Molecular Signatures of CB-6644 Inhibition of the RUVBL1/2 Complex in Multiple Myeloma.

Multiple myeloma is the second most hematological cancer. RUVBL1 and RUVBL2 form a subcomplex of many chromatin remodeling complexes implicated in cancer progression. As an inhibitor specific to the RUVBL1/2 complex, CB-6644 exhibits remarkable anti-tumor activity in xenograft models of Burkitt's lymphoma and multiple myeloma (MM). In this work, we defined transcriptional signatures corresponding to CB-6644 treatment in MM cells and determined underlying epigenetic changes in terms of chromatin accessibility. CB-6644 upregulated biological processes related to interferon response and downregulated those linked to cell proliferation in MM cells. Transcriptional regulator inference identified E2Fs as regulators for downregulated genes and MED1 and MYC as regulators for upregulated genes. CB-6644-induced changes in chromatin accessibility occurred mostly in non-promoter regions. Footprinting analysis identified transcription factors implied in modulating chromatin accessibility in response to CB-6644 treatment, including ATF4/CEBP and IRF4. Lastly, integrative analysis of transcription responses to various chemical compounds of the molecular signature genes from public gene expression data identified CB-5083, a p97 inhibitor, as a synergistic candidate with CB-6644 in MM cells, but experimental validation refuted this hypothesis.

Grainyhead-like-2, an epithelial master programmer, promotes interferon induction and suppresses breast cancer recurrence.

Type-I and -III interferons play a central role in immune rejection of pathogens and tumors, thus promoting immunogenicity and suppressing tumor recurrence. Double strand RNA is an important ligand that stimulates tumor immunity via interferon responses. Differentiation of embryonic stem cells to pluripotent epithelial cells activates the interferon response during development, raising the question of whether epithelial vs. mesenchymal gene signatures in cancer potentially regulate the interferon pathway as well. Here, using genomics and signaling approaches, we show that Grainyhead-like-2 (GRHL2), a master programmer of epithelial cell identity, promotes type-I and -III interferon responses to double-strand RNA. GRHL2 enhanced the activation of IRF3 and relA/NF-kB and the expression of IRF1; a functional GRHL2 binding site in the IFNL1 promoter was also identified. Moreover, time to recurrence in breast cancer correlated positively with GRHL2 protein expression, indicating that GRHL2 is a tumor recurrence suppressor, consistent with its enhancement of interferon responses. These observations demonstrate that epithelial cell identity supports interferon responses in the context of cancer.

Interleukin-1α: Novel functions in cell senescence and antiviral response.

The interleukin-1 proteins are a hub of innate inflammatory signaling that activates diverse aspects of adaptive immunity. Until recently, the IL-1α isoform was relatively incompletely understood compared with IL-1ß. This review briefly summarizes novel and surprising aspects of IL-1α biology. IL-1α localizes to the nucleus, cytoplasm, mitochondria, cell membrane or extracellular space in various contexts, with corresponding distinct functions. In particular, we focus on multiple pathways by which IL-1α promotes the senescent cell phenotype, unexpectedly involving signaling molecules including mTOR, GATA4, mitochondrial cardiolipin and caspases-4/5. Finally, I review a novel pathway by which IL-1α promotes antiviral immunity.

Type I interferons and related pathways in cell senescence.

This review article addresses the largely unanticipated convergence of two landmark discoveries. The first is the discovery of interferons, critical signaling molecules for all aspects of both innate and adaptive immunity, discovered originally by Isaacs and Lindenmann at the National Institute for Medical Research, London, in 1957 (Proceedings of the Royal Society of London. Series B: Biological Sciences, 1957, 147, 258). The second, formerly unrelated discovery, by Leonard Hayflick and Paul Moorhead (Wistar Institute, Philadelphia) is that cultured cells undergo an irreversible but viable growth arrest, termed senescence, after a finite and predictable number of cell divisions (Experimental Cell Research, 1961, 25, 585). This phenomenon was suspected to relate to organismal aging, which was confirmed subsequently (Nature, 2011, 479, 232). Cell senescence has broad-ranging implications for normal homeostasis, including immunity, and for diverse disease states, including cancer progression and response to therapy (Nature Medicine, 2015, 21, 1424; Cell, 2019, 179, 813; Cell, 2017, 169, 1000; Trends in Cell Biology, 2018, 28, 436; Journal of Cell Biology, 2018, 217, 65). Here, we critically address the bidirectional interplay between interferons (focusing on type I) and cell senescence, with important implications for health and healthspan.

