Courtney Schaal

Nicotine-Mediated Cell Proliferation and Tumor Progression in Smoking-Related Cancers

Tobacco smoke contains multiple classes of established carcinogens including benzo(a)pyrenes, polycyclic aromatic hydrocarbons, and tobacco-specific nitrosamines. Most of these compounds exert their genotoxic effects by forming DNA adducts and generation of reactive oxygen species, causing mutations in vital genes such as K-Ras and p53. In addition, tobacco-specific nitrosamines can activate nicotinic acetylcholine receptors (nAChR) and to a certain extent β-adrenergic receptors (β-AR), promoting cell proliferation. Furthermore, it has been demonstrated that nicotine, the major addictive component of tobacco smoke, can induce cell-cycle progression, angiogenesis, and metastasis of lung and pancreatic cancers. These effects occur mainly through the α7-nAChRs, with possible contribution from the β-ARs and/or epidermal growth factor receptors. This review article will discuss the molecular mechanisms by which nicotine and its oncogenic derivatives such as 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and N-nitrosonornicotine induce cell-cycle progression and promote tumor growth. A variety of signaling cascades are induced by nicotine through nAChRs, including the mitogen-activated protein kinase/extracellular signal-regulated kinase pathway, phosphoinositide 3-kinase/AKT pathway, and janus-activated kinase/STAT signaling. In addition, studies have shown that nAChR activation induces Src kinase in a β-arrestin-1-dependent manner, leading to the inactivation of Rb protein and resulting in the expression of E2F1-regulated proliferative genes. Such nAChR-mediated signaling events enhance the proliferation of cells and render them resistant to apoptosis induced by various agents. These observations highlight the role of nAChRs in promoting the growth and metastasis of tumors and raise the possibility of targeting them for cancer therapy. Mol Cancer Res; 12(1); 14–23. ©2014 AACR.

YAP1 Regulates OCT4 Activity and SOX2 Expression to Facilitate Self‐Renewal and Vascular Mimicry of Stem‐Like Cells

Non‐small cell lung cancer (NSCLC) is highly correlated with smoking and has very low survival rates. Multiple studies have shown that stem‐like cells contribute to the genesis and progression of NSCLC. Our results show that the transcriptional coactivator yes‐associated protein 1 (YAP1), which is the oncogenic component of the Hippo signaling pathway, is elevated in the stem‐like cells from NSCLC and contributes to their self‐renewal and ability to form angiogenic tubules. Inhibition of YAP1 by a small molecule or depletion of YAP1 by siRNAs suppressed self‐renewal and vascular mimicry of stem‐like cells. These effects of YAP1 were mediated through the embryonic stem cell transcription factor, Sox2. YAP1 could transcriptionally induce Sox2 through a physical interaction with Oct4; Sox2 induction occurred independent of TEAD2 transcription factor, which is the predominant mediator of YAP1 functions. The binding of Oct4 to YAP1 could be detected in cell lines as well as tumor tissues; the interaction was elevated in NSCLC samples compared to normal tissue as seen by proximity ligation assays. YAP1 bound to Oct4 through the WW domain, and a peptide corresponding to this region could disrupt the interaction. Delivery of the WW domain peptide to stem‐like cells disrupted the interaction and abrogated Sox2 expression, self‐renewal, and vascular mimicry. Depleting YAP1 reduced the expression of multiple epithelial‐mesenchymal transition genes and prevented the growth and metastasis of tumor xenografts in mice; overexpression of Sox2 in YAP1 null cells rescued these functions. These results demonstrate a novel regulation of stem‐like functions by YAP1, through the modulation of Sox2 expression. Stem Cells 2015;33:1705–1718

The Rb–E2F Transcriptional Regulatory Pathway in Tumor Angiogenesis and Metastasis

The retinoblastoma tumor suppressor protein Rb plays a major role in regulating G1/S transition and is a critical regulator of cell proliferation. Rb protein exerts its growth regulatory properties mainly by physically interacting with the transcriptionally active members of the E2F transcription factor family, especially E2Fs 1, 2, and 3. Given its critical role in regulating cell proliferation, it is not surprising that Rb is inactivated in almost all tumors, either through the mutation of Rb gene itself or through the mutations of its upstream regulators including K-Ras and INK4. Recent studies have revealed a significant role for Rb and its downstream effectors, especially E2Fs, in regulating various aspects of tumor progression, angiogenesis, and metastasis. Thus, components of the Rb-E2F pathway have been shown to regulate the expression of genes involved in angiogenesis, including VEGF and VEGFR, genes involved in epithelial-mesenchymal transition including E-cadherin and ZEB proteins, and genes involved in invasion and migration like matrix metalloproteinases. Rb has also been shown to play a major role in the functioning of normal and cancer stem cells; further, Rb and E2F appear to play a regulatory role in the energy metabolism of cancer cells. These findings raise the possibility that mutational events that initiate tumorigenesis by inducing uncontrolled cell proliferation might also contribute to the progression and metastasis of cancers through the mediation of the Rb-E2F transcriptional regulatory pathway. This review highlights these recent studies on tumor promoting functions of the Rb-E2F pathway.

