Comparative Characterization of Different Molecular Formats of Bispecific Antibodies Targeting EGFR and PD-L1.

We generated two IgG1-like bispecific antibodies (BsAbs) with different molecular formats, symmetrical DVD-Ig and asymmetrical knob-in-hole (KIH), targeting the same antigens, EGFR and PD-L1 (designated as anti-EGFR/PD-L1). We performed the physiochemical and biological characterization of these two formats of anti-EGFR/PD-L1 BsAbs and compared some key quality attributes and biological activities of these two formats of BsAbs. Physiochemical binding characterization data demonstrated that both formats bound EGFR and PD-L1. However, the binding affinity of the KIH format was weaker than the DVD-Ig format in Biacore binding assays. In contrast, both DVD-Ig and KIH BsAbs had similar ELISA and cell surface binding activities, comparable to mAbs. Triple-negative breast cancer (TNBC) cells and a xenograft model were used to test the potency of BsAbs and other biological activities. Results showed that anti-EGFR/PD-L1 BsAbs exhibited in vitro and in vivo antitumor proliferation activity, but there was a difference in the potencies of the respective BsAb formats (DVD-Ig and KIH) when different cells or assays were used. This study provides evidence that the potency of the BsAbs targeting the same antigens can be affected by the respective molecular features, and selection of appropriate cell lines and assays is critically important for the assay development and potency testing of BsAbs.

Mechanisms contributing to ado-trastuzumab emtansine-induced toxicities: a gateway to better understanding of ADC-associated toxicities.

In order to improve the safety of novel therapeutic drugs, better understanding of the mechanisms of action is important. Ado-trastuzumab emtansine (also known as T-DM1) is an antibody-drug conjugate (ADC) approved for the treatment of HER2-positive breast cancer. While the treatment with T-DM1 results in significant efficacy in the selected patient population, nonetheless, there are concerns with side effects such as thrombocytopenia and hepatotoxicity. While current understanding of the mechanism of T-DM1-mediated side effects is still incomplete, there have been several reports of HER2-dependent and/or -independent mechanisms that could be associated with the T-DM1-induced adverse events. This review highlights the importance of HER2-independent mechanism of T-DM1 to induce hepatotoxicity, which offers a new insight into a role for CKAP5 in the overall maytansinoid-based ADC (DM1 and DM4)-mediated cytotoxicity. This discovery provides a molecular basis for T-DM1-induced off-target toxicity and opens a new avenue for developing the next generation of ADCs.

Histone deacetylase inhibitors: overview and perspectives.

Histone deacetylase inhibitors (HDACi) comprise structurally diverse compounds that are a group of targeted anticancer agents. The first of these new HDACi, vorinostat (suberoylanilide hydroxamic acid), has received Food and Drug Administration approval for treating patients with cutaneous T-cell lymphoma. This review focuses on the activities of the 11 zinc-containing HDACs, their histone and nonhistone protein substrates, and the different pathways by which HDACi induce transformed cell death. A hypothesis is presented to explain the relative resistance of normal cells to HDACi-induced cell death.

Histone deacetylase inhibitors selectively suppress expression of HDAC7.

There are 18 histone deacetylases (HDAC) generally divided into four classes based on homology to yeast HDACs. HDACs have many protein substrates in addition to histones that are involved in regulation of gene expression, cell proliferation, and cell death. Inhibition of HDACs can cause accumulation of acetylated forms of these proteins, thus altering their function. HDAC inhibitors (HDACi), such as the hydroxamic acid-based vorinostat (suberoylanilide hydroxamic acid), inhibit the zinc-containing classes I, II, and IV, but not the NAD(+)-dependent class III, enzymes. HDACis are a group of novel anticancer agents. Vorinostat is the first HDACi approved for clinical use in the treatment of the cancer cutaneous T-cell lymphoma. Factors affecting expression of HDACs are not well understood. This study focuses on the effect of the HDACi vorinostat on the expression of class I and class II HDACs. We found that vorinostat selectively down-regulates HDAC7 with little or no effect on the expression of other class I or class II HDACs. Fourteen cell lines were examined, including normal, immortalized, genetically transformed, and human cancer-derived cell lines. Down-regulation of HDAC7 by vorinostat is more pronounced in transformed cells sensitive to inhibitor-induced cell death than in normal cells or cancer cells resistant to induced cell death. Modulation of HDAC7 levels by small interfering RNA-mediated knockdown or by HDAC7 overexpression is associated with growth arrest but without detectable changes in acetylation of histones or p21 gene expression. Selective down-regulation of HDAC7 protein may serve as a marker of response of tumors to HDACi.

