RhoA as a Potential Target in Lung Cancer
A dissertation submitted to the Division of Research and Advanced Studies
of the University of Cincinnati
in partial fulfillment of the requirements for the degree of
Doctor of Philosophy (Ph.D.)
in the Department of Molecular and Developmental Biology
of the College of Medicine
2015
by
Inuk Zandvakili
B.M.Sc. University of Western Ontario
Committee Members:
Timothy D. LeCras, PhD
Vladimir V. Kalinichenko, MD, PhD
John C. Morris, MD
James C. Mulloy, PhD
Kathryn A. Wikenheiser-Brokamp, MD, PhD
Yi Zheng, PhD (Chair)
Abstract
Many cancers are driven by oncogenic K-Ras, yet K-Ras has remained largely undruggable. In this dissertation we explore inhibiting K-Ras signaling by targeting downstream signaling pathways, namely the RhoA and RhoC GTPase pathways. Numerous cellular studies have indicated that RhoA signaling is required for oncogenic Ras-induced transformation. To date very limited data exist to genetically attribute RhoA function to Ras-mediated tumorigenesis in mammalian models. In order to assess whether RhoA is required for K-Ras-induced lung cancer initiation, we utilized the K-
RasG12D Lox-Stop-Lox murine lung cancer model in combination with the conditional RhoAflox/flox and
RhoC-/- knockout mouse models. We found that deletion of RhoA, RhoC or both did not adversely affect normal lung development. Moreover, we found that deletion of either RhoA or RhoC alone did not suppress K-RasG12D induced lung adenoma initiation. Rather, deletion of RhoA alone increased lung adenoma formation, whereas dual deletion of RhoA and RhoC together significantly reduced K-
RasG12D induced adenoma formation. Deletion of RhoA appears to induce a compensatory mechanism that exacerbates adenoma formation. The compensatory mechanism is at least partly mediated by RhoC. These results are in contrast to RhoA knockdown experiments we performed in human lung cancer cell lines, which show dramatic inhibition of malignant phenotypes with RhoA loss. Taken together, this dissertation work suggests that targeting of RhoA alone may allow for compensation and a paradoxical exacerbation of neoplasia, while simultaneous targeting of both
RhoA and RhoC is more likely to inhibit oncogenic K-Ras driven lung cancers.
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Notice
No part of this work may be reproduced without the explicit written permission of the author.
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Acknowledgements
There are many people to thank and acknowledge for the successful completion of my PhD.
Foremost, I must thank my advisor Dr Yi Zheng for taking me in his lab and under his wing, for guiding me through all of the pitfalls and dead ends, and who always remains enthusiastic and undeterred. He gave me great intellectual freedom so I could make mistakes and learn. We spent many Sundays together discussing my project when he was trying to have a few distraction-free hours to himself. I would also like to thank my thesis committee and all of my research colleagues for their insightful comments and friendship over the years.
Most importantly, I need to thank my family and friends. Above all, I have to thank my uncle for his foresight in guiding me through my education. Without knowing it at the time, he helped me see over the next hill, when I had trouble seeing past the trees in front of me. His wisdom is the reason I will enjoy a successful career. I have to thank my father for always pushing me to work my hardest
(and smartest), and my mother for balancing me out and reminding me of what is important in life.
I have to thank my brother Arya who always challenges me with his intellect, humbles me with his work ethic, and enlightens me with his moral philosophies. Lastly, I have to thank my wife Christina for her endless love and support as my partner in life and fellow academic. I have been humbled by her sharp wit, her knack for multi-multitasking, and ability to pull seemingly insurmountable projects together in the shortest of timeframes. At the end of a long day, she was always there to nourish my soul so that I could start the next day fresh and as the best person I can be.
