The ubiquitin-proteasome system has recently emerged as a major target for drug development in cancer therapy. The proteasome inhibitor bortezomib has clinical activity in multiple myeloma and mantle cell lymphoma. Here we report that Eeyarestatin I (EerI), a chemical inhibitor that blocks endoplasmic reticulum (ER)-associated protein degradation, has antitumor and biologic activities similar to bortezomib and can synergize with bortezomib. Like bortezomib, EerI-induced cytotoxicity requires the up-regulation of the Bcl-2 homology3 (BH3)-only pro-apoptotic protein NOXA. We further demonstrate that both EerI and bortezomib activate NOXA via an unanticipated mechanism that requires cooperation between two processes. First, these agents elicit an integrated stress response program at the ER to activate the CREB/ATF transcription factors ATF3 and ATF4. We show that ATF3 and ATF4 form a complex capable of binding to the NOXA promoter, which is required for NOXA activation. Second, EerI and bortezomib also block ubiquitination of histone H2A to relieve its inhibition on NOXA transcription. Our results identify a class of anticancer agents that integrate ER stress response with an epigenetic mechanism to induce cell death.bortezomib ͉ cancer therapy ͉ ER stress/UPR ͉ histone H2A ͉ ubiquitin T he ubiquitin proteasome system (UPS) constitutes a major disposal pathway for misfolded proteins at the endoplasmic reticulum (ER) and therefore promotes protein homeostasis. It has been shown that misfolded ER proteins are exported from the ER into the cytosol by a process termed ER-associated protein degradation (ERAD) or retrotranslocation (1, 2). This process requires a cytosolic ATPase named p97, which acts as a ''dislocase'' to extract misfolded proteins from the ER membranes once substrates have undergone polyubiquitination. p97 subsequently hands substrates over to the proteasome for degradation (3). Defects in ERAD cause accumulation of misfolded proteins in the ER and thus trigger ER stress (also called Unfolded Protein Response, UPR).The functional integrity of the UPS is also essential for the survival of cancer cells because their uncontrolled proliferation requires proteasome mediated degradation of many cell cycle factors (4). Accordingly, the proteasome inhibitor bortezomib (Velcade TM ) has cytotoxic activities against a broad range of cancer cell lines and is now used in the clinic to treat multiple myeloma and mantle cell lymphoma (MCL) (5). The precise mechanism by which bortezomib induces cytotoxicity in cancer cells remains elusive. Recent studies showed that the induction of the Bcl-2 homology3 (BH3)-only pro-apoptotic protein NOXA plays an important role (6-12). On a separate note, several other studies suggested that ER stress elicited by bortezomib may account for its cytotoxicity (8, 13). It is currently unclear whether NOXA activation and ER stress are mechanistically connected or independently triggered by bortezomib as a result of stabilization of distinct proteasomal substrates. In addition, although...

Host response to cancer signals has emerged as a key factor in cancer development; however, the underlying molecular mechanism is not well understood. In this report, we demonstrate that activating transcription factor 3 (ATF3), a hub of the cellular adaptive response network, plays an important role in host cells to enhance breast cancer metastasis. Immunohistochemical analysis of patient tumor samples revealed that expression of ATF3 in stromal mononuclear cells, but not cancer epithelial cells, is correlated with worse clinical outcomes and is an independent predictor for breast cancer death. This finding was corroborated by data from mouse models showing less efficient breast cancer metastasis in Atf3-deficient mice than in WT mice. Further, mice with myeloid cell-selective KO of Atf3 showed fewer lung metastases, indicating that host ATF3 facilitates metastasis, at least in part, by its function in macrophage/myeloid cells. Gene profiling analyses of macrophages from mouse tumors identified an ATF3-regulated gene signature that could distinguish human tumor stroma from distant stroma and could predict clinical outcomes, lending credence to our mouse models. In conclusion, we identified ATF3 as a regulator in myeloid cells that enhances breast cancer metastasis and has predictive value for clinical outcomes.