Trastuzumab-mediated cardiotoxicity: current understanding, challenges, and frontiers.

Trastuzumab, an epidermal growth factor receptor 2 (HER2) targeting humanized monoclonal antibody, has been approved for the treatment HER2-positive breast cancer and HER2-positve metastatic gastric cancer. However, cardiotoxicity associated with its clinical application poses challenges for clinicians and patients, mechanisms of which are still evolving. This review will summarize the current mechanistic understanding of trastuzumab-mediated cardiotoxicity, discuss the novel role of DNA topoisomerase IIB as a shared target for enhanced cardiotoxicity induced by trastuzumab and anthracyclines-based combination regimens, and speculate the potential impact of trastuzumab intervention in immune checkpoint inhibitors-based therapies.

Cardiotoxicity of ErbB2-targeted therapies and its impact on drug development, a spotlight on trastuzumab.

INTRODUCTION: Trastuzumab, a therapeutic monoclonal antibody directed against ErbB2, is often noted as a successful example of targeted therapy. Trastuzumab improved outcomes for many patients with ErbB2-positive breast and gastric cancers, however, cardiac side effects [e.g., left ventricular dysfunction and congestive heart failure (CHF)] were reported in the early phase clinical studies. This finding, subsequently corroborated by multiple clinical studies, raised concerns that the observed cardiotoxicity induced by trastuzumab might adversely impact the clinical development of other therapeutics targeting ErbB family members. Areas covered: In this review we summarize both basic research and clinical findings regarding trastuzumab-induced cardiotoxicity and assess if there has been an impact of trastuzumab-induced cardiotoxicity on the development of other agents targeting ErbB family members. Expert opinion: There are a number of scientific gaps that are critically important to address for the continued success of HER2-targeted agents. These include: 1) elucidating the molecular mechanisms contributing to cardiotoxicity; 2) developing relevant preclinical testing systems for predicting cardiotoxicity; 3) developing clinical strategies to identify patients at risk of cardiotoxicity; and 4) enhancing management of clinical symptoms of cardiotoxicity.

VPS34 regulates TSC1/TSC2 heterodimer to mediate RheB and mTORC1/S6K1 activation and cellular transformation.

VPS34 is reported to activate S6K1 and is implicated in regulating cell growth, the mechanisms of which remain elusive. Here, we describe novel mechanisms by which VPS34 upregulates mTOR/S6K1 activity via downregulating TSC2 protein and activating RheB activity. Specifically, upregulation of VPS34 lipid kinase increases local production of ptdins(3)p in the plasma membrane, which recruits PIKFYVE, a FYVE domain containing protein, to ptdins(3)p enriched regions of the plasma membrane, where VPS34 forms a protein complex with PIKFYVE and TSC1. This in turn disengages TSC2 from the TSC1/TSC2 heterodimer, leading to TSC2 ubiquitination and degradation. Downregulation of TSC2 promotes the activation of RheB and mTOR/S6K1. When VPS34 lipid kinase activity is increased by introduction of an H868R mutation, ptdins(3)p production at the plasma membrane is dramatically increased, which recruits more PIKFYVE and TSC1 molecules to the plasma membrane. This results in the enhanced TSC2 ubiquitination and degradation, and subsequent activation of RheB and mTORC1/S6K1, leading to oncogenic transformation. The role played by VPS34 in regulating mTOR/S6K1 activity and cellular transformation is underscored by the fact that the VPS34 kinase dead mutant blocks VPS34-induced recruitment of PIKFYVE and TSC1 to the plasma membrane. This study provides mechanistic insight into the cellular function of VPS34 in regulating oncogenic transformation and important indications for identifying VPS34 specific mutations in human cancers.