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Table of Contents
Abstract ...... ii
Acknowledgements ...... iv
List of Figures and Tables ...... viii
Figures ...... viii
Tables...... ix
Supplemental Figures ...... ix
Abbreviations ...... x
Chapter 1 – Introduction ...... 13
Lung Cancer ...... 14
Epidemiology of Lung Cancer ...... 15
Histology of Lung Adenocarcinoma ...... 16
Molecular Pathogenesis of Lung Adenocarcinoma ...... 17
Diagnosis and Clinical Management of Lung Adenocarcinoma ...... 19
K-Ras ...... 22
Introduction to Rho GTPases ...... 28
Rho GTPases Structure and Function ...... 29
General Considerations of Rho GTPase Function ...... 30
Activation of Rho GTPases and Rho Regulatory Proteins ...... 31
Actin filaments ...... 33 v
Microtubules ...... 35
Cell shape and movement independent affects ...... 35
The Intersection of Rho GTPases and Cancer ...... 36
Pro-neoplastic aspects of Rho A Signaling...... 36
RhoB and RhoC in Cancer ...... 38
Rac and Cdc42 Signaling in Cancer ...... 39
Possible anti-neoplastic aspects of Rho GTPases ...... 40
Chapter 2 – Loss of RhoA Exacerbates, Rather than Dampens, Oncogenic K-Ras Induced Lung
Adenoma Formation in Mice...... 42
Loss of RhoA exacerbates, rather than dampens, oncogenic K-Ras induced lung adenoma
formation in mice...... 43
Abstract ...... 44
Introduction ...... 45
Results ...... 47
Discussion ...... 53
Materials and Methods ...... 57
Acknowledgments ...... 59
Bibliography ...... 60
Figure Legends ...... 61
Supporting Information Captions ...... 65
Tables...... 67
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Figures ...... 68
Supplemental Figures ...... 73
Chapter 3 – RhoA is required for Malignant Phenotypes of Human Lung Adenocarcinoma Cell Lines
...... 78
Introduction ...... 79
Results ...... 79
Discussion ...... 80
Methods ...... 82
Chapter 4 – Summaries and Perspectives ...... 87
Summary of Findings ...... 88
Limitations of this Study and Alternative Approaches ...... 89
Deciphering the Paradoxical increase in Adenoma Formation and Growth in RhoAflox/flox
background mice ...... 93
A Case for Rho GTPase inhibition: Reconciling cell line and in vivo findings ...... 96
Parallels to the Paradoxical Activation of the MAPK Pathway through B-Raf inhibition ...... 97
Final Comments and Future Directions ...... 100
Bibliography ...... 102
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List of Figures and Tables
Figures
Figure 1-1. Overview of Ras signaling ...... 24
Figure 1-2. GTPase activity of Rho GTPases...... 28
Figure 1-3. Homology Relations of Rho GTPase Family Members...... 29
Figure 1-4. Cellular Functions of Rho GTPases...... 31
Figure 1-5. Rho Subfamily of GTPases and Main Effector Pathways...... 33
Figures: Figure 2-1. CCSP-promoter driven deletion of RhoA does not affect normal lung development...... 68
Figure 2-2. RhoA is not essential for CCSP-promoter driven, K-RasG12D-induced, lung adenoma formation in mice...... 69
Figure 2-3. Deletion of RhoA and RhoC together does not impair normal lung development...... 70
Figure 2-4. Neither RhoA nor RhoC is required for K-RasG12D-induced adenoma formation in a CCSP-
Cre model...... 71
Figure 2-5. RhoA and RhoC are important in combination, but not individually, for K-RasG12D- induced sporadic lung adenoma formation...... 72
Figure 3-1. Rho and Ras GTPase Downstream Signaling after RhoA Knockdown ...... 84
Figure 3-2. Cell Proliferation after RhoA Knockdown ...... 85
Figure 3-3. Anchorage-independent Growth after RhoA Knockdown...... 86
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Tables
Table 1-1 Lung Cancer Subtypes ...... 14
Table 1-2. Molecular Genetics of Lung Cancers ...... 18
Table 1-3. Lung Cancer Staging ...... 19
Table 1-4. Definitions for Staging of NSCLCs...... 20
Table 2-1. Breeding Schematic for Transgenic Mice ...... 67
Supplemental Figures
Figure S 2-1. PCR analysis of mouse genotypes and lox deletion bands...... 73
Figure S 2-2. CCSP-promoter driven hyperplastic growths and adenomas...... 74