Grainyhead-like-2 confers NK-sensitivity through interactions with epigenetic modifiers.

Natural Killer (NK) cells suppress tumor initiation and metastasis. Most carcinomas are heterogeneous mixtures of epithelial, mesenchymal and hybrid tumor cells, but the relationships of these phenotypes to NK susceptibility are understood incompletely. Grainyhead-like-2 (GRHL2) is a master programmer of the epithelial phenotype, that is obligatorily down-regulated during experimentally induced Epithelial-Mesenchymal Transition (EMT). Here, we utilize GRHL2 re-expression to discover unifying molecular mechanisms that link the epithelial phenotype with NK-sensitivity. GRHL2 enhanced the expression of ICAM-1, augmenting NK-target cell synaptogenesis and NK killing of target cells. The expression of multiple interferon response genes, including ICAM1, anti-correlated with EMT. We identified two novel GRHL2-interacting proteins, the histone methyltransferases KMT2C and KMT2D. Mesenchymal-epithelial transition, NK-sensitization and ICAM-1 expression were promoted by GRHL2-KMT2C/D interactions and by GRHL2 inhibition of p300, revealing novel and potentially targetable epigenetic mechanisms connecting the epithelial phenotype with target cell susceptibility to NK killing.

Mechanisms and context underlying the role of autophagy in cancer metastasis.

Macroautophagy/autophagy is a fundamental cellular degradation mechanism that maintains cell homeostasis, regulates cell signaling, and promotes cell survival. Its role in promoting tumor cell survival in stress conditions is well characterized, and makes autophagy an attractive target for cancer therapy. Emerging research indicates that autophagy also influences cancer metastasis, which is the primary cause of cancer-associated mortality. However, data demonstrate that the regulatory role of autophagy in metastasis is multifaceted, and includes both metastasis-suppressing and -promoting functions. The metastasis-suppressing functions of autophagy, in particular, have important implications for autophagy-based treatments, as inhibition of autophagy may increase the risk of metastasis. In this review, we discuss the mechanisms and context underlying the role of autophagy in metastasis, which include autophagy-mediated regulation of focal adhesion dynamics, integrin signaling and trafficking, Rho GTPase-mediated cytoskeleton remodeling, anoikis resistance, extracellular matrix remodeling, epithelial-to-mesenchymal transition signaling, and tumor-stromal cell interactions. Through this, we aim to clarify the context-dependent nature of autophagy-mediated metastasis and provide direction for further research investigating the role of autophagy in cancer metastasis.

Grainyhead-like 2 inhibits the coactivator p300, suppressing tubulogenesis and the epithelial-mesenchymal transition.

Developmental morphogenesis and tumor progression require a transient or stable breakdown of epithelial junctional complexes to permit programmed migration, invasion, and anoikis resistance, characteristics endowed by the epithelial-mesenchymal transition (EMT). The epithelial master-regulatory transcription factor Grainyhead-like 2 (GRHL2) suppresses and reverses EMT, causing a mesenchymal-epithelial transition to the default epithelial phenotype. Here we investigated the role of GRHL2 in tubulogenesis of Madin-Darby canine kidney cells, a process requiring transient, partial EMT. GRHL2 was required for cystogenesis, but it suppressed tubulogenesis in response to hepatocyte growth factor. Surprisingly, GRHL2 suppressed this process by inhibiting the histone acetyltransferase coactivator p300, preventing the induction of matrix metalloproteases and other p300-dependent genes required for tubulogenesis. A 13-amino acid region of GRHL2 was necessary for inhibition of p300, suppression of tubulogenesis, and interference with EMT. The results demonstrate that p300 is required for partial or complete EMT occurring in tubulogenesis or tumor progression and that GRHL2 suppresses EMT in both contexts through inhibition of p300.

