Pro-oncogenic Functions Research Articles

A Small Molecule Agonist of EphA2 Receptor Tyrosine Kinase Inhibits Tumor Cell Migration In Vitro and Prostate Cancer Metastasis In Vivo

During tumor progression, EphA2 receptor can gain ligand-independent pro-oncogenic functions due to Akt activation and reduced ephrin-A ligand engagement. The effects can be reversed by ligand stimulation, which triggers the intrinsic tumor suppressive signaling pathways of EphA2 including inhibition of PI3/Akt and Ras/ERK pathways. These observations argue for development of small molecule agonists for EphA2 as potential tumor intervention agents. Through virtual screening and cell-based assays, we report here the identification and characterization of doxazosin as a novel small molecule agonist for EphA2 and EphA4, but not for other Eph receptors tested. NMR studies revealed extensive contacts of doxazosin with EphA2/A4, recapitulating both hydrophobic and electrostatic interactions recently found in the EphA2/ephrin-A1 complex. Clinically used as an α1-adrenoreceptor antagonist (Cardura®) for treating hypertension and benign prostate hyperplasia, doxazosin activated EphA2 independent of α1-adrenoreceptor. Similar to ephrin-A1, doxazosin inhibited Akt and ERK kinase activities in an EphA2-dependent manner. Treatment with doxazosin triggered EphA2 receptor internalization, and suppressed haptotactic and chemotactic migration of prostate cancer, breast cancer, and glioma cells. Moreover, in an orthotopic xenograft model, doxazosin reduced distal metastasis of human prostate cancer cells and prolonged survival in recipient mice. To our knowledge, doxazosin is the first small molecule agonist of a receptor tyrosine kinase that is capable of inhibiting malignant behaviors in vitro and in vivo.

Pro-malignant properties of STAT3 during chronic inflammation

Already in 1863 Rudolph Virchow hypothesized that some classes of irritants, together with tissue injury, enhance cell proliferation and that cancer arises from sites of chronic inflammation. Although now we know that cell proliferation is not a cause of cancer on itself, for the first time his hypothesis suggested a link between cell transformation and inflammation. Many tumors contain activated fibroblasts and macrophages displaying an inflammatory gene expression profile. Interestingly, quantitative aspects of wound repair or inflammatory gene expression often negatively correlate with cancer stage and prognosis: in a sense, tumors act as wounds that fail to heal [1]. Chronic inflammatory states may generate a microenvironment favoring genomic lesions and fostering tumor initiation. The presence of free radicals, such as reactive oxygen intermediates and nitric oxide, leads to oxidative damage and nitration of DNA bases, which in turn increases the risk of mutations. Moreover, the soluble mediators secreted by inflammatory cells such as cytokines and growth factors provide survival and proliferative signals to initiated cells, thereby leading to tumor promotion/progression. More detailed insights into the role of inflammation in tumor promotion come from several studies involving the transcription factor NF-κB. For example, NF-κB inactivation dramatically decreased tumor size of myeloid cells in a colitis-associated cancer model, reducing the expression of pro-inflammatory cytokines that may serve as tumor growth factors [2]. NF-κB is a ubiquitous transcription factor that regulates genes involved in native and adaptive immune responses. Importantly, NF-κB is often aberrantly activated in human cancers, up-regulating genes involved in the control of survival and proliferation, and is thus considered an important target for drug therapies [3]. A prominent NF-κB target is the gene encoding for the pro-inflammatory cytokine IL-6, which directly affects cancer cells growth and survival through the activation of another transcription factor, the Signal Transducer and Activator of Transcription (STAT) 3. Indeed, chronic inflammation initiates a positive loop between the transcription factor NF-κB, IL-6 and STAT3 that is a highly predisposing condition for cancer, particularly in the colon, the liver and the breast [4]. STAT3 is constitutively activated by phosphorylation on tyrosine in many tumors that often become addicted to its activity [5], and is accordingly often referred to as an oncogene, even though activating mutations are rare. Importantly, STAT3 is prominently constitutively activated at sites of chronic inflammation, where IL-6 levels are invariably high. In an effort to characterize the pro-oncogenic functions of continuous, weak STAT3 activation, we have recently generated knock-in mice expressing physiological levels of the constitutively active STAT3C mutant form [6]. Primary mouse embryonic fibroblasts (MEFs) derived from the STAT3C/C mice show pre-malignant features, such as increased glycolysis, resistance to apoptosis and senescence and accelerated proliferation [7]. When challenged with a second ‘random’ mutation induced through the 3T3 spontaneous immortalization protocol, STAT3C/C MEFs become fully transformed and are able to form tumors in immunocompromised mice [8]. STAT3C/C cells display an accelerated cell cycle, protection from apoptosis and enhanced HIF-1α-dependent aerobic glycolysis. HIF-1α silencing normalizes their glycolysis levels, correlating with decreased cell proliferation and growth, both in vitro and in vivo. This finding is of particular relevance for the emerging key role of STAT3 in inflammation-driven cancer. Therefore, in addition to the tumor promotion role described above in coordination with IL-6 and NF-κB, our data suggest that cells exposed to chronic IL-6 signaling, which leads to continuous STAT3 activation like that displayed by the STAT3C/C MEFs, can behave like cells that have undergone a first oncogenic mutation. This first hit provides survival and proliferative signals by inducing pro-proliferative and anti-apoptotic genes and switching cell metabolism towards aerobic glycolysis, believed to sustain the anabolic metabolism required by tumor cells. All these features contribute to a pre-malignant state where a second mutation is sufficient to provide full cell transformation. Interestingly, a state of chronic inflammation with IL-6 accumulation develops with age in mice, primates and humans [9]. This may in turn result in increasingly high chronic STAT3 activation and thus the development of STAT3-dependent tumors. Therapeutic strategies focusing on STAT3 modulation could therefore dramatically decrease the incidence of age-related cancers, lowering the accumulation of the pre-malignant cells pool with aging.

