Interferon-beta represses cancer stem cell properties in triple-negative breast cancer.

Triple-negative breast cancer (TNBC), the deadliest form of this disease, lacks a targeted therapy. TNBC tumors that fail to respond to chemotherapy are characterized by a repressed IFN/signal transducer and activator of transcription (IFN/STAT) gene signature and are often enriched for cancer stem cells (CSCs). We have found that human mammary epithelial cells that undergo an epithelial-to-mesenchymal transition (EMT) following transformation acquire CSC properties. These mesenchymal/CSCs have a significantly repressed IFN/STAT gene expression signature and an enhanced ability to migrate and form tumor spheres. Treatment with IFN-beta (IFN-ß) led to a less aggressive epithelial/non-CSC-like state, with repressed expression of mesenchymal proteins (VIMENTIN, SLUG), reduced migration and tumor sphere formation, and reexpression of CD24 (a surface marker for non-CSCs), concomitant with an epithelium-like morphology. The CSC-like properties were correlated with high levels of unphosphorylated IFN-stimulated gene factor 3 (U-ISGF3), which was previously linked to resistance to DNA damage. Inhibiting the expression of IRF9 (the DNA-binding component of U-ISGF3) reduced the migration of mesenchymal/CSCs. Here we report a positive translational role for IFN-ß, as gene expression profiling of patient-derived TNBC tumors demonstrates that an IFN-ß metagene signature correlates with improved patient survival, an immune response linked with tumor-infiltrating lymphocytes (TILs), and a repressed CSC metagene signature. Taken together, our findings indicate that repressed IFN signaling in TNBCs with CSC-like properties is due to high levels of U-ISGF3 and that treatment with IFN-ß reduces CSC properties, suggesting a therapeutic strategy to treat drug-resistant, highly aggressive TNBC tumors.

The opposing effects of interferon-beta and oncostatin-M as regulators of cancer stem cell plasticity in triple-negative breast cancer.

BACKGROUND: Highly aggressive, metastatic and therapeutically resistant triple-negative breast cancers (TNBCs) are often enriched for cancer stem cells (CSC). Cytokines within the breast tumor microenvironment (TME) influence the CSC state by regulating tumor cell differentiation programs. Two prevalent breast TME cytokines are oncostatin-M (OSM) and interferon-ß (IFN-ß). OSM is a member of the IL-6 family of cytokines and can drive the de-differentiation of TNBC cells to a highly aggressive CSC state. Conversely, IFN-ß induces the differentiation of TNBC, resulting in the repression of CSC properties. Here, we assess how these breast TME cytokines influence CSC plasticity and clinical outcome. METHODS: Using transformed human mammary epithelial cell (HMEC) and TNBC cell models, we assessed the CSC markers and properties following exposure to OSM and/or IFN-ß. CSC markers included CD24, CD44, and SNAIL; CSC properties included tumor sphere formation, migratory capacity, and tumor initiation. RESULTS: There are three major findings from our study. First, exposure of purified, non-CSC to IFN-ß prevents OSM-mediated CD44 and SNAIL expression and represses tumor sphere formation and migratory capacity. Second, during OSM-induced de-differentiation, OSM represses endogenous IFN-ß mRNA expression and autocrine/paracrine IFN-ß signaling. Restoring IFN-ß signaling to OSM-driven CSC re-engages IFN-ß-mediated differentiation by repressing OSM/STAT3/SMAD3-mediated SNAIL expression, tumor initiation, and growth. Finally, the therapeutic use of IFN-ß to treat OSM-driven tumors significantly suppresses tumor growth. CONCLUSIONS: Our findings suggest that the levels of IFN-ß and OSM in TNBC dictate the abundance of cells with a CSC phenotype. Indeed, TNBCs with elevated IFN-ß signaling have repressed CSC properties and a better clinical outcome. Conversely, TNBCs with elevated OSM signaling have a worse clinical outcome. Likewise, since OSM suppresses IFN-ß expression and signaling, our studies suggest that strategies to limit OSM signaling or activate IFN-ß signaling will disengage the de-differentiation programs responsible for the aggressiveness of TNBCs.

