylated epidermal growth factor receptor (EGFR) predicts cancer stem cell targeting by erlotinib in preclinical models of wild-type EGFR lung cancer

Tyrosine kinase inhibitors (TKIs) have shown strong activity against non-small-cell lung cancer (NSCLC) patients harboring activating epidermal growth factor receptor (EGFR) mutations. However, a fraction of EGFR wild-type (WT) patients may have an improvement in terms of response rate and progression-free survival when treated with erlotinib, suggesting that factors other than EGFR mutation may lead to TKI sensitivity. However, at present, no sufficiently robust clinical or biological parameters have been defined to identify WT-EGFR patients with greater chances of response. Therapeutics validation has necessarily to focus on lung cancer stem cells (LCSCs) as they are more difficult to eradicate and represent the tumor-maintaining cell population. Here, we investigated erlotinib response of lung CSCs with WT-EGFR and identified EGFR phosphorylation at tyrosine1068 (EGFR^tyr1068) as a powerful biomarker associated with erlotinib sensitivity both in vitro and in preclinical CSC-generated xenografts. In contrast to the preferential cytotoxicity of chemotherapy against the more differentiated cells, in EGFR^tyr1068 cells, erlotinib was even more active against the LCSCs compared with their differentiated counterpart, acquiring potential value as CSC-directed therapeutics in the context of WT-EGFR lung cancer. Although tumor growth was inhibited to a similar extent during erlotinib or chemotherapy administration to responsive tumors, erlotinib proved superior to chemotherapy in terms of higher tolerability and reduced tumor aggressiveness after treatment suspension, substantiating the possibility of preferential LCSC targeting, both in adenocarcinoma (ADC) and squamous cell carcinoma (SCC) tumors. We conclude that EGFR^tyr1068 may represent a potential candidate biomarker predicting erlotinib response at CSC-level in EGFR-WT lung cancer patients. Finally, besides its invariable association with erlotinib sensitivity in EGFR-WT lung CSCs, EGFR^tyr1068 was associated with EGFR-sensitizing mutations in cell lines and patient tumors, with relevant diagnostic, clinical and therapeutic implications.Non-small-cell lung cancer (NSCLC) accounts for ∼80% of lung cancer subtypes and is the leading cause of cancer-related death worldwide.¹ In recent years, molecular characterization of NSCLC has reached an unprecedented detail and has allowed segregating NSCLC into discrete molecular subgroups, characterized by specific oncogenic drivers, such as epidermal growth factor receptor (EGFR), BRAF, KRAS, epidermal growth factor receptor 2 (HER2) mutations, MET amplification and anaplastic lymphoma kinase gene rearrangements (ALK).^{2, 3} Consequently, the understanding of NSCLC biology has brought two new classes of targeted agents into the clinical setting: EGFR tyrosine kinase inhibitors (TKIs) and ALK inhibitors.^{4, 5} In particular, clinical trials have shown that NSCLC patients whose tumors harbor sensitizing EGFR mutations significantly benefit from the upfront use of an EGFR TKI, rather than conventional chemotherapy.^{6, 7, 8, 9, 10, 11} Although licensed for clinical use in chemotherapy-pretreated patients, regardless of EGFR mutational status, the EGFR TKI erlotinib has limited efficacy when compared with standard chemotherapy in patients with WT-EGFR NSCLC.^{12, 13, 14}However, a fraction of patients on erlotinib treatment may achieve clinically significant objective responses and prolonged disease control, despite the lack of detectable EGFR mutations.¹⁵ Nevertheless, no biomarker investigated so far was felt sufficiently robust to select for the use of erlotinib in the maintenance or refractory setting.¹⁶ Thus, it would be crucial to identify molecular predictors of TKI sensitivity in EGFR wild-type (WT) tumors in order to prospectively select the subgroup of patients who may benefit from erlotinib therapy. Moreover, EGFR TKIs have also shown a modest therapeutic effect in lung squamous cell carcinoma (SCC), where EGFR mutations are very rare and patients have limited therapeutic options in the maintenance and relapsed settings.^{16, 17, 18, 19, 20}Even more importantly, in order to obtain meaningful clinical responses it is crucial to effectively target the population of cells that are able to escape treatment and maintain the growth of a resistant tumor.²¹ Cancer stem cells (CSCs) have been in fact identified within most solid tumors, including lung tumors, and are associated with increased resistance to therapies.^{22, 23, 24, 25, 26, 27, 28, 29, 30} Thus, the efficacy of innovative therapeutic strategies should be validated against these more aggressive, tumor-maintaining cells.^{23, 27, 31} Importantly, TKI response has never been determined at the level of the tumor-maintaining CSCs. Thus, we investigated erlotinib response of EGFR mutation-negative lung cancer stem cells (LCSCs) and LCSC-based xenografts with the attempt to evaluate their sensitivity to the drug and correlate it with their molecular pattern in order to identify potential biomarkers predictive of erlotinib response in a WT-EGFR context at the CSC level.     

