Previous and Ongoing Investigative Work
As a Physician-Scientist, I have been active in both clinical and basic scientific research. Following completion of my post-doctoral training in the laboratory of Ethan Dmitrovsky at Memorial Sloan-Kettering Cancer Center, I was recruited to MD Anderson Cancer Center in 1993 by Waun Ki Hong for the purpose of building a retinoid-based clinical chemoprevention program in lung cancer and a basic science research effort to elucidate the genetic and biochemical basis of lung cancer development. Early discoveries from my laboratory elucidated fundamental mechanisms of cross-talk between MAP kinases and retinoid nuclear receptors that control the sensitivity of the bronchial epithelium to retinoic acid treatment, which informed findings from our retinoid-based lung cancer chemoprevention trials. These studies evolved from interests I developed during post-doctoral training on the transcriptional basis of retinoid-induced tumor differentiation, where we were among the first to translate the discovery of retinoic acid nuclear receptors (RARs), made independently by the Chambon and Evans laboratories, to a cellular differentiation model. Our work subsequently moved into more complex in vivo models to examine signals in the tumor microenvironment that drive lung cancer metastasis, which revealed tumor suppressive microRNAs down-regulated in tumor cells in response to extracellular signals. These findings have been translated into clinical trials in patients with advanced lung cancer.
In 2000, we sought to build on our findings in cellular models through studies on an in vivo model that develops lung cancer owing to the same mutated proto-oncogenes found in airway epithelial cells of smokers. We began to collaborate with Tyler Jacks (MIT), who had developed a mouse that spontaneously develops lung adenocarcinomas owing to expression of a somatically activated KRASG12D allele. Prior to the appearance of adenocarcinomas, multifocal premalignant lesions arise that are histologically similar to the atypical alveolar hyperplasias that precede lung adenocarcinomas in patients. We showed that, as these early lesions enlarge, they recruit macrophages, neutrophils, endothelial cells, and cancer-associated fibroblasts (CAFs). The infiltrating macrophages exhibit M2 polarization and have high expression of mTOR, a downstream mediator of PI3K that was required for macrophage survival and lung tumor outgrowth (1). In contrast, the neutrophilic inflammation and angiogenesis in these early lesions were driven by tumor cell-derived secretion of CXCR2 ligands, which chemoattracted neutrophils and endothelial cells and promoted lung tumor growth (2). The infiltrating CAFs exhibited myofibroblast properties and secreted diverse cytokines and chemokines, including vascular endothelial growth factor-A, which promoted tumor cell invasion owing to high expression of VEGFR1 on tumor cells (3). Illustrated conceptually in Figure 1, these findings demonstrated that cells in the tumor microenvironment play an important role in malignant progression at the earliest stage of lung tumorigenesis and might be targeted in future lung cancer chemoprevention clinical trials.
In 2005, I decided to refine the focus of our laboratory to address what I saw as a large gap in the field of lung cancer research. Metastasis is the primary cause of death in patients with lung cancer, and its genetic and biological bases are poorly understood. Progress in this area was hampered by the lack of in vivo models that faithfully recapitulate genetic and biochemical features of human lung cancer metastasis. Lung adenocarcinomas that develop in mice expressing mutant KRAS alone do not metastasize, and we reasoned that these tumors would progress to more advanced stages through secondary mutations in tumor suppressor genes that are inactivated in lung cancer through genetic or epigenetic mechanisms. Through collaboration with Ignacio Wistuba, a molecular pathologist who characterized the histological features of these models, we found that inactivation of PTEN or MAP2K4 accelerated the growth, increased the multiplicity, and enhanced the inflammation and vascularity of KRAS-mutant lung adenocarcinomas but did not induce distant metastasis (4, 5). In contrast, KP mice, which express KRASG12D and TP53R172H, a mutation found in patients with Li Fraumeni Syndrome and a variety of sporadic tumor types, developed widely metastatic lung adenocarcinomas to distant sites commonly involved in lung adenocarcinoma patients (6). Transcriptional profiling studies revealed that poor-prognosis human lung adenocarcinomas were highly enriched in genes differentially expressed between primary and metastatic tumors in KP mice (7). Thus, secondary oncogenic mutations in lung adenocarcinomas initiated by mutant KRAS led to more advanced disease but differed in the degree to which they promoted disease advancement (Fig. 2).