Breaking the oncostatin M feed-forward loop to suppress metastasis and therapy failure.

Deciphering the complex milieu that makes up the tumor microenvironment (TME) and the signaling engaged by TME cytokines continues to provide novel targets for therapeutic intervention. The IL-6 family member oncostatin M (OSM) has recently emerged as a potent driver of tumorigenesis, metastasis, and therapy failure, molecular programs most frequently attributed to IL-6 itself. In a recent issue of The Journal of Pathology, Kucia-Tran et al describe how elevated oncostatin M receptor (OSMR) expression results in a feed-forward loop involving the de novo production of both OSM and OSMR to facilitate aggressive properties in squamous cell carcinoma (SCC). Here, we discuss how new findings implicating OSM in conferring aggressive cancer cell properties can be leveraged to suppress metastatic outgrowth and therapy failure in SCC as well as other cancers. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

DNMT1-associated long non-coding RNAs regulate global gene expression and DNA methylation in colon cancer.

The cancer epigenome exhibits global loss of DNA methylation, which contributes to genomic instability and aberrant gene expression by mechanisms that are yet to be fully elucidated. We previously discovered over 3300 long non-coding (lnc)RNAs in human cells and demonstrated that specific lncRNAs regulate gene expression via interactions with chromatin-modifying complexes. Here, we tested whether lncRNAs could also associate with DNA methyltransferases to regulate DNA methylation and gene expression. Using RIP-seq, we identified a subset of lncRNAs that interact with the DNA methyltransferase DNMT1 in a colon cancer cell line, HCT116. One lncRNA, TCONS_00023265, which we named DACOR1 (DNMT1-associated Colon Cancer Repressed lncRNA 1), shows high, tissue-specific expression in the normal colon (including colon crypts) but was repressed in a panel of colon tumors and patient-derived colon cancer cell lines. We identified the genomic occupancy sites of DACOR1, which we found to significantly overlap with known differentially methylated regions (DMRs) in colon tumors. Induction of DACOR1 in colon cancer cell lines significantly reduced their ability to form colonies in vitro, suggesting a growth suppressor function. Consistent with the observed phenotype, induction of DACOR1 led to the activation of tumor-suppressor pathways and attenuation of cancer-associated metabolic pathways. Notably, DACOR1 induction resulted in down-regulation of Cystathionine ß-synthase, which is known to lead to increased levels of S-adenosyl methionine-the key methyl donor for DNA methylation. Collectively, our results demonstrate that deregulation of DNMT1-associated lncRNAs contributes to aberrant DNA methylation and gene expression during colon tumorigenesis.

IFNß-dependent increases in STAT1, STAT2, and IRF9 mediate resistance to viruses and DNA damage.

A single high dose of interferon-ß (IFNß) activates powerful cellular responses, in which many anti-viral, pro-apoptotic, and anti-proliferative proteins are highly expressed. Since some of these proteins are deleterious, cells downregulate this initial response rapidly. However, the expression of many anti-viral proteins that do no harm is sustained, prolonging a substantial part of the initial anti-viral response for days and also providing resistance to DNA damage. While the transcription factor ISGF3 (IRF9 and tyrosine-phosphorylated STATs 1 and 2) drives the first rapid response phase, the related factor un-phosphorylated ISGF3 (U-ISGF3), formed by IFNß-induced high levels of IRF9 and STATs 1 and 2 without tyrosine phosphorylation, drives the second prolonged response. The U-ISGF3-induced anti-viral genes that show prolonged expression are driven by distinct IFN stimulated response elements (ISREs). Continuous exposure of cells to a low level of IFNß, often seen in cancers, leads to steady-state increased expression of only the U-ISGF3-dependent proteins, with no sustained increase in other IFNß-induced proteins, and to constitutive resistance to DNA damage.

TGF-beta signaling engages an ATM-CHK2-p53-independent RAS-induced senescence and prevents malignant transformation in human mammary epithelial cells.