Tumors secreting glycoproteins that act as tumor-associated antigens have been described as highly invasive and metastatic. In this study, the consequences of the humoral immune response (HIR) against these antigens were investigated. Using an in vitro model of tumor cell invasion, results indicated that the invasiveness of tumor cells secreting antigenic secreted/shed tumor glycoproteins (STGP) increases in the presence of specific anti-STGP IgG, polymorphonuclear cells and monocytes. This in vitro model showed that the coincidental presence in the matrix of both STGP and specific anti-STGP IgG increases the local release of IL-1beta, IL-6 and vascular endothelial growth factor (VEGF) by stromal cells, but not by tumor cells. Using an in vivo model, the experiments show that immune-competent mice develop an anti-tumor HIR with anti-STGP IgG production. In this model, tumor growth was increased in parallel with the serum concentration of specific anti-STGP IgG. In athymic nude (nu/nu)-beige mice the same trend was observed, suggesting a T-cell-independent tumor-promoting effect induced by anti-STGP IgG. Tumor histology showed intense infiltration of IgG-positive plasma cells and lymphocytes. A severe combined immunodeficient-beige mouse-based in vivo model of tumors, experimentally infiltrated with monoclonal IgG plasmocytoma cells, showed that only specific anti-STGP-IgG-secreting cells could exacerbate tumor invasion, angiogenesis and metastasis. These results suggest that tumors shedding/secreting antigenic STGP can induce a host IgG immune response that can promote invasion and metastasis by inducing tumor infiltrating stromal cells to release proinflammatory cytokines and VEGF.

The expression of Fas ligand (FasL) by tumor cells has been reported to have multiple, conflicting effects on tumor growth. The majority of the data support the theory that FasL expressing tumor cells evade immune surveillance by killing T cells expressing Fas. However, the role of the humoral immune-blockade by FasL expressing tumor cells has not been assessed. Using immune-competent mice, we observed that FasL expressing tumor cells reduced the antitumor antibody production together with the T and B cell content of the spleen in these mice. Further, to determine if the expression of FasL in the environment of the tumor suppresses the humoral antitumor immune response and influences tumor growth, a mouse model lacking T cells was used. To assess whether a local reduction of FasL could reduce tumor progression, a plasmid encoding antisense FasL cDNA was delivered directly into a growing tumor (SW620 colon carcinoma). Intratumoral delivery of the plasmid was able to transfect tumor cells, stromal cells, and peritumoral muscle cells. This antisense FasL tumor tissue transfection persisted for at least 25 days, produced a systemic decrease in soluble FasL, and resulted in a 50% reduction in the rate of tumor growth when compared with tumor tissue of the control groups. These results suggest that direct transfection of antisense FasL cDNA impairs FasL translation in tumor and stromal cells, and can inhibit tumor progression by impairing the FasL-mediated, stromal cell-assisted, tumor counter-attack.

Objective: Cancer progression is facilitated by a complex network of interactions between cancer cells and host-derived components, such as stromal cells and extracellular matrix in the tumor microenvironment. We sought to identify the factors that mediate these dynamic cancer-host interplays. Specifically, we asked how the host responds to signals from the cancer cells. To this end, we investigated the role of ATF3, an adaptive-response gene in the cellular stress response network. Overwhelming evidence indicates that ATF3 is induced by a broad spectrum of extra- and intra-cellular signals in a variety of cell types. As such, it is as an excellent candidate for mediating host responses to cancer cells. Methods: We injected breast cancer cells (MMTV-PyMT cells) into syngeneic wild type (WT) or ATF3 knockout (KO) mice, performed survival surgery to remove the primary tumors, and examined metastasis two months after tumor removal. Results: We found that ATF3 deficiency in the host did not affect primary tumor formation; excitingly, though, it dramatically decreased lung metastasis. Analyses of the circulating tumor cells and lung colonization indicated that both early and late steps in the metastatic cascade were defective in the KO host. Since soluble factors are an integral part of the mechanisms by which the host transmits systemic responses, we analyzed the plasma of normal and tumor-bearing mice by an antibody array. Intriguingly, plasma from tumor-bearing KO mice demonstrated a marked impairment in the abundance of various molecules that are known to play important roles in metastasis, suggesting that ATF3 in the host promotes a systemic environment that enhances cancer metastasis. Since the KO mice are whole body KO, the results above do not indicate the cell type(s) in which ATF3 is playing this critical role. We will present evidence that ATF3 plays an important role, at least in part, in the tumor associated macrophages (TAMs), where it up-regulates MMP9 as a functionally important target gene. Two lines of evidence indicated that our findings on ATF3 have clinical relevance. First, analyses of human tumor microarrays by immunohistochemistry revealed that ATF3 expression in monocytic cells correlated with poor outcome. Second, analyses of the mouse TAMs from the WT and KO host identified ∼400 ATF3-regulated genes. Among these genes, a 60-gene signature was identified that could distinguish the human breast tumor stroma from the normal breast stroma (McGill Breast Stroma dataset). Significantly, this signature predicted outcome in two independent patient cohorts. Conclusion and Significance: We uncovered a previously unknown role for ATF3: it is induced in the host during cancer development and its expression in the host cells, specifically the TAMs, promotes metastasis. This finding is significant because it not only links host stress response to cancer metastasis, it also identified a new gene signature that predicts outcome. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2843. doi:10.1158/1538-7445.AM2011-2843