Ado-Trastuzumab Emtansine Targets Hepatocytes Via Human Epidermal Growth Factor Receptor 2 to Induce Hepatotoxicity.

Ado-trastuzumab emtansine (T-DM1) is an antibody-drug conjugate (ADC) approved for the treatment of HER2-positive metastatic breast cancer. It consists of trastuzumab, a humanized mAb directed against HER2, and a microtubule inhibitor, DM1, conjugated to trastuzumab via a thioether linker. Hepatotoxicity is one of the serious adverse events associated with T-DM1 therapy. Mechanisms underlying T-DM1-induced hepatotoxicity remain elusive. Here, we use hepatocytes and mouse models to investigate the mechanisms of T-DM1-induced hepatotoxicity. We show that T-DM1 is internalized upon binding to cell surface HER2 and is colocalized with LAMP1, resulting in DM1-associated cytotoxicity, including disorganized microtubules, nuclear fragmentation/multiple nuclei, and cell growth inhibition. We further demonstrate that T-DM1 treatment significantly increases the serum levels of aspartate aminotransferase, alanine aminotransferase, and lactate dehydrogenase in mice and induces inflammation and necrosis in liver tissues, and that T-DM1-induced hepatotoxicity is dose dependent. Moreover, the gene expression of TNFα in liver tissues is significantly increased in mice treated with T-DM1 as compared with those treated with trastuzumab or vehicle. We propose that T-DM1-induced upregulation of TNFα enhances the liver injury that may be initially caused by DM1-mediated intracellular damage. Our proposal is underscored by the fact that T-DM1 induces the outer mitochondrial membrane rupture, a typical morphologic change in the mitochondrial-dependent apoptosis, and mitochondrial membrane potential dysfunction. Our work provides mechanistic insights into T-DM1-induced hepatotoxicity, which may yield novel strategies to manage liver injury induced by T-DM1 or other ADCs.

Monitoring Trastuzumab Resistance and Cardiotoxicity: A Tale of Personalized Medicine.

While approval of trastuzumab, a recombinant monoclonal antibody directed against HER2, along with a diagnostic kit to detect breast cancers which are positive for HER2 overexpression, has advanced a new era of stratified and personalized medicine, it also created several challenges to our scientific and clinical practice. These problems include trastuzumab resistance and trastuzumab-induced cardiotoxicity. In this review, we will summarize data from the literature regarding mechanisms of trastuzumab resistance and trastuzumab-induced cardiotoxicity and present some promising model systems that may advance our understanding of these mechanisms. Our discussion will include development of circulating tumor cells and circulating tumor DNA for monitoring tumor burden, of patient-derived xenograft models for preclinical testing of novel therapies, and of novel therapeutic strategies for trastuzumab-resistance and possible integration of these strategies in the design of co-clinical studies for testing in relevant patient subpopulations.

Retinoid-induced growth arrest of breast carcinoma cells involves co-activation of multiple growth-inhibitory genes.

Retinoids are used in leukemia therapy and chemoprevention of cancers. Treatment of MCF-7 breast carcinoma cells with low doses of retinoids induces gradual proliferation arrest with phenotypic markers of senescence. cDNA microarray hybridization and reverse transcription-polymerase chain reaction analysis showed that retinoid-induced growth arrest in MCF-7 cells in associated with strong induction of 13 genes. Four of these genes (IGF-binding protein 3, EPLIN, beta IG-H3 and FAT10) have antiproliferative activity; EPLIN and beta IG-H3 are also known to be selectively inhibited in transformed relative to normal cells. The functions of the induced genes may also account for other cellular effects of retinoids, including the proteasome-mediated protein degradation, increased cell adhesion, and retinoic acid synthesis. Only one of 13 strongly induced genes (ring finger protein TRIM31) contains a putative retinoid response element in its promoter; TRIM31 also shows the most rapid kinetics of induction by retinoids. In contrast, the antiproliferative genes contain no identifiable retinoid response elements in their promoters and show more gradual induction kinetics, suggesting that these genes are indirectly induced by retinoids. Elucidation of the mechanisms that mediate co-induction of growth-inhibitory genes in retinoid-treated cells may suggest an approach to reproducing the growth-inhibitory effect of retinoids in retinoid-insensitive human cancers.