Oncogene-induced senescence (OIS), the proliferative arrest engaged in response to persistent oncogene activation, serves as an important tumor-suppressive barrier. We show here that finite lifespan human mammary epithelial cells (HMEC) undergo a p16/RB- and p53-independent OIS in response to oncogenic RAS that requires TGF-ß signaling. Suppression of TGF-ß signaling by expression of a dominant-negative TGF-ß type II receptor, use of a TGF-ß type I receptor inhibitor, or ectopic expression of MYC permitted continued proliferation upon RAS expression. Surprisingly, unlike fibroblasts, shRNA-mediated knockdown of ATM or CHK2 was unable to prevent RAS-mediated OIS, arguing that the DNA damage response is not required for OIS in HMEC. Abrogation of TGF-ß signaling not only allowed HMEC lacking p53 to tolerate oncogenic RAS but also conferred the capacity for anchorage-independent growth. Thus, the OIS engaged after dysregulated RAS expression provides an early barrier to malignant progression and is mediated by TGF-ß receptor activation in HMEC. Understanding the mechanisms that initiate and maintain OIS in epithelial cells may provide a foundation for future therapies aimed at reengaging this proliferative barrier as a cancer therapy.

Pathogenesis of growth failure and partial reversal with gene therapy in murine and canine Glycogen Storage Disease type Ia.

Glycogen Storage Disease type Ia (GSD-Ia) in humans frequently causes delayed bone maturation, decrease in final adult height, and decreased growth velocity. This study evaluates the pathogenesis of growth failure and the effect of gene therapy on growth in GSD-Ia affected dogs and mice. Here we found that homozygous G6pase (-/-) mice with GSD-Ia have normal growth hormone (GH) levels in response to hypoglycemia, decreased insulin-like growth factor (IGF) 1 levels, and attenuated weight gain following administration of GH. Expression of hepatic GH receptor and IGF 1 mRNAs and hepatic STAT5 (phospho Y694) protein levels are reduced prior to and after GH administration, indicating GH resistance. However, restoration of G6Pase expression in the liver by treatment with adeno-associated virus 8 pseudotyped vector expressing G6Pase (AAV2/8-G6Pase) corrected body weight, but failed to normalize plasma IGF 1 in G6pase (-/-) mice. Untreated G6pase (-/-) mice also demonstrated severe delay of growth plate ossification at 12 days of age; those treated with AAV2/8-G6Pase at 14 days of age demonstrated skeletal dysplasia and limb shortening when analyzed radiographically at 6 months of age, in spite of apparent metabolic correction. Moreover, gene therapy with AAV2/9-G6Pase only partially corrected growth in GSD-Ia affected dogs as detected by weight and bone measurements and serum IGF 1 concentrations were persistently low in treated dogs. We also found that heterozygous GSD-Ia carrier dogs had decreased serum IGF 1, adult body weights and bone dimensions compared to wild-type littermates. In sum, these findings suggest that growth failure in GSD-Ia results, at least in part, from hepatic GH resistance. In addition, gene therapy improved growth in addition to promoting long-term survival in dogs and mice with GSD-Ia.

Regulation of NF-kappaB by NSD1/FBXL11-dependent reversible lysine methylation of p65.

NF-kappaB, a central coordinator of immune and inflammatory responses, must be tightly regulated. We describe a NF-kappaB regulatory pathway that is driven by reversible lysine methylation of the p65 subunit, carried out by a lysine methylase, the nuclear receptor-binding SET domain-containing protein 1 (NSD1), and a lysine demethylase, F-box and leucine-rich repeat protein 11 (FBXL11). Overexpression of FBXL11 inhibits NF-kappaB activity, and a high level of NSD1 activates NF-kappaB and reverses the inhibitory effect of FBXL11, whereas reduced expression of NSD1 decreases NF-kappaB activation. The targets are K218 and K221 of p65, which are methylated in cells with activated NF-kappaB. Overexpression of FBXL11 slowed the growth of HT29 cancer cells, whereas shRNA-mediated knockdown had the opposite effect, and these phenotypes were dependent on K218/K221 methylation. In mouse embryo fibroblasts, the activation of most p65-dependent genes relied on K218/K221 methylation. Importantly, expression of the FBXL11 gene is driven by NF-kappaB, revealing a negative regulatory feedback loop. We conclude that reversible lysine methylation of NF-kappaB is an important element in the complex regulation of this key transcription factor.