Figure S 2-3. Downstream K-Ras and RhoA signaling status in CCSP-promotor driven adenomas. .. 75
Figure S 2-4. Adeno-Cre induced hyperplastic growths and adenomas...... 76
Figure S 2-5. Original Western Blots...... 77
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Abbreviations
AAH: atypical adenomatous hyperplasia DNA: deoxyribonucleic acid
AIS: adenocarcinoma in situ DOCK: dedicator of cytokinesis
ALK: Anaplastic lymphoma kinase EGF: epidermal growth factor
ANOVA: analysis of variance EGFR: epidermal growth factor receptor
APC: adenomatous polyposis coli ERK: Extracellular signal-regulated kinases
ATCC: American Type Culture Collection FAK: focal adhesion kinase
ATP: adenosine triphosphate FRET: fluorescence resonance energy
transfer BCR: breakpoint cluster region
GAP: GTPase-activating protein CCSP: club/clara cell secretory protein
GAPDH: Glyceraldehyde 3-phosphate CMV: cytomegalovirus dehydrogenase CT: computed tomography GBD: GTPase-binding domain DAB: 3,3'-diaminobenzidine GDP: guanosine diphosphate DAG: diacylglycerol GEF: Guanine nucleotide exchange factors DAPI: 4',6-diamidino-2-phenylindole GIMP: GNU Image Manipulation Program Dbl: diffuse B-cell lymphoma GPCR: G-protein coupled receptor DH: Dbl homology GTP: guanosine triphosphate DKO: double-knock out x
HRP: horse-radish peroxidase MMTV: mouse mammary tumor virus
IASLC: International Association for the Study MSK: mitogen- and stress-activated kinase of Lung Cancer MTS: 3-(4,5-dimethyl-2-yl)-5-(3-
JNK: c-Jun N-terminal kinases carboxymethoxyphenyl)-2-(4-sulfophenyl)-
2H-tetrazolium KSR: kinase suppressor of ras
NF: neurofibromin/neurofibromatosis LARG: leukemia-associated RhoGEF
NSCLC: non-small cell lung carcinoma LIM: Lin11, Isl-1 & Mec-3
NT: non-targeting LIMK: LIM domain kinase
PAK: p21-activated kinase G-LISA: GTP-based enzyme-linked immunosorbent assay PBS: phosphate buffered saline
LSL: lox-stop-lox PCR: polymerase chain reaction
MAL: megakaryocytic acute leukemia (MLK1) PET: positron-emission tomography
MAPK: Mitogen-activated protein kinases PFU: plaque forming unit
MEK: MAPK/ERK kinase PH: pleckstrin homology domain
MET: hepatocyte growth factor recptor PI3K: phosphatidylinositol-4,5-bisphosphate
3-kinase MLC: myosin light chain
PKB: protein kinase B (AKT) MLK1: megakaryoblastic leukemia 1 (MAL)
PTB: phosphotyrosine-binding domain MLL: mixed-lineage leukemia
RBD: ras-binding domain xi
RIPA: radioimmunoprecipitation assay buffer
RNA: ribonucleic acid
ROCK: rho-associated protein kinase
ROS: reactive oxygen species
SCLC: small cell lung carcinoma shRNA: short-hairpin ribonucleic acid
SOS: son of sevenless
SPC: surfactant protein C
SRF: serum response factors
TCF: transcription factor
TITF: thyroid transcription factor 1 (NK2 homeobox 1, NKX2-1)
TNM: tumor nodes metastasis
VEGF: vascular endothelial growth factor
WASP: wiskott–aldrich syndrome protein
WAVE: WASP-family verprolin-homologous protein
WT: wildtype
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Chapter 1 – Introduction
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Lung Cancer
In the United States lung cancer kills more people each year than breast, prostate and colon cancer combined (1,2). Moreover, lung cancer is the leading cause of cancer death worldwide and is likely to grow in incidence with increased air pollution and tobacco use throughout the developing world.
There are two major factors that govern why lung cancer kills many more people than more common cancers such as colon and breast cancer. The first important factor is a lack of a cheap and assessable screening tool to catch early stage cancers (3,4). Secondly, there is a dearth of effective therapies to treat lung cancers, as there are few effective targeted therapies for lung cancer as compared to other major cancers (5,6). Due to these two factors, most patients present with advanced stages of the disease and have dismal survival prospects with median overall survival of less than 15% at 5-years.