The critical roles of endoplasmic reticulum chaperones and unfolded protein response in tumorigenesis and anticancer therapies

Cancer progression is characterized by rapidly proliferating cancer cells that are in need of increased protein synthesis. Therefore, enhanced endoplasmic reticulum (ER) activity is required to facilitate the folding, assembly and transportation of membrane and secretory proteins. These functions are carried out by ER chaperones. It is now becoming clear that the ER chaperones have critical functions outside of simply facilitating protein folding. For example, cancer progression requires glucose regulated protein (GRP) 78 for cancer cell survival and proliferation, as well as angiogenesis in the microenvironment. GRP78 can translocate to the cell surface acting as a receptor regulating oncogenic signaling and cell viability. Calreticulin, another ER chaperone, can translocate to the cell surface of apoptotic cancer cells and induce immunogenic cancer cell death and antitumor responses in vivo. Tumor-secreted GRP94 has been shown to elicit antitumor immune responses when used as antitumor vaccines. Protein disulfide isomerase is another ER chaperone that demonstrates pro-oncogenic and pro-survival functions. Because of intrinsic alterations of cellular metabolism and extrinsic factors in the tumor microenvironment, cancer cells are under ER stress, and they respond to this stress by activating the unfolded protein response (UPR). Depending on the severity and duration of ER stress, the signaling branches of the UPR can activate adaptive and pro-survival signals, or induce apoptotic cell death. The protein kinase RNA-like ER kinase signaling branch of the UPR has a dual role in cancer proliferation and survival, and is also required for ER stress-induced autophagy. The activation of the inositol-requiring kinase 1α branch promotes tumorigenesis, cancer cell survival and regulates tumor invasion. In summary, perturbance of ER homeostasis has critical roles in tumorigenesis, and therapeutic modulation of ER chaperones and/or UPR components presents potential antitumor treatments.

Regulation of Tumor Suppressor p53 and HCT116 Cell Physiology by Histone Demethylase JMJD2D/KDM4D

JMJD2D, also known as KDM4D, is a histone demethylase that removes methyl moieties from lysine 9 on histone 3 and from lysine 26 on histone 1.4. Here, we demonstrate that JMJD2D forms a complex with the p53 tumor suppressor in vivo and interacts with the DNA binding domain of p53 in vitro. A luciferase reporter plasmid driven by the promoter of p21, a cell cycle inhibitor and prominent target gene of p53, was synergistically activated by p53 and JMJD2D, which was dependent on JMJD2D catalytic activity. Likewise, overexpression of JMJD2D induced p21 expression in U2OS osteosarcoma cells in the absence and presence of adriamycin, an agent that induces DNA damage. Furthermore, downregulation of JMJD2D inhibited cell proliferation in wild-type and even more so in p53−/− HCT116 colon cancer cells, suggesting that JMJD2D is a pro-proliferative molecule. JMJD2D depletion also induced more strongly apoptosis in p53−/− compared to wild-type HCT116 cells. Collectively, our results demonstrate that JMJD2D can stimulate cell proliferation and survival, suggesting that its inhibition may be helpful in the fight against cancer. Furthermore, our data imply that activation of p53 may represent a mechanism by which the pro-oncogenic functions of JMJD2D become dampened.