Mammalian lysine histone demethylase KDM2A regulates E2F1-mediated gene transcription in breast cancer cells.

It is established that histone modifications like acetylation, methylation, phosphorylation and ubiquitination affect chromatin structure and modulate gene expression. Lysine methylation/demethylation on Histone H3 and H4 is known to affect transcription and is mediated by histone methyl transferases and histone demethylases. KDM2A/JHDM1A/FBXL11 is a JmjC-containing histone demethylase that targets mono- and dimethylated Lys36 residues of Histone H3; its function in breast cancer is not fully understood. Here we show that KDM2A is strongly expressed in myoepithelial cells (MEPC) in breast cancer tissues by immunohistochemistry. Ductal cells from ductal carcinoma in situ (DCIS) and infiltrating ductal carcinoma (IDC) show positive staining for KDM2A, the expression decreases with disease progression to metastasis. Since breast MEPCs have tumor-suppressive and anti-angiogenic properties, we hypothesized that KDM2A could be contributing to some of these functions. Silencing KDM2A with small interfering RNAs demonstrated increased invasion and migration of breast cancer cells by suppressing a subset of matrix metalloproteinases (MMP-2, -9, -14 and -15), as seen by real-time PCR. HUVEC cells showed increased angiogenic tubule formation ability in the absence of KDM2A, with a concomitant increase in the expression of VEGF receptors, FLT-1 and KDR. KDM2A physically bound to both Rb and E2F1 in a cell cycle dependent manner and repressed E2F1 transcriptional activity. Chromatin immunoprecipitation (ChIP) assays revealed that KDM2A associates with E2F1-regulated proliferative promoters CDC25A and TS in early G-phase and dissociates in S-phase. Further, KDM2A could also be detected on MMP9, 14 and 15 promoters, as well as promoters of FLT1 and KDR. KDM2A could suppress E2F1-mediated induction of these promoters in transient transfection experiments. These results suggest a regulatory role for KDM2A in breast cancer cell invasion and migration, through the regulation of E2F1 function.

The Role of nAChR and Calcium Signaling in Pancreatic Cancer Initiation and Progression

Pancreatic cancer shows a strong correlation with smoking and the current therapeutic strategies have been relatively ineffective in improving the survival of patients. Efforts have been made over the past many years to understand the molecular events that drive the initiation and progression of pancreatic cancer, especially in the context of smoking. It has become clear that components of tobacco smoke not only initiate these cancers, especially pancreatic ductal adenocarcinomas (PDACs) through their mutagenic properties, but can also promote the growth and metastasis of these tumors by stimulating cell proliferation, angiogenesis, invasion and epithelial-mesenchymal transition. Studies in cell culture systems, animal models and human samples have shown that nicotinic acetylcholine receptor (nAChR) activation enhances these tumor-promoting events by channeling signaling through multiple pathways. In this context, signaling through calcium channels appear to facilitate pancreatic cancer growth by itself or downstream of nAChRs. This review article highlights the role of nAChR downstream signaling events and calcium signaling in the growth, metastasis as well as drug resistance of pancreatic cancer.

Nicotine-Mediated Regulation of Nicotinic Acetylcholine Receptors in Non-Small Cell Lung Adenocarcinoma by E2F1 and STAT1 Transcription Factors

Cigarette smoking is the major risk factor for non-small cell lung cancer (NSCLC), which accounts for 80% of all lung cancers. Nicotine, the addictive component of tobacco smoke, can induce proliferation, migration, invasion, epithelial-mesenchymal transition (EMT), angiogenesis, and survival in NSCLC cell lines, as well as growth and metastasis of NSCLC in mice. This nicotine-mediated tumor progression is facilitated through activation of nicotinic acetylcholine receptors (nAChRs), specifically the α7 subunit; however, how the α7 nAChR gene is regulated in lung adenocarcinoma is not fully clear. Here we demonstrate that the α7 nAChR gene promoter is differentially regulated by E2F and STAT transcription factors through a competitive interplay; E2F1 induces the promoter, while STAT transcription factors repress it by binding to an overlapping site at a region -294 through -463bp upstream of the transcription start site. Treatment of cells with nicotine induced the mRNA and protein levels of α7 nAChR; this could be abrogated by treatment with inhibitors targeting Src, PI3K, MEK, α7 nAChR, CDK4/6 or a disruptor of the Rb-Raf-1 interaction. Further, nicotine–mediated induction of α7 nAChR was reduced when E2F1 was depleted and in contrast elevated when STAT1 was depleted by siRNAs. Interestingly, extracts from e-cigarettes, which have recently emerged as healthier alternatives to traditional cigarette smoking, can also induce α7 nAChR expression in a manner similar to nicotine. These results suggest an autoregulatory feed-forward loop that induces the levels of α7 nAChR upon exposure to nicotine, which enhances the strength of the signal. It can be imagined that such an induction of α7 nAChR contributes to the tumor-promoting functions of nicotine.