Greater than 95% of lung cancers are carcinomas which have historically been divided into two major types: small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). NSCLCs can be further subdivided into three histological groups: adenocarcinoma, squamous cell carcinoma and large cell carcinoma (7). Though more recently lung cancers have started to be categorized by their genetic basis and susceptibility to targeted therapies (8–11). Overall, lung adenocarcinoma is the most common subtype of lung cancer, accounting for Table 1-1 Lung Cancer Subtypes about 40-50% of all lung cancers. Although lung Small cell lung cancer Non-small cell lung cancer adenocarcinoma is also linked to smoking, it is the most Adenocarcinoma Squamous cell carcinoma common form of lung cancer among never smokers, as Large cell carcinoma Adenosquamous carcinoma well as among women and younger patients (<45 years Other, including old). Carcinoid tumors Sarcomatoid tumors Adapted from Kumar et al. (7)
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Epidemiology of Lung Cancer
Environmental exposure to carcinogens is the by far the most important factor in the development of lung cancers, though germline mutations in genes such as retinoblastoma or EGFR are linked to lung cancer susceptibility (4). In fact, first-degree relatives of lung cancer patients have a 3-fold increased risk of a multitude of cancers, many which are not related to smoking, indicating the likely presence of as of yet unknown susceptibility genes (4).
It is estimated that cigarette smoking is the cause of 80-90% of lung cancers. Indeed, cigarette smoke is associated with a 10-30 fold increase in risk of lung cancer in active smokers over the general population, such that the cumulative lifetime risk of lung cancer among heavy smokers may be as high as 30%, compared with a lifetime risk of lung cancer of <1% in never smokers (12).
Though squamous cell and small cell carcinomas have the strongest association with tobacco smoke exposure, deep sequencing of adenocarcinomas demonstrates an average of 10-fold higher mutation frequency in smokers as compared to never-smokers with adenocarcinoma (9). Physician counsel of patients to stop or at least decrease smoking is very important as it reduces the risk of lung cancer with notable risk improvement within five years. Case-control studies reported by the
Surgeon General of United States Department of Health and Human Services show that smoking cession for more than 15 years reduced the risk of lung cancer by 80-90% as compared to continuous heavy smoking (13). Other risk factors for the development of lung cancers include exposure to asbestos, radon, polycyclic aromatic hydrocarbons (often from air pollution) and certain metals (e.g. arsenic, chromium and nickel). Evidence is also emerging that a diet low in fruits and vegetables is also a contributing factor to lung cancer incidence.
With increased awareness of the link between tobacco smoke and lung cancer, the incidence of lung cancer in men peaked around 1990, but the incidence in women had continued to increase through the 2000s until finally peaking in the last few years (2). However, the statistics are alarming
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worldwide, especially in developing countries such as China where roughly 2/3rds of men smoke at levels of >10 cigarettes per day, which is similar to smoking levels in men in the USA in the 1950s
(14,15). Therefore, it is expected that lung cancer incidence overall will increase and that lung cancer will continue to remain the biggest cancer killer worldwide.
Histology of Lung Adenocarcinoma
Small cell and squamous cell lung carcinomas are typically centrally located masses, whereas adenocarcinomas tend to be peripherally located. Small cell lung carcinomas have invariably metastasized by the time they are detected. These tumors often express neuroendocrine markers and secrete different polypeptide hormones that cause paraneoplastic syndromes. Squamous cell carcinomas, often located near the main bronchus, invade local structures and are often only symptomatic once they start to obstruct distal airways causing infections. As is typical of squamous cancers these cancers can at times be identified by the presence of “keratin pearls”.
In contrast to these two cancers, lung adenocarcinomas are typically peripherally located in the chest. Lung adenocarcinomas are further subdivided and described by histology as acinar, papillary, solid or lepidic morphology and additionally by features such as mucinous (colloid), fetal, signet cell or clear cell (8). Lung adenocarcinomas are thought to arise from precursor lesions called atypical adenomatous hyperplasia (AAH). These lesions then progress to adenocarcinoma in situ (AIS) and then finally to frank cancer. AAH are described as a small (<5mm) but well-demarcated foci of proliferating cuboidal or low-columnar cells demonstrating cytologic atypia (i.e. nuclear hyperchromasia, pleomorphism and prominent nucleoli). AIS, formerly referred to as bronchioloalveolar carcinoma, is a small but slightly larger nodule (<3cm) that grows along preexisting structures and preserves alveolar architecture. Both of these lesions contain precursor lesions such as K-Ras or EGFR mutations (discussed further below).