Grainyhead-like 2 Reverses the Metabolic Changes Induced by the Oncogenic Epithelial-Mesenchymal Transition: Effects on Anoikis.

UNLABELLED: Resistance to anoikis is a prerequisite for tumor metastasis. The epithelial-to-mesenchymal transition (EMT) allows tumor cells to evade anoikis. The wound-healing regulatory transcription factor Grainyhead-like 2 (GRHL2) suppresses/reverses EMT, accompanied by suppression of the cancer stem cell (CSC) phenotype and by resensitization to anoikis. Here, the effects of GRHL2 upon intracellular metabolism in the context of reversion of the EMT/CSC phenotype, with a view toward understanding how these effects promote anoikis sensitivity, were investigated. EMT enhanced mitochondrial oxidative metabolism. Although this was accompanied by higher accumulation of superoxide, the overall level of reactive oxygen species (ROS) declined, due to decreased hydrogen peroxide. Glutamate dehydrogenase 1 (GLUD1) expression increased in EMT, and this increase, via the product α-ketoglutarate (α-KG), was important for suppressing hydrogen peroxide and protecting against anoikis. GRHL2 suppressed GLUD1 gene expression, decreased α-KG, increased ROS, and sensitized cells to anoikis. IMPLICATIONS: These results demonstrate a mechanistic role for GRHL2 in promoting anoikis through metabolic alterations. Mol Cancer Res; 14(6); 528-38. ©2016 AACR.

CD44S-hyaluronan interactions protect cells resulting from EMT against anoikis.

The detachment of normal epithelial cells from matrix triggers an apoptotic response known as anoikis, during homeostatic turnover. Metastatic tumor cells evade anoikis, by mechanisms that are only partly characterized. In particular, the epithelial-mesenchymal transition (EMT) in a subset of invasive tumor cells confers anoikis-resistance. In some cases, EMT up-regulates the cancer stem cell marker CD44S and the enzyme hyaluronic acid synthase-2 (HAS2). CD44S is the major receptor for hyaluronan in the extracellular matrix. Herein, we demonstrate that CD44S, unlike the CD44E isoform expressed in normal epithelial cells, contributes to the protection against anoikis. This protection requires the interaction of CD44S with hyaluronan (HA). CD44S-HA interaction is proposed to play an important role in tumor metastasis through enhanced cell survival under detached conditions.

The wind god promotes lung cancer.

In this issue of Cancer Cell, Li and colleagues demonstrate that the hematopoietic transcription factor Aiolos (named after the Wind God of Greek mythology) confers anoikis resistance in lung tumor cells through repression of cell adhesion-related genes including the mechanosensor p66Shc.

Epithelial-mesenchymal transition and tumor suppression are controlled by a reciprocal feedback loop between ZEB1 and Grainyhead-like-2.

Epithelial-mesenchymal transition (EMT) in carcinoma cells enhances malignant progression by promoting invasion and survival. EMT is induced by microenvironmental factors, including TGF-ß and Wnt agonists, and by the E-box-binding transcription factors Twist, Snail, and ZEB. Grainyhead-like-2 (GRHL2), a member of the mammalian Grainyhead family of wound-healing regulatory transcription factors, suppresses EMT and restores sensitivity to anoikis by repressing ZEB1 expression and inhibiting TGF-ß signaling. In this study, we elucidate the functional relationship between GRHL2 and ZEB1 in EMT/MET and tumor biology. At least three homeodomain proteins, Six1, LBX1, and HoxA5, transactivated the ZEB1 promoter, in the case of Six1, through direct protein-promoter interaction. GRHL2 altered the Six1-DNA complex, inhibiting this transactivation. Correspondingly, GRHL2 expression prevented tumor initiation in xenograft assays, sensitized breast cancer cells to paclitaxel, and suppressed the emergence of CD44(high)CD24(low) cells (defining the cancer stem cell phenotype in the cell type studied). GRHL2 was downregulated in recurrent mouse tumors that had evolved to an oncogene-independent, EMT-like state, supporting a role for GRHL2 downregulation in this phenotypic transition, modeling disease recurrence. The combination of TGF-ß and Wnt activation repressed GRHL2 expression by direct interaction of ZEB1 with the GRHL2 promoter, inducing EMT. Together, our observations indicate that a reciprocal feedback loop between GRHL2 and ZEB1 controls epithelial versus mesenchymal phenotypes and EMT-driven tumor progression.