Key contribution of CPEB4-mediated translational control to cancer progression.

Malignant transformation, invasion and angiogenesis rely on the coordinated reprogramming of gene expression in the cells from which the tumor originated. Although deregulated gene expression has been extensively studied at genomic and epigenetic scales, the contribution of the regulation of mRNA-specific translation to this reprogramming is not well understood. Here we show that cytoplasmic polyadenylation element binding protein 4 (CPEB4), an RNA binding protein that mediates meiotic mRNA cytoplasmic polyadenylation and translation, is overexpressed in pancreatic ductal adenocarcinomas and glioblastomas, where it supports tumor growth, vascularization and invasion. We also show that, in pancreatic tumors, the pro-oncogenic functions of CPEB4 originate in the translational activation of mRNAs that are silenced in normal tissue, including the mRNA of tissue plasminogen activator, a key contributor to pancreatic ductal adenocarcinoma malignancy. Taken together, our results document a key role for post-transcriptional gene regulation in tumor development and describe a detailed mechanism for gene expression reprogramming underlying malignant tumor progression.

3′UTR elements inhibit Ras-induced C/EBPβ post-translational activation and senescence in tumour cells

C/EBPβ is an auto-repressed protein that becomes post-translationally activated by Ras-MEK-ERK signalling. C/EBPβ is required for oncogene-induced senescence (OIS) of primary fibroblasts, but also displays pro-oncogenic functions in many tumour cells. Here, we show that C/EBPβ activation by H-Ras(V12) is suppressed in immortalized/transformed cells, but not in primary cells, by its 3' untranslated region (3'UTR). 3'UTR sequences inhibited Ras-induced cytostatic activity of C/EBPβ, DNA binding, transactivation, phosphorylation, and homodimerization, without significantly affecting protein expression. The 3'UTR suppressed induction of senescence-associated C/EBPβ target genes, while promoting expression of genes linked to cancers and TGFβ signalling. An AU-rich element (ARE) and its cognate RNA-binding protein, HuR, were required for 3'UTR inhibition. These components also excluded the Cebpb mRNA from a perinuclear cytoplasmic region that contains activated ERK1/2, indicating that the site of C/EBPβ translation controls de-repression by Ras signalling. Notably, 3'UTR inhibition and Cebpb mRNA compartmentalization were absent in primary fibroblasts, allowing Ras-induced C/EBPβ activation and OIS to proceed. Our findings reveal a novel mechanism whereby non-coding mRNA sequences selectively regulate C/EBPβ activity and suppress its anti-oncogenic functions.

Response to Thum et al.