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Lastly, large cell carcinomas are composed of undifferentiated malignant cells that lack specific histologies of the other subtypes of lung cancer.
Molecular Pathogenesis of Lung Adenocarcinoma
Knowledge of the molecular pathogenesis of lung cancers has increased dramatically over the past ten years, but fundamental knowledge of the molecular drivers in the field still lags behind many other forms of cancer. Deep sequencing of adenocarcinomas and squamous cell carcinomas has greatly aided our understanding (9,10). These studies and many others have validated long understood genetic drivers, but have also revealed rare, but specific cancer drivers. Paramount above all, oncogenic mutation of the KRAS gene is the by far the most common driver of lung adenocarcinoma and can be appreciated in precursor lesions such as AAH and AIS (16). As outlined in Table 1-2, oncogenic mutation of KRAS is found in about 25-30% of lung adenocarcinomas and is more common among smokers. The next most commonly mutated lung cancer driver is EGFR, which occurs in about ~15% of lung adenocarcinomas (including amplifications, kinase domain mutation and extracellular domain deletions). Other important genetic events that are almost certainly cancer initiating events are chromosome 3p deletion and EML4-ALK gene fusion; supported by the fact that 3p deletions are seen in almost all lung cancers of all types whereas
EGFR, KRAS and EML4-ALK mutations are mutually exclusive mutations. Chromosome 3p deletions are very interesting as several regions of interest in this chromosome arm map to tumor suppressors as well as microRNAs that regulate EGFR expression.
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Table 1-2. Molecular Genetics of Lung Cancers
Abnormality Non–Small-Cell Lung Cancer Small-Cell Lung Cancer Squamous-Cell Adenocarcinoma Carcinoma
Precursor Lesions Lesion type Dysplasia Atypical adenomatous Possible hyperplasia (neuroendocrine field) Drivers PIK3CA KRAS mutation (in - smokers), EGFR kinase mut (in nonsmokers) Chromosome 3p del. ~100% ~90% ~100%
Cancer KRAS mutation Very rare 20 - 30% Very rare BRAF mutation 3% 2% Very rare
EGFR Rare mutation, and ~15% Very rare (mut or amp) <10% amplification
HER2 (mut or amp) Very rare ~5% Very rare
ALK fusion Very rare 7-15% Very rare (including EML4-ALK)
MET Mutation 12% 14% 13% Amplification 21% 20% Not known
TTF-1 amplification 15% 15% Very rare p53 mutation ~70% 50-70% 75% LKB1 mutation 19% 34% Very rare
PIK3CA Mutation 2% 2% Very rare Amplification 33% 6% 4%
Rb family members ~20% ~20% ~90% p16/CDKN2A ~50% ~50% ~10%
Adapted from Herbst et al. Lung Cancer. NEJM 2008 (4) with added information from several other sources (9–11,17)
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Diagnosis and Clinical Management of Lung Adenocarcinoma
Lung cancer should be considered in any older patients or any smokers presenting with new onset symptoms of coughing or hemoptysis. The following are the most common presenting symptoms of lung cancer along with the frequency: cough (50-75%), hemoptysis (25-50%), dyspnea (25%) and chest pain (20%) (19). However, these signs and symptoms are highly non-specific, and should be followed with a complete history, physical and imaging, preferably chest CT (or alternatively a chest x-ray). Imaging may reveal a mass, which must then be followed to determine its etiology which may be primary lung cancer, metastasis to the lung, benign tumor, infectious granuloma (e.g. from histoplasmosis or tuberculosis), non-infectious granuloma (e.g. inflammatory diseases), bacterial infection, vascular malformation or other causes. Often, solitary pulmonary nodules are found incidentally on chest x-ray ordered for other reasons (e.g. pneumonia). These must be evaluated based on size, contour, calcification and lung Table 1-3. Lung Cancer Staging Stage T N M cancer risk profile of the patient. For example, history of Ia T1a N0 M0 T1b smoking, patient age over 60 and nodule size >20mm all Ib T2a increase the likelihood of malignancy considerably (4,5). IIa T1a N1 T1b N1 Lab studies are also useful to evaluate the risk of T2a N1 T2b N0 malignancy and elucidate paraneoplastic processes or IIb T2b N1 T3 N0 other causes of the respiratory symptoms. IIIa T1 N2 T2 N2 T3 N2 If there is a moderate to high suspicion that a nodule is T3 N1 T4 N0 cancerous this should be followed by PET imaging and/or T4 N1 IIIb T4 N2 subsequent biopsy to determine the subtype of lung T1 N3 T2 cancer and confirm the diagnosis. Full body CT or PET T3 T4 provides the most important data guiding the patient’s IV Any Any M1a/b Adapted from Goldstraw et al. (18) prognosis and treatment options by staging the cancer.