Contrast Agents for Quantitative MicroCT of Lung Tumors in Mice

The identification and quantitative evaluation of lung tumors in mouse models is challenging and an unmet need in preclinical arena. In this study, we developed a noninvasive contrast-enhanced microCT (μCT) method to longitudinally evaluate and quantitate lung tumors in mice. Commercially available μCT contrast agents were compared to determine the optimal agent for visualization of thoracic blood vessels and lung tumors in naïve mice and in non-small-cell lung cancer models. Compared with the saline control, iopamidol and iodinated lipid agents provided only marginal increases in contrast resolution. The inorganic nanoparticulate agent provided the best contrast and visualization of thoracic vascular structures; the density contrast was highest at 15 min after injection and was stable for more than 4 h. Differential contrast of the tumors, vascular structures, and thoracic air space by the nanoparticulate agent enabled identification of tumor margins and accurate quantification. μCT data correlated closely with traditional histologic measurements (Pearson correlation coefficient, 0.995). Treatment of ELM4–ALK mice with crizotinib yielded 65% reduction in tumor size and thus demonstrated the utility of quantitative μCT in longitudinal preclinical trials. Overall and among the 3 agents we tested, the inorganic nanoparticulate product was the best commercially available contrast agent for visualization of thoracic blood vessels and lung tumors. Contrast-enhanced μCT imaging is an excellent noninvasive method for longitudinal evaluation during preclinical lung tumor studies.Abbreviations: μCT, microCT, HU, Hounsfield units, RECIST, response evaluation criteria in solid tumorsLung cancer is the leading cause of cancer death worldwide, and non-small–cell lung cancer is the most common form of lung cancer that is diagnosed.¹⁹ Various animal models have been used to mimic these cancers, understand their biology, and evaluate potential therapeutics.^14,20,27 Traditionally, the method to study the disease in vivo has been to inject human tumor derived cell lines subcutaneously in immunodeficient mice (xenografts) or to orthotopically implant tumors in tissues of interest. Xenografts, despite being of human origin, are not ideal models because tumor cell–stroma interactions cannot be reconstituted in the system. Moreover, the tumor microenvironment has been shown to be essential in predicting cancer cell survival, progression, metastasis, and response to therapy.^14,20,27 To overcome these issues, several investigators have propagated tumors orthotopically by either direct injection of tumor cells into the organ of choice, intravenous injection of tumor cell lines, or implantation of patient-derived tumor biopsy samples into immunodeficient mice. These models simulate the tumor microenvironment and bear a closer resemblance to clinical cancer than do xenografts.^11,27 In the past decade, tremendous progress has been made in development of genetically engineered mouse models that reconstitute facets of human disease in the organ of choice. In these models, the tumors are developed in immunocompetent hosts with intact tumor–stroma interactions, and the tumors can be controlled temporally and spatially.^7,14,25,26Despite the many advantages of genetically engineered and orthotopic models, their use has been limited, primarily due to the heterogeneity and technical difficulties associated with monitoring disease progression.¹⁷ Conventional optical imaging is not widely used with genetically engineered and orthotopic models, because these modalities provide low spatial resolution, limited tissue penetration,^1,17 and rarely accomdate reporter genes like luciferase or fluorescent proteins.³⁰ Nuclear imaging (for example, positron-emission tomography and MRI) has been used in evaluating lung tumor models,^7,13,33 but these modalities are not readily available in all facilities, require specialized laboratories (for example, radionucleotide synthesis), and do not achieve accurate quantification of nodular tumors.