Regulation of anoikis by deleted in breast cancer-1 (DBC1) through NF-κB.

Anoikis-resistance of tumor cells is critical for anchorage-independent growth and metastasis. The inflammatory-response transcription factor NF-κB contributes to anoikis-resistance and tumor progression through mechanisms that are understood incompletely. Deleted in breast cancer-1 (DBC1) protein (KIAA1967) is over-expressed in several tumor types, and correlates with a poorer prognosis in some cases. We report here that DBC1 suppressed anoikis in normal epithelial and breast cancer cell lines. DBC1 interacted with IKK-ß, stimulating its kinase activity, promoting NF-κB transcriptional activity through the phosphorylation of relA serine-536 and enhancing the expression of the NF-κB target genes, c-FLIP and bcl-xl. Our results indicate that DBC1 is an important co-factor for the control of the IKK-ß-NF-κB signaling pathway that regulates anoikis.

Mechanisms that link the oncogenic epithelial-mesenchymal transition to suppression of anoikis.

The oncogenic epithelial-mesenchymal transition (EMT) contributes to tumor progression in various context-dependent ways, including increased metastatic potential, expansion of cancer stem cell subpopulations, chemo-resistance and disease recurrence. One of the hallmarks of EMT is resistance of tumor cells to anoikis. This resistance contributes to metastasis and is a defining property not only of EMT but also of cancer stem cells. Here, we review the mechanistic coupling between EMT and resistance to anoikis. The discussion focuses on several key aspects. First, we provide an update on new pathways that lead from the loss of E-cadherin to anoikis resistance. We then discuss the relevance of transcription factors that are crucial in wound healing in the context of oncogenic EMT. Next, we explore the consequences of the breakdown of cell-polarity complexes upon anoikis sensitivity, through the Hippo, Wnt and transforming growth factor ß (TGF-ß) pathways, emphasizing points of crossregulation. Finally, we summarize the direct regulation of cell survival genes through EMT-inducing transcription factors, and the roles of the tyrosine kinases focal adhesion kinase (FAK) and TrkB neurotrophin receptor in EMT-related regulation of anoikis. Emerging from these studies are unifying principles that will lead to improvements in cancer therapy by reprogramming sensitivity of anoikis.

Suppression of the epithelial-mesenchymal transition by Grainyhead-like-2.

Grainyhead genes are involved in wound healing and developmental neural tube closure. In light of the high degree of similarity between the epithelial-mesenchymal transitions (EMT) occurring in wound-healing processes and the cancer stem cell-like compartment of tumors, including TGF-ß dependence, we investigated the role of the Grainyhead gene, Grainyhead-like-2 (GRHL2) in oncogenic EMT. GRHL2 was downregulated specifically in the claudin-low subclass breast tumors and in basal-B subclass breast cancer cell lines. GRHL2 suppressed TGF-ß-induced, Twist-induced or spontaneous EMT, enhanced anoikis sensitivity, and suppressed mammosphere generation in mammary epithelial cells. These effects were mediated in part by suppression of ZEB1 expression via direct repression of the ZEB1 promoter. GRHL2 also inhibited Smad-mediated transcription and it upregulated mir-200b/c as well as the TGF-ß receptor antagonist, BMP2. Finally, ectopic expression of GRHL2 in MDA-MB-231 breast cancer cells triggered an MET and restored sensitivity to anoikis. Taken together, our findings define a major role for GRHL2 in the suppression of oncogenic EMT in breast cancer cells.

A pathway for the control of anoikis sensitivity by E-cadherin and epithelial-to-mesenchymal transition.