Given their transcriptional and biological similarities to human lung adenocarcinoma, KP mice provided a useful platform on which to investigate the mechanistic basis of human lung cancer metastasis, which has been the focus of my laboratory since 2008. To facilitate these studies, we derived a panel of lung adenocarcinoma cell lines (KP cells) from primary and metastatic tumors in KP mice that are either highly or poorly metastatic following injection into syngeneic wild-type mice. Global transcriptional profiling studies revealed evidence of epithelial-to-mesenchymal transition (EMT) in highly but not poorly metastatic KP cells (8). Through collaborations with Dr. Greg Goodall (University of Adelaide, Australia), whose laboratory was one of the first to discover that the transcriptional repressor ZEB1 is a miR-200 target, we found that metastasis-prone KP cells form polarized epithelial spheres that undergo EMT in response to extracellular cues, a process driven by ZEB1 and inhibited by the microRNA-200 (miR-200) family (ref. 8 and Fig. 3). These cells were marked by high expression of prominin-1 (CD133), and transcriptional profiling of KP cells sorted on the basis of CD133 expression (high versus low) revealed that CD133high tumor cells have increased expression of multiple Notch receptors and their ligands (9). Functional studies revealed that one of the Notch ligands (jagged2) triggers EMT in these cells through up-regulation of a Notch-dependent transcriptional repressor (GATA3) that binds to promoter elements in the miR-200b/c/429 cluster (ref. 9 and Fig. 3). These findings became the basis for an NCI-sponsored (R21 CA8508) clinical trial to target Notch with gamma secretase inhibitors in patients with lung adenocarcinoma.
We found that ZEB1 drives pro-metastatic actin cytoskeletal remma.odeling by downregulating the expression of multiple microRNAs, including miR-34a and miR-148a, which mediate Zeb1-induced filopodia formation and a collective-to-amoeboid migratory switch (10). On the basis of our observation that the polarity of metastatic tumor cells is regulated by signals emanating from the extracellular matrix (Fig. 4), we posited that miR-200 regulates tumor cell sensitivity to pro-invasive extracellular cues. Using a Boyden chamber co-culture assay in which metastasis-prone KP cells invade in response to paracrine factors secreted by CAFs and lose their invasive properties following forced miR-200 expression, we found that multiple cytokines and chemokines were secreted in response to paracrine signals between tumor cells and CAFs. KP cells expressed receptors for those ligands, one of which (Flt1/VEGFR1) was found to be a bonafide miR-200 target gene that regulates tumor cell sensitivity to VEGF-A, a pro-invasive ligand secreted by CAFs (3). Collectively, these findings support the hypothesis that miR-200 regulates tumor cell sensitivity to pro-invasive extracellular cues and raise the possibility that, in addition to inhibiting angiogenesis, VEGF antagonists may directly exert anti-tumor effects.
Given the importance of extracellular signals in governing tumor cell plasticity, we posited a feed-forward model in which miR-200 functions as both sensor and regulator of the tumor extracellular matrix (ECM). To test this hypothesis, we collaborated with Sam Hanash (MD Anderson) to perform LC-MS/MS analysis on metastasis-prone KP cells following forced expression of the miR-200b/c/429 cluster. We found that miR-200 regulates the expression of multiple integrin receptors, their ligands (fibronectin and multiple laminins), and lysyl oxidase collagen cross-linking enzymes that govern tissue stiffness (11). To determine whether secreted ECM molecules regulate sphere formation, we collaborated with Jennifer West (Duke U), a bioengineer who has developed synthetic 3-D polyethylene glycol (PEG)-based hydrogels that mimic specific properties of native tissue ECM. KP cells efficiently formed tumor spheres in defined hydrogels, and changes in matrix stiffness or laminin peptide concentrations led to changes in sphere diameter and lumen formation efficiency (12), supporting a feed-forward model in which miR-200 regulates tumor cell secretion of factors that control ECM biochemical and biophysical properties, which, in turn, govern tumor cell polarity (Fig. 5).
On the basis of the scientific foundation laid by the above multi-disciplinary, collaborative studies, we have built a larger research effort to more fully explore the role of miR-200 and its targets as regulators of the tumor ECM and metastasis. Our team includes Jennifer West (Duke U.), Ignacio Wistuba (MD Anderson), Don Gibbons (MD Anderson), Mary Dickinson (BCM), Ken Scott (BCM), and Chad Creighton (BCM). We recently received a Multiple Investigator Research Award (MIRA) on which I serve as overall Principal Investigator from the Cancer Prevention and Research Institute of Texas (CPRIT).
Our central hypothesis is that miR-200 down-regulation in tumor cells controls pro-metastatic processes in the tumor microenvironment by regulating tumor cell sensitivity to pro-invasive ligands secreted by CAFs and by controlling tumor cell secretion of ECM and pro-angiogenic molecules. By using KP mice and in vitro models derived from them, we will screen candidate miR-200 target genes for their capacity to drive tumor cell invasion and EMT, promote vascular tube formation, and enhance metastasis. The prognostic value of genes uncovered by these gain-of-function assays will be examined in our bank of human lung cancer specimens annotated for somatic mutations, transcriptomic changes, and histopathological and clinical data. This research infrastructure will serve as a springboard for the studies proposed herein on miR-34a and facilitate the translation of miR-34a and its targets to the clinical setting.