How cancer cells make and respond to interferon-I.

Acute exposure of cancer cells to high concentrations of type I interferon (IFN-I) drives growth arrest and apoptosis, whereas chronic exposure to low concentrations provides important prosurvival advantages. Tyrosine-phosphorylated IFN-stimulated gene (ISG) factor 3 (ISGF3) drives acute deleterious responses to IFN-I, whereas unphosphorylated (U-)ISGF3, lacking tyrosine phosphorylation, drives essential constitutive prosurvival mechanisms. Surprisingly, programmed cell death-ligand 1 (PD-L1), often expressed on the surfaces of tumor cells and well recognized for its importance in inactivating cytotoxic T cells, also has important cell-intrinsic protumor activities, including dampening acute responses to cytotoxic high levels of IFN-I and sustaining the expression of the low levels that benefit tumors. More thorough understanding of the newly recognized complex roles of IFN-I in cancer may lead to the identification of novel therapeutic strategies.

Oncostatin-M and OSM-Receptor Feed-Forward Activation of MAPK Induces Separable Stem-like and Mesenchymal Programs.

Patients diagnosed with pancreatic ductal adenocarcinoma (PDAC) frequently present with advanced metastatic disease and exhibit a poor response to therapy, resulting in poor outcomes. The tumor microenvironment cytokine Oncostatin-M (OSM) initiates PDAC plasticity, inducing the reprogramming to a stem-like/mesenchymal state, which enhances metastasis and therapy resistance. Using a panel of PDAC cells driven through epithelial-mesenchymal transition (EMT) by OSM or the transcription factors ZEB1 or SNAI1, we find that OSM uniquely induces tumor initiation and gemcitabine resistance independently of its ability to induce a CD44HI/mesenchymal phenotype. In contrast, while ZEB1 and SNAI1 induce a CD44HI/mesenchymal phenotype and migration comparable with OSM, they are unable to promote tumor initiation or robust gemcitabine resistance. Transcriptomic analysis identified that OSM-mediated stemness requires MAPK activation and sustained, feed-forward transcription of OSMR. MEK and ERK inhibitors prevented OSM-driven transcription of select target genes and stem-like/mesenchymal reprogramming, resulting in reduced tumor growth and resensitization to gemcitabine. We propose that the unique properties of OSMR, which hyperactivates MAPK signaling when compared with other IL6 family receptors, make it an attractive therapeutic target, and that disrupting the OSM-OSMR-MAPK feed-forward loop may be a novel way to therapeutically target the stem-like behaviors common to aggressive PDAC. IMPLICATIONS: Small-molecule MAPK inhibitors may effectively target the OSM/OSMR-axis that leads to EMT and tumor initiating properties that promote aggressive PDAC.

Validation-based insertional mutagenesis identifies lysine demethylase FBXL11 as a negative regulator of NFkappaB.

We describe a highly efficient use of lentiviral validation-based insertional mutagenesis (VBIM) to generate large populations of mammalian cells in which a strong promoter is inserted into many different genomic loci, causing greatly increased expression of downstream sequences. Many different selections or screens can follow, to isolate dominant mutant clones with a desired phenotypic change. The inserted promoter can be excised or silenced at will, to prove that the insertion caused the mutation. Cloning DNA flanking the insertion site identifies the locus precisely. VBIM virus particles are pseudotyped with VSV G protein, allowing efficient infection of most mammalian cell types, including non-dividing cells, and features are included that give high yields of stable virus stocks. In several different selections, useful mutants have been obtained at frequencies of approximately 10(-6) or higher. We used the VBIM technique to isolate mutant human cells in which the F-box leucine-rich protein 11 (FBXL11), a histone H3K36 demethylase, is shown to be a negative regulator of NFkappaB. High levels of FBXL11 block the ability of NFkappaB to bind to DNA or activate gene expression, and siRNA-mediated reduction of FBXL11 expression has the opposite effects. The H212A mutation of FBXL11 abolishes both its histone H3K36 demethylase activity and its ability to inhibit NFkappaB. Thus, we have used a powerful tool for mutagenesis of mammalian cells to reveal an aspect of the complex regulation of NFkappaB-dependent signaling.