Trastuzumab-induced recruitment of Csk-homologous kinase (CHK) to ErbB2 receptor is associated with ErbB2-Y1248 phosphorylation and ErbB2 degradation to mediate cell growth inhibition.

The inhibitory effect of trastuzumab, a humanized monoclonal antibody directed against the extracellular domain of ErbB2, is associated with its ability to induce ErbB2-Y1248 phosphorylation, and the status of phosphorylated ErbB2-Y1248 (ErbB2-pY1248) may correlate with the sensitivity of breast cancers to trastuzumab. The mechanisms of which remain unclear. Here, we show that binding of trastuzumab to ErbB2 activates ErbB2 kinase activity and enhances ErbB2-Y1248 phosphorylation in trastuzumab-sensitive breast cancer cells. This in turn increases the interaction between ErbB2 and non-receptor Csk-homologous kinase (CHK), leading to growth inhibition of breast cancer cells. Overexpression of CHK mimics trastuzumab treatment to mediate ErbB2-Y1248 phosphorylation, Akt downregulation, and growth inhibition of trastuzumab-sensitive breast cancer cells. CHK overexpression combined with trastuzumab exerts an additive effect on cell growth inhibition. We further demonstrate that positive ErbB2-pY1248 staining in ErbB2-positive breast cancer biopsies correlates with the increased trastuzumab response in trastuzumab neoadjuvant settings. Collectively, this study highlights an important role for ErbB2-pY1248 in mediating trastuzumab-induced growth inhibition and trastuzumab-induced interactions between CHK and ErbB2-pY1248 is identified as a novel mechanism of action that mediates the growth inhibition of breast cancer cells. The novel mechanistic insights into trastuzumab action revealed by this study may impact the design of next generation of therapeutic monoclonal antibodies targeting receptor tyrosine kinases, as well as open new avenues to identify novel targets for the treatment of ErbB2-positive cancers.

Insulin activation of vacuolar protein sorting 34 mediates localized phosphatidylinositol 3-phosphate production at lamellipodia and activation of mTOR/S6K1.

The class III phosphatidylinositol 3-kinase, VPS34, phosphorylates the D3 hydroxyl of inositol generating phosphatidylinositol 3-phosphate (ptdins(3)p). Initial studies suggested that ptdins(3)p solely functioned as a component of vesicular and endosomal membranes and that VPS34 did not function in signal transduction. However, VPS34 has recently been shown to be required for insulin-mediated activation of S6 kinase 1 (S6K1). Whether VPS34 activity is directly regulated by insulin is unclear. It is also not known whether VPS34 activity can be spatially restricted in response to extracellular stimuli. Data presented here demonstrate that in response to insulin, VPS34 is activated and translocated to lamellipodia where it produces ptdins(3)p. The localized production of ptdins(3)p is dependent on Src phosphorylation of VPS34. In cells expressing VPS34 with mutations at Y231 or Y310, which are Src-phosphorylation sites, insulin-stimulated VPS34 translocation to the plasma membrane and lamellipodia formation are blocked. mTOR also colocalizes with VPS34 and ptdins(3)p at lamellipodia following insulin-stimulation. In cells expressing the VPS34-Y231F mutant, which blocks lamellipodia formation, mTOR localization at the plasma membrane and insulin-mediated S6K1 activation are reduced. This suggests that mTOR localization at lamellipodia is important for full activation of S6K1 induced by insulin. These data demonstrate that insulin can spatially regulate VPS34 activity through Src-mediated tyrosine phosphorylation and that this membrane localized activity contributes to lamellipodia formation and activation of mTOR/S6K1signaling.

Trastuzumab alters the expression of genes essential for cardiac function and induces ultrastructural changes of cardiomyocytes in mice.