Thum et al. conclude that microRNA-21 (miR-21) is essential for cardiac hypertrophy and fibrosis in response to pressure overload (1). They also claim that our failure to observe a blockade to these processes in mice treated with an 8-mer locked nucleic acid–modified oligonucleo­tide against miR-21 (called Anti-21) (2) is due to the ineffectiveness of such inhibitors. We wish to point out several caveats to their study regarding the role of miR-21 in cardiac hypertrophy and their conclusions regarding the efficacy of the Anti-21 oligonucleotide. First, we find that Anti-21 inhibits miR-21 with a half-maximal inhibitory concentration of 0.9 nM, indicating the efficacy of Anti-21. Second, Thum et al. do not state the method they used to measure miR-21 inhibition, though we assume it to be quantitative PCR (qPCR). In our hands, qPCR alone is unreliable for measuring miRNA inhibition, especially for 8-mer inhibitors, since they may be displaced during qPCR and thereby give an underrepresentation of miRNA inhibition. To demonstrate functional inhibition of a miRNA, it is important to show data from multiple assays, such as small RNA Northern blots, luciferase reporter assays, and target derepression, as shown in our study (2). Such data are lacking in the Thum et al. rebuttal, which makes comparison of the different chemistries impossible. Thum et al. also state that we measured miR-21 inhibition on day 2 after dosing with Anti-21, when in fact we measured inhibition 3 weeks after dosing. At this time point, we observed inhibition of miR-21 in pressure-overloaded hearts at a level significantly below that of control mice. Thus, their approach for inhibition and/or measurement of miR-21 by their 8-mer inhibitors differs markedly from ours, since we observed robust miR-21 inhibition 3 weeks after injection, as demonstrated by multiple readouts (2). Using mismatched oligonucleotide controls is also important for interpreting miRNA inhibition studies in vivo, as described in our paper, rather than using PBS as a control, as reported by Thum et al. (1). Finally, Thum et al. postulate that constitutive genetic deletion of miR-21 in mice may not reveal the functions of miR-21 in cardiac disease because of compensatory events that mask such functions. If such compensation occurs, it must be specific for the cardiac functions of miR-21, since miR-21 null mice are resistant to lung tumorigenesis (3), consistent with the documented pro-oncogenic functions of miR-21. To further address the possibility of genetic compensation, we have deleted a floxed miR-21 allele immediately prior to thoracic aortic constriction in mice using a ubiquitously expressed tamoxifen-regulated Cre transgene. These animals show cardiac hypertrophy and fibrosis comparable to that of their Cre-negative littermates. Genetic compensation therefore cannot account for the normal pathological cardiac remodeling response in miR-21 null mice. Moreover, functions of other miRNAs in heart disease can be revealed by genetic deletion in mice, as shown for miR-208 (4). Thus, while 22-mer oligonucleotide inhibitors against miR-21 are efficacious in inhibiting cardiac hypertrophy (1), other loss-of-function approaches appear ineffective. We remain enthusiastic about miRNAs as therapeutic targets and welcome such dialog.

SnoN in mammalian development, function and diseases

SnoN (Ski-novel protein) was discovered as a nuclear proto-oncogene on the basis of its ability to induce transformation of chicken and quail embryonic fibroblasts. As a crucial negative regulator of transforming growth factor-β (TGF-β) signaling and also an activator of p53, it plays an important role in regulating cell proliferation, senescence, apoptosis, and differentiation. Recent studies of its expression patterns and functions in mouse models and mammalian cells have revealed important functions of SnoN in normal epithelial development and tumorigenesis. Evidence suggests that SnoN has both pro-oncogenic and anti-oncogenic functions by modulating multiple signaling pathways. These studies suggest that SnoN may have broad functions in the development and homeostasis of embryonic and postnatal tissues.

The zinc-finger protein KCMF1 is overexpressed during pancreatic cancer development and downregulation of KCMF1 inhibits pancreatic cancer development in mice

Potassium channel modulatory factor 1 (KCMF1) was found upregulated in a differential screen in the metaplastic epithelium in the pancreas of transforming growth factor (TGF)-alpha transgenic mice. Expression analysis indicated broad overexpression in human cancer tissues. Therefore, we investigated the hypothesis that KCMF1 promotes metaplastic changes and tumor development. KCMF1 represents an evolutionarily highly conserved protein with a 95% identity between human and zebrafish. KCMF1 is expressed during embryonic development and in the majority of adult tissues investigated. Upregulation of nuclear KCMF1 expression is evident in preneoplastic lesions and in several epithelial malignancies, such as pancreatic cancer in mice and humans. In cell culture and in the chicken chorioallantoic membrane model, KCMF1 enhances proliferation, migration and invasion of HEK-293 and Panc1 cells. In crossbreeding experiments, KCMF1-knockdown gene trap mice showed a reduced number and size of premalignant lesions and absence of pancreatic cancer formation in TGF-alpha transgenic mice. This effect is related to the decreased expression of G1 to S cell-cycle regulators such as cyclin D and cyclin-dependent kinase (CDK) 4. Our data support the hypothesis that KCMF1 mediates pro-oncogenic functions in vitro and in vivo and downregulation of KCMF1 results in the inhibition of pancreatic cancer formation in mice. These effects are mediated through downregulation of cell-cycle control genes such as cyclin D and CDK4.