A major focus of ongoing collaborative work in our multi-investigator studies is to understand the functional role of CAFs in lung cancer progression. The unique biological features of this population of cells include its expression of Thy-1, secretion of diverse pro-angiogenic and pro-inflammatory cytokines, potent interactions with tumor cells, and striking differences from normal lung fibroblasts with respect to its ability to activate integrins and form focal adhesions (Fig. 6). We are actively studying how CAFs and tumor cells promote metastasis by creating a stiff tumor matrix through the secretion of paracrine factors that stimulate the expression of collagen modifying enzymes (Fig. 7).
To date 26 post-docs and 3 Ph.D. candidates have completed their training in my laboratory. MD Anderson has an active undergraduate summer student internship program through which I have mentored 22 students. My philosophy is to provide a rigorous training experience for a relatively small number of trainees at any given time. Two post-doctoral fellows, Ho-Young Lee and Naoko Sueoka, carried out the bulk of our early work on retinoid biology and currently hold independent faculty positions at Peking University and Saga University, respectively, and have achieved their own academic distinctions in elucidating the roles of insulin-like growth factor binding proteins and heterogeneous nuclear ribonucleoproteins, respectively, in lung cancer. Our work on the tumor microenvironment was initiated by a pulmonologist, Marie Wislez, who came to my lab after making the first observation that neutrophilic inflammation in bronchial secretions is a poor prognostic marker in patients with bronchoalveolar cell carcinoma. After extensively characterizing and interrogating the tumor microenvironment in mouse models of lung adenocarcinoma in my lab, she returned to her independent faculty position in France and continues to use our mouse model in translational research studies on this disease. Our entry into the microRNA field was initiated by two medical oncology fellows working in my lab, Don Gibbons and Wei Lin, who are now investigators in academia (MD Anderson) and industry (Genentech), respectively. I am the scientific mentor on an NIH K08 award that Don Gibbons received while working in my lab. My exploration of upstream regulators and downstream mediators of the miR-200/ZEB1 axis was carried out by two post-doctoral fellows, one of whom (Yanan Yang) now holds an independent faculty position at the Mayo Clinic, and the other (Young-ho Ahn) is still in my laboratory and has begun to interview for independent positions. During their tenures in my lab, my trainees have received an NIH Award (Jon Roybal); numerous Young Investigator Awards from ASCO (Anne Tsao, Don Gibbons, Wei Lin, Erminia Massarelli, and Yanis Boumber), AACR (Wei Lin), and the International Association for the Study of Lung Cancer (Don Gibbons); and 3 Keystone Symposia travel awards (Jon Roybal). This year, MD Anderson recognized my ability to mentor young scientists with the Division of Cancer Medicine’s “Mentor of the Year” award.
A unique aspect of the training program I have created for students and fellows in my laboratory is intensive tutoring in writing skills, which are a key to success in research and are frequently deficient in trainees. Most writing courses do not provide sufficient instruction to address these needs. When initiating the writing of a paper or grant, each trainee receives a copy of William Strunk’s “The Elements of Style” and is encouraged to read it from cover-to-cover before composing the first sentence. Throughout the writing process, trainees meet one-on-one on a weekly basis with tutors from the Ph.D. program in English Literature at Rice University, whom I hire on an hourly basis using my limited philanthropic funds, to go over their composition line-by-line, an editing process that helps them to understand their writing problems and how to fix them. This practice is extremely popular among both national and international trainees and was featured in OSP INSIGHT, an MD Anderson Office of Sponsored Programs quarterly publication.
References (Literature Citations)
1. Wislez, M.; Spencer, M.L.; Izzo, J.G.; Juroske, D.M.; Balhara, K.; Cody, D.D.; Price, R.E.; Hittelman, W.N.; Wistuba, II; Kurie, J.M. Inhibition of mammalian target of rapamycin reverses alveolar epithelial neoplasia induced by oncogenic K-ras. Cancer Res. 2005 Apr 15;65(8):3226-3235.
2. Wislez, M.; Fujimoto, N.; Izzo, J.G.; Hanna, A.E.; Cody, D.D.; Langley, R.R.; Tang, H.; Burdick, M.D.; Sato, M.; Minna, J.D.; Mao, L.; Wistuba, I.; Strieter, R.M.; Kurie, J.M. High expression of ligands for chemokine receptor CXCR2 in alveolar epithelial neoplasia induced by oncogenic kras. Cancer Res. 2006 Apr 15;66(8):4198-4207.
