Zachary C. Hartman

A SUMOylation-Dependent Transcriptional Subprogram Is Required for Myc-Driven Tumorigenesis

Taking the Myc Despite nearly 30 years of research into the mechanisms by which Myc oncogene dysregulation contributes to tumorigenesis, there are still no effective therapies that inhibit Myc activity. Kessler et al. (p. 348, published online 8 December; see the Perspective by Evan) searched for gene products that support Myc-driven tumorigenesis. One pharmacologically tractable target that emerged from the screen was the SUMO-activating enzyme complex SAE1/2, which catalyzes a posttranslational modification (SUMOylation) that alters protein behavior and function. SUMOylation was found to control the Myc transcriptional response, and its inhibition caused mitotic defects and apoptosis in Myc-dependent breast cancer cells. An RNA interference screen identifies a “druggable” enzyme whose inhibition halts tumor cell growth. Myc is an oncogenic transcription factor frequently dysregulated in human cancer. To identify pathways supporting the Myc oncogenic program, we used a genome-wide RNA interference screen to search for Myc–synthetic lethal genes and uncovered a role for the SUMO-activating enzyme (SAE1/2). Loss of SAE1/2 enzymatic activity drives synthetic lethality with Myc. Inactivation of SAE2 leads to mitotic catastrophe and cell death upon Myc hyperactivation. Mechanistically, SAE2 inhibition switches a transcriptional subprogram of Myc from activated to repressed. A subset of these SUMOylation-dependent Myc switchers (SMS genes) is required for mitotic spindle function and to support the Myc oncogenic program. SAE2 is required for growth of Myc-dependent tumors in mice, and gene expression analyses of Myc-high human breast cancers suggest that low SAE1 and SAE2 abundance in the tumors correlates with longer metastasis-free survival of the patients. Thus, inhibition of SUMOylation may merit investigation as a possible therapy for Myc-driven human cancers.

Growth of Triple-Negative Breast Cancer Cells Relies upon Coordinate Autocrine Expression of the Proinflammatory Cytokines IL-6 and IL-8

Triple-negative breast cancers (TNBC) are aggressive with no effective targeted therapies. A combined database analysis identified 32 inflammation-related genes differentially expressed in TNBCs and 10 proved critical for anchorage-independent growth. In TNBC cells, an LPA-LPAR2-EZH2 NF-κB signaling cascade was essential for expression of interleukin (IL)-6, IL-8, and CXCL1. Concurrent inhibition of IL-6 and IL-8 expression dramatically inhibited colony formation and cell survival in vitro and stanched tumor engraftment and growth in vivo. A Cox multivariable analysis of patient specimens revealed that IL-6 and IL-8 expression predicted patient survival times. Together these findings offer a rationale for dual inhibition of IL-6/IL-8 signaling as a therapeutic strategy to improve outcomes for patients with TNBCs.

Adenovirus Infection Triggers a Rapid, MyD88-Regulated Transcriptome Response Critical to Acute-Phase and Adaptive Immune Responses In Vivo

ABSTRACT Nearly 50 years ago, the discovery of interferon prompted the notion that host cells innately respond to viral invasion. Since that time, technological advances have allowed this response to be extensively characterized and dissected in vitro. However, these advances have only recently been applied to highly complex, in vivo biological systems. To this end, we exploited high-titer adenovirus (Ad) vectors to globally investigate the innate immune response to nonenveloped viral infection in vivo. Our results indicated a potent cellular transcriptome response shortly after infection, with global assessments revealing significant dysregulation in ∼15% of the measured transcripts derived from Ad vector-transduced tissue. Bioinformatics-based transcriptome analysis revealed a complex innate response to Ad infection, with induction of proinflammatory responses (and suppression of metabolism and mitochondrial genes) akin to those observed when mice are challenged with lipopolysaccharide. Despite this commonality, there were many unique aspects of the Ad-dependent transcriptome response, including the upregulation of several RNA regulatory mechanisms and apoptosis-related pathways, accompanied by the suppression of lysosomal and endocytic genes. Our results also implicated the Toll-like receptors (TLRs) in these responses, prompting specific investigations into this pathway. By using MyD88KO mice, our results confirmed that Ad-induced dysregulation of five functionally related gene clusters are significantly dependent on this TLR adaptor gene. MyD88 deficiency also resulted in significantly diminished, although not abolished, adaptive and acute-phase immune responses to Ad, confirming the transcriptome data, as well as specifically identifying MyD88 as a significant Ad immunity amplifier and regulator in vivo.