Abstract 2487: Nicotine-mediated regulation of Sox2 and stemness in non-small cell lung cancer

Lung cancer is the leading cause of cancer deaths in both men and women, and is highly correlated with cigarette smoking. Nicotine, the addictive component of tobacco smoke, cannot initiate tumors, but can promote proliferation, migration, and invasion of cells in vitro and promote growth and metastasis of tumors in vivo. More recently, nicotine has been implicated in the promoting self-renewal of stem-like side-population cells from lung cancers. This subpopulation of cancer stem-like cells has been implicated in tumor initiation, generation of entire heterogeneous tumor population, metastasis, dormancy, and drug resistance. We have shown that nicotine promotes the expression of genes that promote stemness, such as Stem Cell Factor (SCF/c-kit ligand), in a specific receptor dependent fashion. Here we demonstrate that nicotine can induce the expression of embryonic stem cell factor Sox2, which is indispensable for self-renewal and maintenance of stem cell properties in non-small cell lung adenocarcinoma cells. High level of Sox2 expression also correlates with poor patient survival outcome in lung cancer, and metastatic lung adenocarcinoma shows higher levels of Sox2 expression than primary tumor or normal tissue, implicating Sox2 in this disease. Nicotine was found to induce Sox2 in A549 cells after 18 and 24 hours of stimulation, an effect which was diminished by 48 hours. This was seen at the mRNA and protein level as assessed by transient transfection and luciferase assay, real-time PCR, western blot, and immunofluorescence respectively. Experiments were conducted to elucidate the mechanism by which nicotine induces Sox2; it was found that siRNA mediated depletion of α7 nicotinic acetylcholine receptor (nAChR), the Hippo pathway effector Yap1, or the transcriptional repressor ID1 could abrogate nicotine-mediated induction of Sox2. Further, treatment with inhibitors of Src kinase or PI3K additionally abrogated the effect, suggesting these proteins play a role in nicotine-mediate induction of Sox2. Interestingly, nicotine could induce Sox2 expression in human mesenchymal stem cells (hMSCs) after 24 hours of stimulation as seen by immunofluorescence and real-time PCR, and this effect was abrogated by treatment with inhibitors to Src kinase, PI3K, Yap1, or α7 nAChR. Additionally, studies are under way to elucidate whether e-cigarettes or flavored tobacco, newer and less studied alternatives to traditional cigarettes, have similar effects on stemness. These studies can be expected to have a direct impact on our understanding of the molecular mechanisms involved in the initiation, growth, and metastasis of non-small cell lung cancer, especially in smokers and tobacco users. Citation Format: Courtney Schaal, Srikumar Chellappan. Nicotine-mediated regulation of Sox2 and stemness in non-small cell lung cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2487.

Abstract 1803: Transcriptional regulation of nicotinic acetylcholine receptors (nAChRs) by E2F family transcription factors in non-small cell lung cancer.

Nicotine, the addictive component of cigarette smoke, has been shown to promote cell proliferation, migration, invasion, and angiogenesis in multiple cancer types. Earlier studies from our lab had shown that nicotine can promote the growth and metastasis of non-small cell lung carcinoma (NSCLC) in mouse models. While a broad range of nAChRs have been found to be expressed on NSCLC cell lines, nicotine-mediated proliferation, invasion, and migration are facilitated predominantly through the α7 subunit. Consistent with this, α7 nAChR levels are elevated in NSCLC in mice that were administered nicotine. Stimulation of α7 nAChR with nicotine has been reported to activate Src, resulting in inactivation of the retinoblastoma (Rb) tumor suppressor protein and enhancing E2F-mediated transcription. The Rb-E2F transcriptional regulation pathway is known to induce genes involved in cell cycle progression, angiogenesis, and metastasis implicating it in tumor survival and progression. Given this background, attempts were made to elucidate whether nAChR genes are regulated by the Rb-E2F pathway. Analysis of a 2000bp promoter region of the human α7 gene revealed the presence of multiple E2F binding sites. Transient transfection experiments showed α7 to be responsive to multiple E2Fs. E2F1 was found to associate with the α7 promoter via chromatin immunoprecipitation assays. Depletion of E2F genes via small interfering RNA demonstrated a differential regulation of α7 by E2F family members 1-5. These results raise the possibility that exposure to nicotine stimulates the α7 signaling cascade resulting in elevated E2F1-mediated activation of various proliferative promoters, including that of α7 itself in a positive feedback mechanism. In addition to the α7 nAChR, recent genome wide association studies (GWAS) have identified a susceptibility locus for human lung cancer at 15q25.1 which encodes for nAChR subunits α3 and α5. Promoter analysis has revealed that both α3 and α5 have multiple potential E2F binding sites, as well. Preliminary knock down of E2F family members via small interfering RNA has suggested these subunits may also be differentially regulated by the E2F family of transcription factors. Further studies are under way to elucidate the role of E2F in the regulation of nAChR α3 and α5, and how this impacts nicotine signaling as well as growth and progression of NSCLC. Citation Format: Courtney M. Schaal, Smitha Pillai, Jackie L. Johnson, Srikumar Chellappan. Transcriptional regulation of nicotinic acetylcholine receptors (nAChRs) by E2F family transcription factors in non-small cell lung cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1803. doi:10.1158/1538-7445.AM2013-1803