Treatment with trastuzumab, a humanized monoclonal antibody directed against the extracellular domain of Human Epidermal Growth Factor Receptor 2 (HER2), very successfully improves outcomes for women with HER2-positive breast cancer. However, trastuzumab treatment was recently linked to potentially irreversible serious cardiotoxicity, the mechanisms of which are largely elusive. This study reports that trastuzumab significantly alters the expression of myocardial genes essential for DNA repair, cardiac and mitochondrial functions, which is associated with impaired left ventricular performance in mice coupled with significant ultrastructural alterations in cardiomyocytes revealed by electron microscopy. Furthermore, trastuzumab treatment also promotes oxidative stress and apoptosis in myocardium of mice, and elevates serum levels of cardiac troponin-I (cTnI) and cardiac myosin light chain-1 (cMLC1). The elevated serum levels of cMLC1 in mice treated with trastuzumab highlights the potential that cMLC1 could be a useful biomarker for trastuzumab-induced cardiotoxicity.

Small GTPase Rho regulates R-cadherin through Dia1/profilin-1.

R-cadherin is a member of the classical cadherins. Though much is known about E-cadherin in adherens junction formation in epithelial cells, the role of R-cadherin in epithelial cells remains elusive. This study examines regulation of R-cadherin adherens junctions by the small GTPase Rho and its downstream effectors in MDA-MB-231 breast cancer cells, MDA-MB-231 cells stably expressing the N-terminus of c-Cbl, and MCF10A normal breast epithelial cells. We find that the small GTPase Rho regulates R-cadherin adherens junction formation via Dia1 (also known as p140mDia) and profilin-1-mediated signaling pathway. The role played by Rho in regulating R-cadherin is underscored by the fact that constitutively active RhoA(Q63L) induces R-cadherin junction formation in MDA-MB-231 cells. Importantly, R-cadherin adherens junction formation facilitates a mesenchymal to epithelial-like transition in MDA-MB-231 cells. Additionally, our data suggest an inverse relationship between EGFR signaling and R-cadherin adherens junction formation. Taken together, results from this study indicate that R-cadherin is a critical regulator of epithelial phenotype.

Trastuzumab regulates IGFBP-2 and IGFBP-3 to mediate growth inhibition: implications for the development of predictive biomarkers for trastuzumab resistance.

Activation of insulin-like growth factor-I receptor (IGF-IR) signaling is an important mechanism for trastuzumab resistance. IGF-binding proteins (IGFBP) modulate IGF-IR signaling and play important roles in the control of breast cancer progression. In this article, we report that trastuzumab treatment enhances the expression and secretion of IGFBP-3 in SKBR3 cells, a trastuzumab-sensitive breast cancer cell line, and that this upregulation of IGFBP-3 induced by trastuzumab correlates with trastuzumab-mediated growth inhibition. We describe a new role for IGFBP-3 in the regulation of IGF-I-mediated cross-talk between IGF-IR and ErbB2 signaling pathways. In particular, treatment of SKBR3 cells with recombinant IGFBP-3 blocks IGF-I-induced activation of IGF-IR and ErbB2, and stable expression of IGFBP-3 inhibits SKBR3 cell growth. We find an inverse relationship in the levels of secreted IGFBP-3 such that high levels of IGFBP-3 are associated with trastuzumab-sensitive breast cancer cells (SKBR3 and BT-474), whereas low levels of IGFBP-3 are found in trastuzumab-resistant cells (clone 3 and JIMT-1). In contrast to IGFBP-3, the secretion and expression of IGFBP-2 are upregulated in trastuzumab-resistant SKBR3 cells. Furthermore, we show that IGFBP-2 stimulates activation of ErbB2 and that trastuzumab reduces IGFBP-2-stimulated ErbB2 activation. Based on our data, we propose a novel mechanism of action whereby trastuzumab enhances the expression and secretion of IGFBP-3, which interferes with IGF-I-mediated mitogenic signaling via autocrine and paracrine mechanisms and reduces IGFBP-2-induced ErbB2 activation to mediate growth inhibition. Changes in secretion profiles of IGFBP-2 and IGFBP-3 in trastuzumab-sensitive and trastuzumab-resistant cells may promote the development of IGFBP-2 and IGFBP-3 as predictive biomarkers for trastuzumab resistance.

pp60c-Src phosphorylates and activates vacuolar protein sorting 34 to mediate cellular transformation.