HER2 Overexpression Elicits a Proinflammatory IL-6 Autocrine Signaling Loop That Is Critical for Tumorigenesis

HER2 overexpression occurs in approximately 25% of breast cancers, where it correlates with poor prognosis. Likewise, systemic inflammation in breast cancer correlates with poor prognosis, although the process is not understood. In this study, we explored the relationship between HER2 and inflammation, comparing the effects of overexpressing wild-type or mutated inactive forms of HER2 in primary human breast cells. Wild-type HER2 elicited a profound transcriptional inflammatory profile, including marked elevation of interleukin-6 (IL-6) expression, which we established to be a critical determinant of HER2 oncogenesis. Mechanistic investigations revealed that IL-6 secretion induced by HER2 overexpression activated Stat3 and altered gene expression, enforcing an autocrine loop of IL-6/Stat3 expression. Both mouse and human in vivo models of HER2-amplified breast carcinoma relied critically on this HER2-IL-6-Stat3 signaling pathway. Our studies offer the first direct evidence linking HER2 to a systemic inflammatory mechanism that orchestrates HER2-mediated tumor growth. We suggest that the HER2-IL-6-STAT3 signaling axis we have defined in breast cancer could prompt new therapeutic or prevention strategies for treatment of HER2-amplified cancers.

The deacetylase HDAC4 controls myocyte enhancing factor-2-dependent structural gene expression in response to neural activity.

Histone deacetylase 4 (HDAC4) binds and inhibits activation of the critical muscle transcription factor myocyte enhancer factor‐2 (MEF2). However, the physiological significance of the HDAC4‐MEF2 complex in skeletal muscle has not been established. Here we show that in skeletal muscle, HDAC4 is a critical modulator of MEF2‐dependent structural and contractile gene expression in response to neural activity. We present evidence that loss of neural input leads to concomitant nuclear accumulation of HDAC4 and transcriptional reduction of MEF2‐regulated gene expression. Cell‐based assays show that HDAC4 represses structural gene expression via direct binding to AT‐rich MEF2 response elements. Notably, using both surgical denervation and the neuromuscular disease amyotrophic lateral sclerosis (ALS) model, we found that elevated levels of HDAC4 are required for efficient repression of MEF2‐dependent structural gene expression, indicating a link between the patho‐logical induction of HDAC4 and subsequent MEF2 target gene suppression. Supporting this supposition, we show that ectopic expression of HDAC4 in muscle fibers is sufficient to induce muscle damage in mice. Our study identifies HDAC4 as an activity‐dependent regulator of MEF2 function and suggests that activation of HDAC4 in response to chronically reduced neural activity suppresses MEF2‐dependent gene expression and contributes to progressive muscle dysfunction observed in neuromuscular diseases.—Cohen, T. J., Barrientos, T., Hartman, Z. C., Garvey, S. M., Cox, G. A., Yao, T.‐P. The deacetylase HDAC4 controls myocyte enhancing factor‐2 dependent structural gene expression in response to neural activity. FASEB J. 23, 99‐106 (2009)

HER2 stabilizes EGFR and itself by altering autophosphorylation patterns in a manner that overcomes regulatory mechanisms and promotes proliferative and transformation signaling.

One of the causes of breast cancer is overexpression of the human epidermal growth factor receptor 2 (HER2). Enhanced receptor autophosphorylation and resistance to activation-induced downregulation have been suggested as mechanisms for HER2-induced sustained signaling and cell transformation. However, the molecular mechanisms underlying these possibilities remain incompletely understood. In the current report, we present evidence that show that HER2 overexpression does not lead to receptor hyper-autophosphorylation, but alters patterns in a manner that favors receptor stability and sustained signaling. Specifically, HER2 overexpression blocks epidermal growth factor receptor (EGFR) tyrosine phosphorylation on Y1045 and Y1068, the known docking sites of c-Cbl and Grb2, respectively, whereas promoting phosphorylation on Y1173, the known docking site of the Gab adaptor proteins and phospholipase C gamma. Under these conditions, HER2 itself is phosphorylated on Y1221/1222, with no known role, and on Y1248 that corresponds to Y1173 of EGFR. Interestingly, suppressed EGFR autophosphorylation on the Grb2 and c-Cbl-binding sites correlated with receptor stability and sustained signaling, suggesting that HER2 accomplishes these tasks by altering autophosphorylation patterns. In conformity with these findings, mutation of the Grb2-binding site on EGFR (Y1068F–EGFR) conferred resistance to ligand-induced degradation, which in turn induced sustained signaling, and increased cell proliferation and transformation. These findings suggest that the Grb2-binding site on EGFR is redundant for signaling, but critical for receptor regulation. On the other hand, mutation of the putative Grb2-binding site in HER2 (Y1139) did not affect stability, signaling or transformation, suggesting that Y1139 in HER2 may not serve as a Grb2-binding site. In agreement with the role of EGFR in HER2 signaling, inhibition of EGFR expression reduced HER2-induced anchorage-independent growth and tumorigenesis. These results imply that complementing HER2-targeted therapies with anti-EGFR drugs may be beneficial in HER2-positive breast cancer.

Optimization of vaccine responses with an E1, E2b and E3-deleted Ad5 vector circumvents pre-existing anti-vector immunity

Recombinant serotype 5 adenovirus (Ad5) vectors lacking E1 expression induce robust immune responses against encoded transgenes in pre-clinical models, but have muted responses in human trials because of widespread pre-existing anti-adenovirus immunity. Attempts to circumvent Ad5-specific immunity by using alternative serotypes or modifying capsid components have not yielded profound clinical improvement. To address this issue, we explored a novel alternative strategy, specifically reducing the expression of structural Ad5 genes by creating E1 and E2b deleted recombinant Ad5 vectors. Our data show that [E1−, E2b−]vectors retaining the Ad5 serotype are potent immunogens in pre-clinical models despite the presence of significant Ad5-specific immunity, in contrast to [E1−] vectors. These pre-clinical studies with E1 and E2b-deleted recombinant Ad5 vectors suggest that anti-Ad immunity will no longer be a limiting factor, and that clinical trials to evaluate their performance are warranted.