3. Roybal, J.D.; Zang, Y.; Ahn, Y.H.; Yang, Y.; Gibbons, D.L.; Baird, B.N.; Alvarez, C.; Thilaganathan, N.; Liu, D.D.; Saintigny, P.; Heymach, J.V.; Creighton, C.J.; Kurie, J.M. miR-200 Inhibits lung adenocarcinoma cell invasion and metastasis by targeting Flt1/VEGFR1. Molecular cancer research : MCR. 2011 Jan;9(1):25-35.
4. Iwanaga, K.; Yang, Y.; Raso, M.G.; Ma, L.; Hanna, A.E.; Thilaganathan, N.; Moghaddam, S.; Evans, C.M.; Li, H.; Cai, W.W.; Sato, M.; Minna, J.D.; Wu, H.; Creighton, C.J.; Demayo, F.J.; Wistuba, II; Kurie, J.M. Pten inactivation accelerates oncogenic K-ras-initiated tumorigenesis in a mouse model of lung cancer. Cancer Res. 2008 Feb 15;68(4):1119-1127.
5. Ahn, Y.H.; Yang, Y.; Gibbons, D.L.; Creighton, C.J.; Yang, F.; Wistuba, II; Lin, W.; Thilaganathan, N.; Alvarez, C.A.; Roybal, J.; Goldsmith, E.J.; Tournier, C.; Kurie, J.M. Map2k4 functions as a tumor suppressor in lung adenocarcinoma and inhibits tumor cell invasion by decreasing peroxisome proliferator-activated receptor gamma2 expression. Molecular and cellular biology. 2011 Nov;31(21):4270-4285.
6. Zheng, S.; El-Naggar, A.K.; Kim, E.S.; Kurie, J.M.; Lozano, G. A genetic mouse model for metastatic lung cancer with gender differences in survival. Oncogene. 2007 Oct 18;26(48):6896-6904.
7. Gibbons, D.L.; Lin, W.; Creighton, C.J.; Zheng, S.; Berel, D.; Yang, Y.; Raso, M.G.; Liu, D.D.; Wistuba, II; Lozano, G.; Kurie, J.M. Expression signatures of metastatic capacity in a genetic mouse model of lung adenocarcinoma. PLoS One. 2009;4(4):e5401.
8. Gibbons, D.L.; Lin, W.; Creighton, C.J.; Rizvi, Z.H.; Gregory, P.A.; Goodall, G.J.; Thilaganathan, N.; Du, L.; Zhang, Y.; Pertsemlidis, A.; Kurie, J.M. Contextual extracellular cues promote tumor cell EMT and metastasis by regulating miR-200 family expression. Genes Dev. 2009 Sep 15;23(18):2140-2151.
9. Yang, Y.; Ahn, Y.H.; Gibbons, D.L.; Zang, Y.; Lin, W.; Thilaganathan, N.; Alvarez, C.A.; Moreira, D.C.; Creighton, C.J.; Gregory, P.A.; Goodall, G.J.; Kurie, J.M. The Notch ligand Jagged2 promotes lung adenocarcinoma metastasis through a miR-200-dependent pathway in mice. The Journal of clinical investigation. 2011 Apr;121(4):1373-1385.
10. Ahn, Y.H.; Gibbons, D.L.; Chakravarti, D.; Creighton, C.J.; Rizvi, Z.H.; Adams, H.P.; Pertsemlidis, A.; Gregory, P.A.; Wright, J.A.; Goodall, G.J.; Flores, E.R.; Kurie, J.M. ZEB1 drives prometastatic actin cytoskeletal remodeling by downregulating miR-34a expression. The Journal of clinical investigation. 2012 Sep 4;122(9):3170-3183.
11. Schliekelman, M.J.; Gibbons, D.L.; Faca, V.M.; Creighton, C.J.; Rizvi, Z.H.; Zhang, Q.; Wong, C.H.; Wang, H.; Ungewiss, C.; Ahn, Y.H.; Shin, D.H.; Kurie, J.M.; Hanash, S.M. Targets of the tumor suppressor miR-200 in regulation of the epithelial-mesenchymal transition in cancer. Cancer Res. 2011 Dec 15;71(24):7670-7682.
12. Gill, B.J.; Gibbons, D.L.; Roudsari, L.C.; Saik, J.E.; Rizvi, Z.H.; Roybal, J.D.; Kurie, J.M.; West, J.L. A synthetic matrix with independently tunable biochemistry and mechanical properties to study epithelial morphogenesis and EMT in a lung adenocarcinoma model. Cancer Res. 2012 Sep 4.