³³ X-ray CT has been used in human clinical practice to assess lung tumor nodules to predict likelihood of malignancy and to monitor the response of tumors to treatment.^18,23 Response Evaluation Criteria in Solid Tumors (RECIST) scores based on CT imaging data are used routinely in clinical trials and practice.³² Similarly, high-resolution microCT (μCT) scanners have been used successfully to image lung tumors^{6,15,17,24,25,34} in small animals such as rodents. The investigators in the aforementioned studies took advantage of the natural air–tissue contrast within the thorax to identify tumors. However, this method was unable to distinguish tumor and soft tissue from nearby vascular structures.^25,29 This limitation decreased the accuracy of tumor margin demarcation and tumor volumetric measurements.The goal of our study was to compare 3 commercially available μCT contrast agents (products containing iopamidol, iodinated lipid, and inorganic nanoparticulate) to evaluate and accurately quantify lung tumors in preclinical models. Our results showed that, among those we tested, the nanoparticulate product was the best contrast agent for tumor visualization and quantitation. In addition, we demonstrated the utility of contrast-enhanced μCT in a preclinical evaluation of the efficacy of crizotinib (PF-2341066), a small-molecule multikinase inhibitor,^9,10 in a genetically modified mouse model of lung cancer.     

New-Onset Diabetes Mellitus After Transplantation in a Cynomolgus Macaque (Macaca fasicularis)

A 5.5-y-old intact male cynomolgus macaque (Macaca fasicularis) presented with inappetence and weight loss 57 d after heterotopic heart and thymus transplantation while receiving an immunosuppressant regimen consisting of tacrolimus, mycophenolate mofetil, and methylprednisolone to prevent graft rejection. A serum chemistry panel, a glycated hemoglobin test, and urinalysis performed at presentation revealed elevated blood glucose and glycated hemoglobin (HbA1c) levels (727 mg/dL and 10.1%, respectively), glucosuria, and ketonuria. Diabetes mellitus was diagnosed, and insulin therapy was initiated immediately. The macaque was weaned off the immunosuppressive therapy as his clinical condition improved and stabilized. Approximately 74 d after discontinuation of the immunosuppressants, the blood glucose normalized, and the insulin therapy was stopped. The animal''s blood glucose and HbA1c values have remained within normal limits since this time. We suspect that our macaque experienced new-onset diabetes mellitus after transplantation, a condition that is commonly observed in human transplant patients but not well described in NHP. To our knowledge, this report represents the first documented case of new-onset diabetes mellitus after transplantation in a cynomolgus macaque.Abbreviations: NODAT, new-onset diabetes mellitus after transplantationNew-onset diabetes mellitus after transplantation (NODAT, formerly known as posttransplantation diabetes mellitus) is an important consequence of solid-organ transplantation in humans.^{7-10,15,17,19,21,25-28,31,33,34,37,38,42} A variety of risk factors have been identified including increased age, sex (male prevalence), elevated pretransplant fasting plasma glucose levels, and immunosuppressive therapy.^{7-10,15,17,19,21,25-28,31,33,34,37,38,42} The relationship between calcineurin inhibitors, such as tacrolimus and cyclosporin, and the development of NODAT is widely recognized in human medicine.^{7-10,15,17,19,21,25-28,31,33,34,37,38,42} Cynomolgus macaques (Macaca fasicularis) are a commonly used NHP model in organ transplantation research. Cases of natural and induced diabetes of cynomolgus monkeys have been described in the literature;^14,43,45 however, NODAT in a macaque model of solid-organ transplantation has not been reported previously to our knowledge.     