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Table 1-4. Definitions for Staging of NSCLCs. Primary tumor (T) T0/X No primary tumor or tumor cannot be assessed Tis Carcinoma in situ T1 Tumor ≤ 3 cm in greatest dimension, surrounded by lung or visceral pleura, and no invasion or spreading T1a Tumor ≤ 1 cm in the greatest dimension T1b Tumor > 1 cm but ≤ 2 cm in the greatest dimension T1c Tumor > 2 cm but ≤ 3 cm in the greatest dimension T2 Tumor > 3 cm but ≤ 5 cm or tumor with any of the following: Involves the main bronchus or carina but without invasion of the carina Associated with atelectasis or obstructive pneumonitis T2a Tumor > 3 cm but ≤ 4 cm in the greatest dimension T2b Tumor > 4 cm but ≤ 5 cm in the greatest dimension T3 Tumor > 5 cm but ≤ 7 cm or one that directly invades any of the following: Chest wall (including superior sulcus tumors), phrenic nerve, or parietal pericardium;
T4 Tumor > 7 cm or of any size that invades any of the following: mediastinum, heart, great vessels, trachea, recurrent laryngeal nerve, esophagus, vertebral body, diaphragm, or carina; or separate tumor nodule(s) in a different ipsilateral lobe Regional lymph nodes (N) N0 No regional node metastasis N1 Metastasis in ipsilateral peribronchial and/or ipsilateral hilar lymph nodes and intrapulmonary nodes, including involvement by direct extension N2 Metastasis in the ipsilateral mediastinal and/or subcarinal lymph node(s) N3 Metastasis in the contralateral mediastinal, contralateral hilar, ipsilateral or contralateral scalene, or supraclavicular lymph nodes Distant metastasis (M) M0 No distant metastasis M1 Distant metastasis M1a Separate tumor nodule(s) in a contralateral lobe; tumor with pleural nodules or malignant pleural (or pericardial) effusion M1b Distant metastasis Adapted from Rami-Porta et al. The IASLC Lung Cancer Staging Project Proposals for the Revisions of the T Descriptors in the Forthcoming Eighth Edition of the TNM Classification for Lung Cancer (18,20)
First it must be determined whether the patient has SCLC or NSCLC and the cancer stage. The cancer stage is assessed by the TNM system which integrates the tumor size/characteristics (T), spread to lymph nodes (N) and metastasis (M), as outlined for NSCLC in Tables 2 and 3 (18).
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Patients with low stage NSCLC (stage I to IIIA) may benefit from surgery (pneumonectomy or lobectomy along with mapping of cancer spread to regional lymph nodes). Radiotherapy may also be helpful to treat primary tumors as well as regional lymph nodes to prevent disease spread (5).
Only in the last two years has there been validation and approval by the United States Task Force on Preventive Services to routinely use low-dose CT is a screening tool in high risk populations to diagnose lung cancers early when chances of favorably outcomes are highest (21,22). Solitary pulmonary nodules or other suspicious lesions and surrounding lymph nodes can be biopsied using video-assisted thoracoscopic surgery to assess definitively whether a lesion is cancerous, the type of cancer and also to stage the patient’s cancer. Lower stage cancers may be treated with surgical excision such as a lobar pneumonectomy.