Validation-Based Insertional Mutagenesis (VBIM), A Powerful Forward Genetic Screening Strategy.

Forward genetics begins with a biological phenotype and attempts to identify genetic changes that influence that phenotype. These changes can be induced in a selected group of genes, for instance, by using libraries of cDNAs, shRNAs, CRISPR guide RNAs, or genetic suppressor elements (GSEs), or randomly throughout the genome using chemical or insertional mutagens, with each approach creating distinct genetic changes. The Validation-Based Insertional Mutagenesis (VBIM) strategy utilizes modified lentiviruses as insertional mutagens, placing strong promoters throughout the genome. Generating libraries with millions of cells carrying one or a few VBIM promoter insertions is straightforward, allowing selection of cells in which overexpression of VBIM-driven RNAs or proteins promote the phenotype of interest. VBIM-driven RNAs may encode full-length proteins, truncated proteins (which may have wild-type, constitutive, or dominant-negative activity), or antisense RNAs that can disrupt gene expression. The diversity in VBIM-driven changes allows for the identification of both gain-of-function and loss-of-function mutations in a single screen. Additionally, VBIM can target any genomic locus, regardless of whether it is expressed in the cells under study or known to have a biological function, allowing for true whole-genome screens without the complication and cost of constructing, maintaining, and delivering a comprehensive library. Here, we review the VBIM strategy and discuss examples in which VBIM has been successfully used in diverse screens to identify novel genes or novel functions for known genes. In addition, we discuss considerations for transitioning the VBIM strategy to in vivo screens. We hope that other laboratories will be encouraged to use the VBIM strategy to identify genes that influence their phenotypes of interest. © 2022 Wiley Periodicals LLC.

DNA synthesis from unbalanced nucleotide pools causes limited DNA damage that triggers ATR-CHK1-dependent p53 activation.

p53-dependent G(1) and G(2) cell cycle checkpoints are activated in response DNA damage that help to maintain genomic stability. p53 also helps to protect cells from damage that occurs during S phase, for example, when the cells are starved for DNA precursors or irradiated with a low dose of UV. p53 is activated in normal cells starved for pyrimidine nucleotides by treatment with N-(phosphonacetyl)-l-aspartate (PALA). The treated cells progress through a first S phase with kinetics similar to those of untreated cells. However, the DNA of the treated cells begins to become damaged rapidly, within 12 h, as revealed by a comet assay, which detects broken DNA, and by staining for phosphorylated histone H2AX, which accumulates at sites of DNA damage. Because the cells survive, the damage must be reversible, suggesting single-strand breaks or gaps as the most likely possibility. The transiently damaged DNA stimulates activation of ATR and CHK1, which in turn catalyze the phosphorylation and accumulation of p53. Although PALA-induced DNA damage occurs only in dividing cells, the p53 that is activated is only competent to transcribe genes such as p21 and macrophage inhibitory cytokine 1 (whose products regulate G(2) and G(1) or S phase checkpoints, respectively) after the cells have exited the S phase during which damage occurs. We propose that p53 is activated by stimulation of mismatch repair in response to the misincorporation of deoxynucleotides into newly synthesized DNA, long before the lack of pyrimidine nucleoside triphosphates causes the rate of DNA synthesis to slow appreciably.

MYO10 drives genomic instability and inflammation in cancer.

Genomic instability is a hallmark of human cancer; yet the underlying mechanisms remain poorly understood. Here, we report that the cytoplasmic unconventional Myosin X (MYO10) regulates genome stability, through which it mediates inflammation in cancer. MYO10 is an unstable protein that undergoes ubiquitin-conjugating enzyme H7 (UbcH7)/ß-transducin repeat containing protein 1 (ß-TrCP1)­dependent degradation. MYO10 is upregulated in both human and mouse tumors and its expression level predisposes tumor progression and response to immune therapy. Overexpressing MYO10 increased genomic instability, elevated the cyclic GMP-AMP synthase (cGAS)/stimulator of interferon genes (STING)­dependent inflammatory response, and accelerated tumor growth in mice. Conversely, depletion of MYO10 ameliorated genomic instability and reduced the inflammation signaling. Further, inhibiting inflammation or disrupting Myo10 significantly suppressed the growth of both human and mouse breast tumors in mice. Our data suggest that MYO10 promotes tumor progression through inducing genomic instability, which, in turn, creates an immunogenic environment for immune checkpoint blockades.