Spontaneous Osteoblastic Osteosarcoma in a Mongolian Gerbil (Meriones unguiculatus)

Spontaneous neoplasms in Mongolian gerbils have an incidence of 20% to 26.8%, but osteosarcomas occur at a much lower rate. Here we report a 1-y-old Mongolian gerbil with a spontaneous osteosarcoma at the level of the proximal tibia, with metastases to the pectoral muscles and lungs. Grossly, the tibial mass obliterated the tibia and adjacent muscles, and an axillary mass with a bloody, cavitary center expanded the pectoral muscles. Microscopically, the tibial mass was an infiltrative, osteoblastic mesenchymal neoplasm, and the axillary mass was an anaplastic mesenchymal neoplasm with hemorrhage. The lung contained multiple metastatic foci. Immunohistochemistry for osteonectin was strongly positive in the tibial, axillary, and pulmonary metastases. Although osteosarcoma is the most common primary malignant bone neoplasm that occurs spontaneously in all laboratory and domestic animal species and humans, it arises less frequently than does other neoplasms. The current case of spontaneous osteoblastic osteosarcoma of the proximal tibia and metastases to the pectoral muscles and lung in a Mongolian gerbil is similar in presentation, histology, and predilection site of both osteoblastic and telangiectatic osteosarcomas in humans. In addition, this case is an unusual manifestation of osteosarcoma in the appendicular skeleton of a Mongolian gerbil.Mongolian gerbils are used frequently in biologic research,^{1,2,4,9,10,12-14} particularly in oncogenic studies and filariasis research studying Brugia malayi.² There have been several reports^{1,6,10,11,13-15} of spontaneous neoplasms, particularly in gerbils 2 y of age and older, typically occurring with the highest incidences in the skin, reproductive tract, and adrenal glands; however, neoplasms have also been reported in the thyroid, thymus, liver, kidney, pancreas, and bone.^{1,6,10,11,13-15} The incidence of spontaneous neoplasms occurring in the subfamily Gerbillinae ranges from 20% to 26.8%,^{1,6,10,11,13-15} depending on the study, age, and sex of the animals.With a lower incidence than those reported for other neoplasms, osteosarcomas in gerbils have been described in the ramus of the mandible and as an extraskeletal mass throughout the peritoneum.^10,11 The usual age of onset for osteosarcomas in Mongolian gerbils is approximately 3 y (36 to 39 mo); however, no tumor type has been reported at less than 2 y of age in this species.^10,11 Here we report a spontaneous osteosarcoma that occurred at the level of the proximal tibia, with metastases to the pectoral muscles and lung, in a 1-y-old Mongolian gerbil.     