Targeting the transforming growth factor-β signaling pathway in human cancer

The transforming growth factor-ß (TGF-β) signaling pathway plays a pivotal role in diverse cellular processes. TGF-β switches its role from a tumor suppressor in normal or dysplastic cells to a tumor promoter in advanced cancers. It is widely believed that the Smad-dependent pathway is involved in TGF-β tumor-suppressive functions, whereas activation of Smad-independent pathways, coupled with the loss of tumor-suppressor functions of TGF-β, is important for its pro-oncogenic functions. TGF-β signaling has been considered a useful therapeutic target. The discovery of oncogenic actions of TGF-β has generated a great deal of enthusiasm for developing TGF-β signaling inhibitors for the treatment of cancer. The challenge is to identify the group of patients where targeted tumors are not only refractory to TGF-β-induced tumor suppressor functions but also responsive to the tumor-promoting effects of TGF-β. TGF-β pathway inhibitors, including small and large molecules, have now entered clinical trials. Preclinical studies with these inhibitors have shown promise in a variety of different tumor models. Here, we focus on the mechanisms of signaling and specific targets of the TGF-β pathway that are critical effectors of tumor progression and invasion. This report also examines the therapeutic intervention of TGF-ß signaling in human cancers.

Conditional overexpression of Stat3α in differentiating myeloid cells results in neutrophil expansion and induces a distinct, antiapoptotic and pro-oncogenic gene expression pattern

Normal neutrophil development requires G-CSF signaling, which includes activation of Stat3. Studies of G-CSF-mediated Stat3 signaling in cell culture and transgenic mice have yielded conflicting data regarding the role of Stat3 in myelopoiesis. The specific functions of Stat3 remain unclear, in part, because two isoforms, Stat3alpha and Stat3beta, are expressed in myeloid cells. To understand the contribution of each Stat3 isoform to myelopoiesis, we conditionally overexpressed Stat3alpha or Stat3beta in the murine myeloid cell line 32Dcl3 (32D) and examined the consequences of overexpression on cell survival and differentiation. 32D cells induced to overexpress Stat3alpha, but not Stat3beta, generated a markedly higher number of neutrophils in response to G-CSF. This effect was a result of decreased apoptosis but not of increased proliferation. Comparison of gene expression profiles of G-CSF-stimulated, Stat3alpha-overexpressing 32D cells with those of cells with normal Stat3alpha expression revealed novel Stat3 gene targets, which may contribute to neutrophil expansion and improved survival, most notably Slc28a2, a purine nucleoside transporter, which is critical for maintenance of intracellular nucleotide levels and prevention of apoptosis, and Gpr65, an acid-sensing, G protein-coupled receptor with pro-oncogenic and antiapoptotic functions.

The Role of TGF-β in Epithelial Malignancy and its Relevance to the Pathogenesis of Oral Cancer (Part II)

The role of transforming growth factor-beta (TGF-beta) in epithelial malignancy is complex, but it is becoming clear that, in the early stages of carcinogenesis, the protein acts as a potent tumor suppressor, while later, TGF-beta can function to advance tumor progression. We review the evidence to show that the pro-oncogenic functions of TGF-beta are associated with (1) a partial loss of response to the ligand, (2) defects of components of the TGF-beta signal transduction pathway, (3) over-expression and/or activation of the latent complex, (4) epithelial-mesenchymal transition, and (5) recruitment of signaling pathways which act in concert with TGF-beta to facilitate the metastatic phenotype. These changes are viewed in the context of what is known about the pathogenesis of oral cancer and whether this knowledge can be translated into the development of new therapeutic modalities.

A mutant P53 can activate apoptosis through a mechanism distinct from those induced by wild type P53

A common mutation in P53 protein occurs at amino acid residue 281 in the DNA binding domain (P53 gly(281)), which results in loss of transcriptional regulation of P53 target genes and has been reported to gain pro-oncogenic functions. In the present study, we investigated the activity of P53 gly(281) in P53-null PC3 human prostate cancer cells and found that the P53 gly(281) induced apoptosis as efficiently as the wild-type P53 (wtP53). However, in contrast to wtP53-induced apoptosis, the P53 gly(281)-induced apoptosis was insensitive to overexpression of bcl-2. Thus, our findings indicate that while a mutation in the DNA binding domain of p53 may result in a more oncogenic form of the protein, it may also paradoxically result in the ‘gain’ of a new, alternative pathway for apoptosis.