Vacuolar protein sorting 34 (VPS34) contributes to the regulation of the mammalian target of rapamycin complex 1/S6 kinase 1 pathway downstream of nutrient signaling. However, intracellular mechanisms leading to VPS34 activation remain unclear. Here, we report that Src directly phosphorylates VPS34, and that this phosphorylation activates VPS34 lipid kinase activity, leading to Src-Y527F-mediated cellular transformation. Silencing endogenous VPS34 specifically inhibits Src-Y527F-induced colony formation in soft agar, but not Ras-G12V-induced colony formation. We have identified two novel hVPS34 mutations, which either eliminate lipid kinase activity (kinase-dead mutant) or reduce tyrosine phosphorylation by Src-Y527F. When kinase-dead mutant of hVPS34 is stably expressed in Src-Y527F-transformed cells, transformation activities are blocked, indicating that the lipid kinase activity of hVPS34 is essential for Src-mediated cellular transformation. Furthermore, stable expression of this hVPS34 kinase-dead mutant causes an increased number of binucleate and multinucleate cells, suggesting that the kinase activity of hVPS34 is also required for cytokinesis. Moreover, when the hVPS34 mutant that has reduced tyrosine phosphorylation by Src is stably expressed in Src-Y527F-transformed cells, Src-Y527F-stimulated colony formation is also reduced. Data presented here provide important evidence that VPS34 lipid kinase activity could be positively regulated by Src-mediated tyrosine phosphorylation in mammalian cells. This finding highlights a previously unappreciated relationship between VPS34, a class III phosphatidylinositol-3-kinase, and Src non-receptor tyrosine kinase. Additionally, we find that the levels of VPS34 expression and tyrosine phosphorylation are correlated with the tumorigenic activity of human breast cancer cells, indicating that Src to VPS34 signaling warrants further investigation as a pathway contributing to the development and progression of human cancers.

Rac1 contributes to trastuzumab resistance of breast cancer cells: Rac1 as a potential therapeutic target for the treatment of trastuzumab-resistant breast cancer.

Although treatment with trastuzumab improves outcomes for women with ErbB2-positive breast cancer, many patients who achieve an initial response to trastuzumab subsequently acquire resistance within 1 year. Rac1, a Ras-like small GTPase, has been implicated in the control of cell growth and morphology and is believed to be associated with breast cancer progression and metastasis. Here, we show that when parental SKBR3 cells become resistant to trastuzumab, Rac1 activity is increased, leading to altered cell morphology, which is accompanied by significant cytoskeleton disorganization. Furthermore, both trastuzumab-mediated down-regulation of ErbB2 and epidermal growth factor-induced down-regulation of epidermal growth factor receptor are impaired in the trastuzumab-resistant SKBR3 cells, indicating that the endocytic down-regulation of ErbB receptors is compromised in the resistant cells. This results in an aberrant accumulation of ErbB2 on the cell surface and enhanced ErbB2 and extracellular signal-regulated kinase activity in trastuzumab-resistant SKBR3 cells. Additionally, overexpression of constitutively active Rac1G12V in parental SKBR3 cells reduces sensitivity to trastuzumab. After reduction of Rac1 activity by NSC23766, a specific Rac1 inhibitor, trastuzumab-resistant SKBR3 cells display a cellular morphology similar to parental SKBR3 cells. Moreover, we show that NSC23766 restores trastuzumab-mediated endocytic down-regulation of ErbB2 and reduces extracellular signal-regulated kinase activity in resistant SKBR3 cells. Our findings highlight an important role for Rac1 in trastuzumab resistance of human breast cancer cells and identify the impaired trastuzumab-mediated endocytic down-regulation of ErbB2 as a novel mechanism of trastuzumab resistance. The significant effects of NSC23766 on trastuzumab-resistant SKBR3 cells warrant further study of NSC23766 as a potential treatment of trastuzumab-resistant breast cancers.