Daytime Blue Light Enhances the Nighttime Circadian Melatonin Inhibition of Human Prostate Cancer Growth

Light controls pineal melatonin production and temporally coordinates circadian rhythms of metabolism and physiology in normal and neoplastic tissues. We previously showed that peak circulating nocturnal melatonin levels were 7-fold higher after daytime spectral transmittance of white light through blue-tinted (compared with clear) rodent cages. Here, we tested the hypothesis that daytime blue-light amplification of nocturnal melatonin enhances the inhibition of metabolism, signaling activity, and growth of prostate cancer xenografts. Compared with male nude rats housed in clear cages under a 12:12-h light:dark cycle, rats in blue-tinted cages (with increased transmittance of 462–484 nm and decreased red light greater than 640 nm) evinced over 6-fold higher peak plasma melatonin levels at middark phase (time, 2400), whereas midlight-phase levels (1200) were low (less than 3 pg/mL) in both groups. Circadian rhythms of arterial plasma levels of linoleic acid, glucose, lactic acid, pO₂, pCO₂, insulin, leptin, and corticosterone were disrupted in rats in blue cages as compared with the corresponding entrained rhythms in clear-caged rats. After implantation with tissue-isolated PC3 human prostate cancer xenografts, tumor latency-to-onset of growth and growth rates were markedly delayed, and tumor cAMP levels, uptake–metabolism of linoleic acid, aerobic glycolysis (Warburg effect), and growth signaling activities were reduced in rats in blue compared with clear cages. These data show that the amplification of nighttime melatonin levels by exposing nude rats to blue light during the daytime significantly reduces human prostate cancer metabolic, signaling, and proliferative activities.Abbreviations: A-V, arterial–venous difference, ipRGC, intrinsically photosensitive retinal ganglion cell, LA, linoleic acid, 13-HODE, 13-hydroxyoctadecadienoic acid, TFA, total fatty acidsLight profoundly influences circadian, neuroendocrine, and neurobehavioral regulation in all mammals and is essential to life on our planet.^{2,15,28, 40} The light–dark cycle entrains the master biologic clock, located in the suprachiasmatic nucleus of the brain, in an intensity-, duration-, and wavelength-dependent manner.^8-13 Photobiologic responses, including circadian rhythms of metabolism and physiology, are mediated by organic molecules called ‘chromophores,’ which are contained within a small subset of retinal cells, called the intrinsically sensitive retinal ganglion cells (ipRGC).^{16,29,31,36,41,49,53,59} In humans and rodents light quanta are detected by the chromophore melanopsin, which detects light quanta in principally the short-wavelength, blue-appearing portion of the spectrum (446 to 477 nm), and transmits its photic information via the retinohypothalamic tract to the ‘molecular clock’ of the suprachiasmatic nucleus. This region of the brain regulates the daily pineal gland production of the circadian neurohormone melatonin (N-acetyl-5-methoxytryptamine), which results in high levels produced at night and low levels during daytime.^38,54 The daily, rhythmic melatonin signal provides temporal coordination of normal behavioral and physiologic functions including chronobiologic rhythms of locomotor activity,² sleep-wake cycle,^2,14 dietary and water intake,^2,51 hormone secretion and metabolism.^5,44,47,61 Alterations in light intensity, duration, and spectral quality at a given time of day,^{8-13,17,19-22,24,61} such as occurs in night-shift workers exposed to light at night,^26,34,46,57 acutely suppresses endogenous melatonin levels in most mammalian species^{9,11,44,45,54,55} and may lead to various disease states, including metabolic syndrome^5,61 and carcinogenesis.^4-7,17,18Recent studies from our laboratory^{5,20,23-25,60,61} have demonstrated that relatively small changes in the spectral transmittance (color) of light passing through translucent amber (>590 nm), blue (>480 nm), and red-tinted (>640 nm) polycarbonate laboratory rodent cages, compared with standard polycarbonate clear cages (390 to 700 nm), during the light phase markedly influenced the normal nighttime melatonin signal and disrupted temporal coordination of metabolism and physiology.