Unfortunately, most patients present with advanced stages of lung cancer (stage III or higher), and in this case chemotherapy +/- targeted therapy is the mainstay of treatment, though surgery is often an option when regional lymph node involvement is limited (2,23). For this reason, biopsy specimens are important to determine if the patient’s cancer has any “driver mutations” that can inhibited with targeted therapies. There is no standard testing method currently but multiplexed genotyping of a panel of mutations with either prognostic or management value appears to be the most popular. Currently, validated targets include EGFR mutation and ALK fusions. EGFR mutant cancers can be treated with TKIs such as erlotinib, gefitinib and afatinib (6). ALK gene fusions can be treated with the TKIs crizotinib or ceritinib (19). Additionally, bevacizumab, a monoclonal antibody directed against VEGF, can be helpful in advanced stage non-squamous NSCLCs. Finally, traditional chemotherapy with a platinum-based agent and either vinorelbine or docetaxel is the mainstay of therapy (19,23). The most common platinum-based agents are cisplatin or carboplatin, which damage DNA and replication by causing intrastrand crosslinks and adducts. Vinorelbine and docetaxel, though different in their mechanism of action, both interfere with microtubule assembly and thus with mitosis. Additionally, pemetrexed, an antifolate agent which inhibits nucleotide
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synthesis has also been shown to be effective for treating NSCLC (23). Finally, for patients with unresectable stage IIIb disease or higher, and who cannot tolerate surgery or aggressive therapy, less aggressive chemotherapy measures along with palliative radiation and comfort measures also exist (24).
K-Ras
K-Ras is a small GTPase (~21 kDa) that is the protypic member of the Ras superfamily of GTPases.
Along with its highly homologues family proteins H-Ras, and N-Ras, this trio make up among the most important proteins in all of cancer. Ras GTPases are signaling proteins that transmit predominantly extracellular signals through to a multitude of downstream signaling pathways that regulate important cellular processes such as proliferation, differentiation, motility and apoptosis.
The most prominent signaling pathway downstream of K-Ras is the MAPK pathway leading to activation of proliferation programs. The Ras superfamily has nine families: Ras, Rad, Rab, Rap, Ran,
Rho, Rheb, Rit, and Arf. These families are further categorized into subfamilies (25).
All signaling GTPases share commonalities in their structure and how they signal. When bound to
GTP, GTPases possess an active conformation that allows them to bind to effector proteins. These effector proteins then relay the active Ras signal to other downstream mediators. Being GTPases, the proteins eventually hydrolyze the GTP into GDP causing a change of conformation into an inactive state such that it can no longer bind effector proteins and thus signaling is shut off.
However, the intrinsic GTPase activity of these Ras superfamily of GTPases is relatively low, and the process of GTP hydrolysis is greatly accelerated by GTPase-activating proteins (GAPs). Two well- known Ras GAPs include NF1 and p120GAP. Once the GTP is hydrolyzed to GDP, GDP must then be exchanged with a new GTP in order to re-establish active signaling. This process is catalyzed by guanine nucleotide exchange factors (GEFs). As an example, son of sevenless (SOS1) is a well-
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known and characterized Ras GEF. Thus, together GAPs and GEFs regulate the activity of the
GTPase.
Classically, activation of K-Ras occurs as a result of activation of upstream receptor tyrosine kinases
(RTKs) such as the epidermal growth factor receptor (EGFR). However, many upstream signals can contribute to K-Ras activation including signaling from integrins and all flavors of g-protein coupled receptors (GPCRs, including Gi, Gs and Gq types) (26). The most classic form of K-Ras activation starts with the binding of EGF to the EGFR receptor. RTKs such as EGFR are transmembrane proteins that exist as inactive monomers that activate by dimerization following ligand binding.
Dimerization of the two receptor halves results in close approximation of the intracellular domains and reciprocal phosphorylation of tyrosine residues in each dimer half. This autophosphorylation process allows proteins containing Src homology 2 (SH2) or phosphotyrosine-binding (PTB) domains to bind the intracellular aspects of the receptor (27). One such protein is GRB2, which is able to bind to the phosphorylated tyrosines on EGFR via SH2 domains. GRB2 functions as an adaptor protein to recruit SOS1 (28). As was mentioned, SOS1 is a Ras GEF, which allows for activation of K-Ras via GDP to GTP exchange (28). SOS also contains important plasma membrane binding domains called Pleckstrin homology (PH) domains that allow it to localize to the plasma membrane where MAPK most signaling takes place.