Aberrant Induction of a Mesenchymal/Stem Cell Program Engages Senescence in Normal Mammary Epithelial Cells.

Although frequently associated with tumor progression, inflammatory cytokines initially restrain transformation by inducing senescence, a key tumor-suppressive barrier. Here, we demonstrate that the inflammatory cytokine, oncostatin M, activates a mesenchymal/stem cell (SC) program that engages cytokine-induced senescence (CIS) in normal human epithelial cells. CIS is driven by Snail induction and requires cooperation between STAT3 and the TGFß effector, SMAD3. Importantly, as cells escape CIS, they retain the mesenchymal/SC program and are thereby bestowed with a set of cancer SC (CSC) traits. Of therapeutic importance, cells that escape CIS can be induced back into senescence by CDK4/6 inhibition, confirming that the mechanisms allowing cells to escape senescence are targetable and reversible. Moreover, by combining CDK4/6 inhibition with a senolytic therapy, mesenchymal/CSCs can be efficiently killed. Our studies provide insight into how the CIS barriers that prevent tumorigenesis can be exploited as potential therapies for highly aggressive cancers. IMPLICATIONS: These studies reveal how a normal cell's arduous escape from senescence can bestow aggressive features early in the transformation process, and how this persistent mesenchymal/SC program can create a novel potential targetability following tumor development.

SLX4IP and telomere dynamics dictate breast cancer metastasis and therapeutic responsiveness.

Metastasis is the leading cause of breast cancer-related death and poses a substantial clinical burden owing to a paucity of targeted treatment options. The clinical manifestations of metastasis occur years-to-decades after initial diagnosis and treatment because disseminated tumor cells readily evade detection and resist therapy, ultimately giving rise to recurrent disease. Using an unbiased genetic screen, we identified SLX4-interacting protein (SLX4IP) as a regulator of metastatic recurrence and established its relationship in governing telomere maintenance mechanisms (TMMs). Inactivation of SLX4IP suppressed alternative lengthening of telomeres (ALT), coinciding with activation of telomerase. Importantly, TMM selection dramatically influenced metastatic progression and survival of patients with genetically distinct breast cancer subtypes. Notably, pharmacologic and genetic modulation of TMMs elicited telomere-dependent cell death and prevented disease recurrence by disseminated tumor cells. This study illuminates SLX4IP as a potential predictive biomarker for breast cancer progression and metastatic relapse. SLX4IP expression correlates with TMM identity, which also carries prognostic value and informs treatment selection, thereby revealing new inroads into combating metastatic breast cancers.

Inflammation mobilizes copper metabolism to promote colon tumorigenesis via an IL-17-STEAP4-XIAP axis.

Copper levels are known to be elevated in inflamed and malignant tissues. But the mechanism underlying this selective enrichment has been elusive. In this study, we report a axis by which inflammatory cytokines, such as IL-17, drive cellular copper uptake via the induction of a metalloreductase, STEAP4. IL-17-induced elevated intracellular copper level leads to the activation of an E3-ligase, XIAP, which potentiates IL-17-induced NFκB activation and suppresses the caspase 3 activity. Importantly, this IL-17-induced STEAP4-dependent cellular copper uptake is critical for colon tumor formation in a murine model of colitis-associated tumorigenesis and STEAP4 expression correlates with IL-17 level and XIAP activation in human colon cancer. In summary, this study reveals a IL-17-STEAP4-XIAP axis through which the inflammatory response induces copper uptake, promoting colon tumorigenesis.

p53-mediated growth suppression in response to Nutlin-3 in cyclin D1 transformed cells occurs independently of p21.