^19,24,61 Most notable was our discovery that, in both male and female pigmented nude rats maintained in blue-tinted rodent cages, nighttime melatonin levels were as much as 7 times higher than normal nighttime peak levels in animals maintained in all other cage types.¹⁹ An earlier study in human subjects diagnosed with midwinter insomnia coupled with low nighttime melatonin levels demonstrated that daily exposure to intense morning bright polychromatic light therapy for up to one week resulted in a restoration of nocturnal melatonin levels to those of control subjects.³⁵ In another study, exposure to blue-tinted (470 nm) LED light (100 lx) for approximately 20 min in the morning after 2 sleep-restricted (6 h) nights led to earlier onset of the melatonin surge at nighttime.³⁰In the United States alone this year, approximately 240,000 men will be diagnosed with prostate cancer, and nearly 30,000 will die from this disease (National Cancer Institute; www.cancer.gov/). Epidemiologic studies have shown that night shift work, which involves circadian disruption, including nocturnal melatonin suppression, markedly increases prostate cancer risk in men.^{26,34,46,57,58} Both in vitro and in vivo studies have demonstrated that melatonin inhibits human prostate cancer growth, including that of androgen-receptor–negative, castration-resistant PC3 human prostate cancer cells.^20,29,42,56 Cancer cells depend primarily on aerobic glycolysis (Warburg effect) over oxidative phosphorylation to meet their bioenergetic needs supporting biomass formation.⁵ The Warburg effect is characterized by increased cellular uptake of glucose and production of lactate despite an abundance of oxygen. Investigations have shown that signal transduction pathways that include AKT, MEK, NFκB, GS3Kβ, and PDK1 drive the Warburg effect.^5,61 In addition, cancer cells rely on increased uptake of the ω6 fatty acid linoleic acid (LA), which is prevalent in the western diet.^4-6 In most cancers, LA uptake occurs through a cAMP-dependent transport mechanism, and LA is metabolized to the mitogenic agent 13-hydroxyoctadecadienoic acid (13-HODE). In most tumors, 13-HODE plays an important role in enhancing downstream phosphorylation of ERK 1/2, AKT, and activation of the Warburg effect, thereby leading to increased cell proliferation and tumor growth.^4-6 Melatonin, the principal neurohormone of the pineal gland and whose production is regulated by the suprachiasmatic nucleus,^4,5 modulates processes of tumor initiation, progression, and growth in vivo.⁵ The circadian nocturnal melatonin signal not only inhibits LA uptake and metabolism, the Warburg effect in human cancer xenografts, and ultimately tumor growth, but it actually drives circadian rhythms in tumor metabolism, signal transduction activity, and cell proliferation. These effects are extinguished when melatonin production is suppressed by light exposure at night.⁵In the present investigation, we examined the hypothesis that the spectral transmittance (color) of short-wavelength (480 nm) bright light passing through blue-tinted standard laboratory rodent cages during the light phase not only amplifies the normal circadian nocturnal melatonin signal but also enhances the inhibition of the metabolism, signaling activity, and growth progression of human PC3 androgen-receptor–negative human prostate cancer xenografts in male nude rats.     

Caspase-3 feedback loop enhances Bid-induced AIF/endoG and Bak activation in Bax and p53-independent manner

Chemoresistance in cancer has previously been attributed to gene mutations or deficiencies. Bax or p53 deficiency can lead to resistance to cancer drugs. We aimed to find an agent to overcome chemoresistance induced by Bax or p53 deficiency. Here, we used immunoblot, flow-cytometry analysis, gene interference, etc. to show that genistein, a major component of isoflavone that is known to have anti-tumor activities in a variety of models, induces Bax/p53-independent cell death in HCT116 Bax knockout (KO), HCT116 p53 KO, DU145 Bax KO, or DU145 p53 KO cells that express wild-type (WT) Bak. Bak knockdown (KD) only partially attenuated genistein-induced apoptosis. Further results indicated that the release of AIF and endoG also contributes to genistein-induced cell death, which is independent of Bak activation. Conversely, AIF and endoG knockdown had little effect on Bak activation. Knockdown of either AIF or endoG alone could not efficiently inhibit apoptosis in cells treated