Truncated ErbB2 Expressed in Tumor Cell Nuclei Contributes to Acquired Therapeutic Resistance to ErbB2 Kinase Inhibitors

ErbB2 tyrosine kinase inhibitors (TKI) block tyrosine autophosphorylation and activation of the full-length transmembrane ErbB2 receptor (p185ErbB2). In addition to p185ErbB2, truncated forms of ErbB2 exist in breast cancer cell lines and clinical tumors. The contribution of these truncated forms, specifically those expressed in tumor cell nuclei, to the development of therapeutic resistance to ErbB2 TKIs has not been previously shown. Here, we show that expression of a 95-kDa tyrosine phosphorylated form of ErbB2, herein referred to as p95L (lapatinib-induced p95) was increased in ErbB2+ breast cancer cells treated with potent ErbB2 TKIs (lapatinib, GW2974). Expressed in tumor cell nuclei, tyrosine phosphorylation of p95L was resistant to inhibition by ErbB2 TKIs. Furthermore, the expression of p95L was increased in ErbB2+ breast cancer models of acquired therapeutic resistance to lapatinib that mimic the clinical setting. Pretreatment with proteasome inhibitors blocked p95L induction in response to ErbB2 TKIs, implicating the role of the proteasome in the regulation of p95L expression. In addition, tyrosine phosphorylated C-terminal fragments of ErbB2, generated by alternate initiation of translation and similar in molecular weight to p95L, were expressed in tumor cell nuclei, where they too were resistant to inhibition by ErbB2 TKIs. When expressed in the nuclei of lapatinib-sensitive ErbB2+ breast cancer cells, truncated ErbB2 rendered cells resistant to lapatinib-induced apoptosis. Elucidating the function of nuclear, truncated forms of ErbB2, and developing therapeutic strategies to block their expression and/or activation may enhance the clinical efficacy of ErbB2 TKIs. Mol Cancer Ther; 10(8); 1367–74. ©2011 AACR.

Ligand-Independent Toll-like Receptor Signals Generated by Ectopic Overexpression of MyD88 Generate Local and Systemic Antitumor Immunity

Although critical for initiating and regulating immune responses, the therapeutic use of individual cytokines as anticancer immunotherapeutic agents has achieved only modest clinical success. Consequently, many current strategies have focused on the use of specific immunotherapeutic agonists that engage individual receptors of innate immune networks, such as the Toll-like receptor (TLR) system, each resulting in specific patterns of gene expression, cytokine production, and inflammatory outcome. However, these immunotherapeutics are constrained by variable cellular TLR expression and responsiveness to particular TLR agonists, as well as the specific cellular context of different tumors. We hypothesized that overexpression of MyD88, a pivotal regulator of multiple TLR signaling pathways, could circumvent these constraints and mimic coordinated TLR signaling across all cell types in a ligand-independent fashion. To explore this hypothesis, we generated an adenoviral vector expressing MyD88 and show that Ad-MyD88 infection elicits extensive Th1-specific transcriptional and secreted cytokine signatures in all murine and human cell types tested in vitro and in vivo. Importantly, in vivo intratumoral injection of Ad-MyD88 into established tumor masses enhanced adaptive immune responses and inhibited local tumor immunosuppression, resulting in significantly inhibited local and systemic growth of multiple tumor types. Finally, Ad-MyD88 infection of primary human dendritic cells, tumor-associated fibroblasts, and colorectal carcinoma cells elicited significant Th1-type cytokine responses, resulting in enhanced tumor cell lysis and expansion of human tumor antigen-specific T cells. Thus, Ad-MyD88 initiated robust antitumor activity in established murine tumor microenvironments and in human contexts, suggesting its potential effectiveness as a clinical immunotherapeutic strategy.

Replication-attenuated Human Adenoviral Type 4 vectors elicit capsid dependent enhanced innate immune responses that are partially dependent upon interactions with the complement system

Human Adenovirus Type 4 (HAdV-4) is responsible for epidemic outbreaks of Acute Respiratory Disease (especially in military recruits), and is known to cause significant morbidity with several reported cases of mortality. However, we do not understand why this serotype causes such high morbidity, and have little insight into the immunobiology of HAdV-4 infections. We have now developed a replication attenuated HAdV-4 vector system, and through it, demonstrate that HAdV-4 virions have enhanced infectivity of certain cell types and reveal aspects of the serotype-specific heightened innate immunogenicity of infectious HAdV-4 capsids both in vitro and in vivo. We further found that elements of this serotype-specific immunogenicity were dependent upon interactions with the complement system. These findings provide insights into the mechanisms possibly underlying the known morbidity accompanying wild-type HAdV-4 infections as well as highlight important considerations when considering development of alternative serotype vectors.