Interaction of cyclin D1 with cyclin-dependent kinases (CDK) results in the hyperphosphorylation of the RB family of proteins, thereby inactivating the tumor-suppressive function of RB. Our previous findings suggest that constitutive cyclin D1/CDK activity inhibits p53-mediated gene repression by preventing the appropriate regulation of CDK activity by the CDK inhibitor p21, a transcriptional target of p53. To study the role of cyclin D1 in driving human mammary cell transformation, we expressed a constitutively active cyclin D1-CDK fusion protein (D1/CDK) in immortalized human mammary epithelial cells. D1/CDK-expressing human mammary epithelial cells grew anchorage-independently in the presence of wild-type p53, consistent with the idea that D1/CDK disrupts downstream p53 signaling. Using this transformation model, we examined the sensitivity of the D1/CDK-expressing cells to Nutlin-3, an HDM2 antagonist that activates p53. Surprisingly, treatment of D1/CDK-transformed cells with Nutlin-3 prevented their anchorage-independent growth. The Nutlin-3-induced growth arrest was enforced in D1/CDK-expressing cells despite the presence of hyperphosphorylated RB implicating a p53-dependent, RB-independent mechanism for growth suppression. Further analysis identified that CDC2 and cyclin B1, key cell cycle regulators, were stably down-regulated following p53 stabilization by Nutlin-3, consistent with direct interaction between p53 and the CDC2 and cyclin B1 promoters, leading to the repression of transcription by methylation. In contrast to D1/CDK expression, direct inactivation of p53 resulted in no repression of CDC2 and no cell cycle arrest. We conclude that induction of p53 by Nutlin-3 is a viable therapeutic strategy in cancers with constitutive CDK signaling due to the direct repression of specific p53 target genes.

Nutlin3 blocks vascular endothelial growth factor induction by preventing the interaction between hypoxia inducible factor 1alpha and Hdm2.

Hdm2 is elevated in numerous types of malignancies and is thought to impede the function of wild-type p53. Reactivation of p53 by disrupting the association with Hdm2 was the impetus for the development of Nutlin3. Although regulation of p53 has been the central focus of Hdm2 activity, it also binds other proteins through its p53-binding domain. Here, we show that hypoxia-inducible factor 1alpha (HIF1alpha) binds to Hdm2 in the domain designated to bind p53. HIF1alpha and p53 share a conserved motif that is required to bind Hdm2. Distinct complexes form between Hdm2-HIF1alpha and Hdm2-p53 as determined by immunoprecipitation of nuclear extracts and in vitro. The Hdm2 antagonist Nutlin3 prevents the association between Hdm2 and HIF1alpha. The vascular endothelial growth factor (VEGF) gene is a transcriptional target of HIF1alpha, and under normoxic or hypoxic conditions, Hdm2 increases HIF1alpha activity to induce VEGF production. Blocking the association of Hdm2 and HIF1alpha by Nutlin3, or ablating Hdm2 expression, diminished the level of VEGF under conditions of normoxia or hypoxia. Our findings establish a unique role for Nutlin3 in attenuating VEGF induction by preventing the association of Hdm2 with HIF1alpha.

Balancing STAT Activity as a Therapeutic Strategy.

Driven by dysregulated IL-6 family member cytokine signaling in the tumor microenvironment (TME), aberrant signal transducer and activator of transcription (STAT3) and (STAT5) activation have been identified as key contributors to tumorigenesis. Following transformation, persistent STAT3 activation drives the emergence of mesenchymal/cancer-stem cell (CSC) properties, important determinants of metastatic potential and therapy failure. Moreover, STAT3 signaling within tumor-associated macrophages and neutrophils drives secretion of factors that facilitate metastasis and suppress immune cell function. Persistent STAT5 activation is responsible for cancer cell maintenance through suppression of apoptosis and tumor suppressor signaling. Furthermore, STAT5-mediated CD4+/CD25+ regulatory T cells (Tregs) have been implicated in suppression of immunosurveillance. We discuss these roles for STAT3 and STAT5, and weigh the attractiveness of different modes of targeting each cancer therapy. Moreover, we discuss how anti-tumorigenic STATs, including STAT1 and STAT2, may be leveraged to suppress the pro-tumorigenic functions of STAT3/STAT5 signaling.