Detachment of epithelial cells from matrix or attachment to an inappropriate matrix engages an apoptotic response known as anoikis, which prevents metastasis. Cellular sensitivity to anoikis is compromised during the oncogenic epithelial-to-mesenchymal transition (EMT), through unknown mechanisms. We report here a pathway through which EMT confers anoikis resistance. NRAGE (neurotrophin receptor-interacting melanoma antigen) interacted with a component of the E-cadherin complex, ankyrin-G, maintaining NRAGE in the cytoplasm. Oncogenic EMT downregulated ankyrin-G, enhancing the nuclear localization of NRAGE. The oncogenic transcriptional repressor protein TBX2 interacted with NRAGE, repressing the tumor suppressor gene p14ARF. P14ARF sensitized cells to anoikis; conversely, the TBX2/NRAGE complex protected cells against anoikis by downregulating this gene. This represents a novel pathway for the regulation of anoikis by EMT and E-cadherin.

Caspase-8: fly or die.

Recent studies have revealed that procaspase-8 has an important function in cell adhesion and motility. Src phosphorylation controls this function by preventing the conversion of procaspase-8, which is an adhesion/migration factor, to mature caspase-8, which is an apoptosis-inducing factor. This provides a mechanism to switch these opposing functions. In its migratory role, procaspase-8 interacts with the phosphatidylinositol-3-OH kinase regulatory subunit p85alpha and c-src to modulate signaling by Rac and extracellular signal-regulated kinase, and promote calpain activation. Here, I survey the findings of these studies and discuss potential mechanisms and ramifications for cancer prognosis and therapy.

Caspase-8 interacts with the p85 subunit of phosphatidylinositol 3-kinase to regulate cell adhesion and motility.

Cell migration plays an important role in tumor cell invasion and metastasis. Previously, we reported that caspase-8 contributes to cell migration and adhesion, a novel nonapoptotic function of an established apoptotic factor. Herein, we report that pro-caspase-8 is capable of restoring cell migration/adhesion to caspase-8-null cells, establishing the first biological function of a pro-caspase. The catalytic activity of caspase-8 was not required for cell motility. Stimulation of motility with epidermal growth factor induced the phosphorylation of caspase-8 on tyrosine-380 and the interaction of caspase-8 with the p85 alpha subunit of phosphatidylinositol 3-kinase. Tyrosine-380 was required for the restoration of cell motility and cell adhesion in caspase-8-null cells, demonstrating the importance of the caspase-8-p85 interaction for these nonapoptotic functions. These results suggest that caspase-8 phosphorylation converts it from a proapoptotic factor to a cell motility factor that, through tyrosine-380, interacts with p85, an established cell migration component.

Blockade of tumor necrosis factor-induced Bid cleavage by caspase-resistant Rb.

Tumor necrosis factor-alpha (TNF) activates caspase-8 to cleave effector caspases or Bid, resulting in type-1 or type-2 apoptosis, respectively. We show here that TNF also induces caspase-8-dependent C-terminal cleavage of the retinoblastoma protein (Rb). Interestingly, fibroblasts from Rb(MI/MI) mice, in which the C-terminal caspase cleavage site is mutated, exhibit a defect in Bid cleavage despite caspase-8 activation. Recent results suggest that TNF receptor endocytosis is required for the activation of caspase-8. Consistent with this notion, inhibition of V-ATPase, which plays an essential role in acidification and degradation of endosomes, specifically restores Bid cleavage in Rb(MI/MI) cells. Inhibition of V-ATPase sensitizes Rb(MI/MI) but not wild-type fibroblasts to TNF-induced apoptosis and stimulates inflammation-associated colonic apoptosis in Rb(MI/MI) but not wild-type mice. These results suggest that Rb cleavage is required for Bid cleavage in TNF-induced type-2 apoptosis, and this requirement can be supplanted by the inhibition of V-ATPase.

Caspase-8 promotes cell motility and calpain activity under nonapoptotic conditions.

Significant caspase-8 activity has been found in normal and certain tumor cells, suggesting that caspase-8 possesses an alternative, nonapoptotic function that may contribute to tumor progression. In this article, we report that caspase-8 promotes cell motility. In particular, caspase-8 is required for the optimal activation of calpains, Rac, and lamellipodial assembly. This represents a novel nonapoptotic function of caspase-8 acting at the intersection of the caspase-8 and calpain proteolytic pathways to coordinate cell death versus cell motility signaling.