Agonist and antagonist of retinoic acid receptors cause similar changes in gene expression and induce senescence-like growth arrest in MCF-7 breast carcinoma cells.

Biological effects of retinoids are mediated via retinoic acid (RA) receptors (RAR) and retinoid X receptors (RXR). The best-characterized mechanism of retinoid action is stimulation of transcription from promoters containing RA response elements (RARE). Retinoids induce senescence-like growth arrest in MCF-7 breast carcinoma cells; this effect is associated with the induction of several growth-inhibitory genes. We have now found that these genes are induced by RAR-specific but not by RXR-specific ligands. Genome-scale microarray analysis of gene expression was used to compare the effects of two pan-RAR ligands, one of which is a strong agonist of RARE-dependent transcription, whereas the other induces such transcription only weakly and antagonizes the inducing effect of RAR agonists. Both RAR ligands, however, produced very similar effects on gene expression in MCF-7 cells, suggesting that RARE-dependent transcription is only a minor component of retinoid-induced changes in gene expression. The effects of RAR ligands on gene expression parallel changes associated with damage-induced senescence, and both ligands induced G(1) arrest and the senescent phenotype in MCF-7 cells. The RAR ligands up-regulated many tumor-suppressive genes and down-regulated multiple genes with oncogenic activities. Genes that are strongly induced by RAR ligands encode secreted bioactive proteins, including several tumor-suppressing factors. In agreement with these observations, retinoid-treated MCF-7 cells inhibited the growth of retinoid-insensitive MDA-MB-231 breast carcinoma cells in coculture. These results indicate that RARE-independent transcriptional effects of RAR ligands lead to senescence-like growth arrest and paracrine growth-inhibitory activity in MCF-7 breast carcinoma cells.

Intrinsic apoptotic and thioredoxin pathways in human prostate cancer cell response to histone deacetylase inhibitor.

There is a great need to develop better mechanism-based therapies for prostate cancer. In this investigation, we studied four human prostate cancer cell lines, LNCaP, DU145, LAPC4, and PC3, which differ in response to the histone deacetylase inhibitor, suberoylanilide hydroxamic acid (vorinostat), a new anticancer drug. Examining the role of intrinsic mitochondrial caspase-dependent apoptosis and caspase-independent, reactive oxygen species (ROS) facilitated cell death, has provided an understanding of mechanisms that may determine the varied response to the histone deacetylase inhibitor. We found striking differences among these cancer cells in constitutive expression and response to suberoylanilide hydroxamic acid in levels of antiapoptotic and proapoptotic proteins, mitochondria membrane integrity, activation of caspases, ROS accumulation, and expression of thioredoxin, the major scavenger of ROS. Identifying these differences can have predictive value in assessing therapeutic response and identifying targets to enhance therapeutic efficacy.

Histone sumoylation is a negative regulator in Saccharomyces cerevisiae and shows dynamic interplay with positive-acting histone modifications.

Covalent histone post-translational modifications such as acetylation, methylation, phosphorylation, and ubiquitylation play pivotal roles in regulating many cellular processes, including transcription, response to DNA damage, and epigenetic control. Although positive-acting post-translational modifications have been studied in Saccharomyces cerevisiae, histone modifications that are associated with transcriptional repression have not been shown to occur in this yeast. Here, we provide evidence that histone sumoylation negatively regulates transcription in S. cerevisiae. We show that all four core histones are sumoylated and identify specific sites of sumoylation in histones H2A, H2B, and H4. We demonstrate that histone sumoylation sites are involved directly in transcriptional repression. Further, while histone sumoylation occurs at all loci tested throughout the genome, slightly higher levels occur proximal to telomeres. We observe a dynamic interplay between histone sumoylation and either acetylation or ubiquitylation, where sumoylation serves as a potential block to these activating modifications. These results indicate that sumoylation is the first negative histone modification to be identified in S. cerevisiae and further suggest that sumoylation may serve as a general dynamic mark to oppose transcription.