with genistein, whereas an AIF, endoG, and Bak triple knockdown almost completely attenuated apoptosis. Next, we found that the Akt-Bid pathway mediates Bak-induced caspase-dependent and AIF- and endoG-induced caspase-independent cell death. Moreover, downstream caspase-3 could enhance the release of AIF and endoG as well as Bak activation via a positive feedback loop. Taken together, our data elaborate the detailed mechanisms of genistein in Bax/p53-independent apoptosis and indicate that caspase-3-enhanced Bid activation initiates the cell death pathway. Our results also suggest that genistein may be an effective agent for overcoming chemoresistance in cancers with dysfunctional Bax and p53.Mammalian cell death proceeds through a highly regulated program called apoptosis that is highly dependent on the mitochondria.¹ Mitochondrial outer membrane (MOM) multiple apoptotic stresses permeabilize the MOM, resulting in the release of apoptogenic factors including cytochrome c, Smac, AIF, and endoG.^{2, 3, 4} Released cytochrome c activates Apaf-1, which assists in caspase activation. Then, activated caspases cleave cellular proteins and contribute to the morphological and biochemical changes associated with apoptosis. Bcl-2 family proteins control a crucial apoptosis checkpoint in the mitochondria.^{2, 5, 6, 7} Multidomain proapoptotic Bax and Bak are essential effectors responsible for the permeabilization of the MOM, whereas anti-apoptotic Bcl-2, Bcl-xL, and Mcl-1 preserve mitochondrial integrity and prevent cytochrome c efflux triggered by apoptotic stimuli. The third Bcl-2 subfamily of proteins, BH3-only molecules (BH3s), promotes apoptosis by either activating Bax/Bak or inactivating Bcl-2/Bcl-xL/Mcl-1.^{8, 9, 10, 11, 12} Upon apoptosis, the ‘activator'' BH3s, including truncated Bid (tBid), Bim, and Puma, activate Bax and Bak to mediate cytochrome c efflux, leading to caspase activation.^{8, 11, 12} Conversely, antiapoptotic Bcl-2, Bcl-xL, and Mcl-1 sequester activator BH3s into inert complexes, which prevents Bax/Bak activation.^{8, 9} Although it has been proposed that Bax and Bak activation occurs by default as long as all of the anti-apoptotic Bcl-2 proteins are neutralized by BH3s,¹³ liposome studies clearly recapitulate the direct activation model in which tBid or BH3 domain peptides derived from Bid or Bim induce Bax or Bak oligomerization and membrane permeabilization.^{12, 14, 15}Numerous studies have demonstrated a critical role for Bax in determining tumor cell sensitivity to drug induction and in tumor development. Bax has been reported to be mutated in colon^{16, 17} and prostate cancers,^{18, 19} contributing to tumor cell survival and promoting clonal expansion. Bax has been shown to restrain tumorigenesis²⁰ and is necessary for tBid-induced cancer cell apoptosis.²¹ Loss of Bax has been reported to promote tumor development in animal models.²² Bax knockout (KO) renders HCT116 cells resistant to a series of apoptosis inducers.^{23, 24, 25} p53 has been reported to be a tumor suppressor,²⁶ and its mutant can cause chemoresistance in cancer cells.^{27, 28, 29} Moreover, p53 is often inactivated in solid tumors via deletions or point mutations.^{30, 31} Thus, it is necessary to find an efficient approach or agent to overcome chemoresistance caused by Bax and/or p53 mutants.Few studies have focused on the role of Bak in tumor cell apoptosis and cancer development. Bak mutations have only been shown in gastric and colon cancer cells.³² Some studies have revealed that Bak is a determinant of cancer cell apoptosis.^{33, 34} Some studies have even demonstrated that Bak renders Bax KO cells sensitive to drug induction.^{33, 35} In this study, we are the first group to show that tBid induces Bak activation and the release of AIF and endoG in colon cancer cells, which causes cellular apoptosis independent of Bax/p53. We also found that caspase-3 is activated in apoptosis. Interestingly, downstream caspase-3 can strengthen Bak activation and the release of AIF and endoG during apoptosis via a feedback loop. Furthermore, we reveal that Akt upregulates apoptosis progression. These results will help us to better understand the function of mitochondrial apoptotic protein members in apoptosis and cancer therapies. Furthermore, our experiments may provide a theoretical basis for overcoming chemoresistance in cancer cells.     

Effects of 4-Vinylcyclohexene Diepoxide on Peripubertal and Adult Sprague�CDawley Rats: Ovarian, Clinical, and Pathologic Outcomes

Young rats treated daily with intraperitoneal 4-vinylcyclohexene diepoxide (VCD) undergo selective destruction of primordial follicles, resulting in gradual ovarian failure resembling the menopausal transition in women. To determine whether VCD has similar effects on ovaries of older rats, adult and peripubertal Sprague–Dawley rats were injected intraperitoneally daily for 30 d with vehicle or VCD at 40 or 80 mg/kg. Body weight, food intake, complete blood counts, and markers of liver injury and renal function were measured during VCD treatment. Complete gross necropsy and microscopic observations were performed on day 31, and ovarian follicles were counted. At 80 mg/kg, VCD destroyed primordial and primary follicles to a similar extent in both adult and peripubertal animals, although adult rats likely started with fewer follicles and therefore approached follicle depletion. Treatment with VCD did not affect body weight, but food intake was reduced in both adult and peripubertal rats treated with 80 mg/kg VCD. Adult rats treated with 80 mg/kg VCD had neutrophilia and increased BUN and creatinine; in addition, 4 of these rats were euthanized on days 25 or 26 due to peritonitis. VCD treatment did not increase alanine aminotransferase levels, a marker of liver injury, although the 80-mg/kg dose increased liver weights. In conclusion, VCD effectively destroys small preantral follicles in adult Sprague–Dawley rats, making them a suitable model of the menopausal transition of women. However, because adult rats were more sensitive to the irritant properties of VCD, the use of a lower dose should be considered.Abbreviations: VCD, 4-vinylcyclohexene diepoxideStudies attempting to model the human menopause have relied heavily on using animals from which the ovaries have been removed surgically (ovariectomy). This approach has important limitations because women who enter natural menopause still have ovaries, which continue to produce hormones. Therefore, studies using ovariectomized animals cannot model the hormonal changes associated with the menopausal transition and postmenopausal period. However, rodent models of the menopausal transition and menopause that more closely mimic those of women have recently been developed.^32,33,36 Mice or rats treated with daily intraperitoneal injections of the chemical 4-vinylcyclohexene diepoxide (VCD) undergo selective destruction of primordial and primary follicles.²⁵ This treatment results in a gradual onset of ovarian failure because remaining larger follicles continue to develop and then ovulate or undergo atresia until they are depleted.³⁶ These studies also demonstrate that the length of time to ovarian failure is dependent on VCD dose and duration of treatment.^33,37 Moreover, in VCD-treated mice, the resulting follicle-depleted, stroma-intact ovary retains the ability to produce androgens.³⁶ Therefore, taken together, these characteristics indicate that VCD-treated animals could be used to model the menopausal transition of women and enable research on diseases affecting women postmenopausally.The ability of VCD to destroy preantral follicles in rats by repeated dosing has been well documented.^16,23,24,37 However, to our knowledge, all of the VCD studies using rats that have been published to date have used peripubertal or young (28 to 58 d) Fisher 344 rats. Although younger animals have been useful in separating the effect of age from the effect of hormonal changes associated with VCD-induced ovarian failure,^22,27,32,37 the use of older rodents may provide a more appropriate model for studying the combined effects of aging and hormonal aspects of menopause (for example, osteoporosis, cognitive decline, ovarian cancer).Both young and adult Sprague–Dawley rats have been used extensively to model menopausal effects on osteoporosis,^3,4,13,38,49 brain and cognitive functioning,^{2,14,15,29,34} lipids and cardiovascular health,^30,35,53 bladder health and incontinence,^6,21,31 and breast cancer.^8,18,43,44 These studies used ovariectomized Sprague–Dawley rats ranging in age from 42 to 210 d. The use of this chemically induced model of menopause would be enhanced by determining whether VCD affects Sprague–Dawley rats differently and whether VCD has deleterious effects on nonovarian tissues. Furthermore, although more than a dozen publications have reported that repeated VCD dosing does not adversely affect young rodents,^{19,32,33,36,56} similar data have not been reported for adult Sprague–Dawley rats. The purpose of this study was to determine whether VCD affects the ovaries of peripubertal (28 d) and adult Sprague–Dawley rats differently.