Since the majority of Ras signaling originates extracellularly, Ras typically anchors to the inner surface of the plasma membrane. Anchoring is achieved via aliphatic additions during post- translation modifications. These additions occur at the C-terminal end especially at a special “CAAX” motif (C denotes cysteine, A is usually an aliphatic amino acid and X may be any amino acid, but often a serine or methionine). Additions occur in a step-wise fashion and include farnesylation or geranylgeranylation. These additions generally occur in the endoplasmic reticulum after which the
“AAX” amino acids are removed from the CAAX motif and the cysteine is methylated and may be
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palmitoylated (29). Rho GTPases also contain CAAX and can undergo similar post-translational modifications.
Figure 1-1. Overview of Ras signaling
Once activated, K-Ras can signal through a variety of downstream mediators, the most important and well-studied of these pathways being the Raf-MEK-ERK flavor of MAPK pathways (other MAPK pathways exist). Raf, MEK and ERK are serine-threonine kinase, where each phosphorylates its downstream mediator in the signaling cascade.
Beginning with the first of these serine-threonine kinase, is Raf, of which there are three isoforms.
The first discovered was c-Raf (Raf-1), and then later B-Raf and A-Raf. c-Raf was the primary focus of most studies until about a decade ago when it was discovered that activating mutations of B-Raf
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are common in certain cancers such as melanoma, but A-Raf and c-Raf mutations are rare (30). In fact, B-Raf mutations are common in melanomaand thyroid cancer. B-Raf is thought to be the prototypic Raf as it shares the most homology with the Raf isoform expressed in lower organisms such as Drosophila or C. elegans (31). Embryonic deletion of either B-Raf or c-Raf is incompatible with life, though deletion of A-Raf, in at least some mouse models, does produce viable mice (31).
Relatively little is known about A-Raf although it is known to be widely expressed and not essential for Erk phosphorylation (32). Erk phosphorylation is deceased with either B-Raf and c-Raf deletion and the two isoforms compensate for loss of the other (31). Activation of Raf by Ras is complex and not fully understood but is known to require membrane translocation, phosphorylation, conformational changes, and homo-/hetero-dimerization events.
Raf activity is tightly regulated as a consequence of the protein’s interesting structure. Raf contains a C-terminal kinase domain that is constitutively inhibited by the protein’s own N-terminal end. The
N-terminal end contains Ras-binding domain (RBD), which specifically binds K-Ras:GTP, and, upon binding K-Ras:GTP, releases inhibition of the kinase domain allowing the Raf-MEK-ERK phosphorylation cascade to proceed (33). Interactions between Ras and Raf are mediated via the scaffolding protein kinase suppressor of RAS 1 (KSR1). Recent evidence has accumulated indicating the importance of B-Raf heterodimerization with c-Raf to activate MEK. Although B-Raf and c-Raf can both homodimerize, evidence suggests that most downstream signaling through MEK occurs through heterodimerization in which B-Raf activates c-Raf, which in turn phosphorylates MEK (34).
These complex heterodimerization events appear to be important for paradoxical increases in MEK phosphorylation with the use of B-Raf inhibitors (more on this subject in Chapter 4). Next, MEK phosphorylates and activates it’s only know target: ERK.
The final step of this process occurs when ERK interacts with, and phosphorylates, transcription factors (i.e. c-myc, Elk1, Ets1, Fos), chromatin remodeling factors (i.e. MSK, Rsk2) and regulators of
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translation (i.e. p90Rsk, Mnk1) (35). Importantly, ERK also phosphorylates upstream substrates of the Raf-MEK-ERK pathway resulting in negative feedback (i.e. phosphorylation of Sos1, B-Raf and c-
Raf).
In addition to the Raf-MEK-ERK pathway, there are several other important signaling pathways downstream of K-Ras, most notably the PI3K-AKT pathway. The prominent form of PI3K is composed of a p110 catalytic subunit and a p85 regulatory. This kinase phosphorylates phosphatidylinositol – a component of the plasma membrane – allowing proteins with pleckstrin homology domains to be recruited from the cytosol to the plasma membrane, the most important of these proteins being Akt (also known as protein kinase B, PKB). Upon recruitment to the cytoplasm,
Akt is then phosphorylated at threonine 308 by phosphoinositide dependent kinase 1 (PDPK1) and at serine 473 by mTORC2 (a complex composed of mTOR and Rictor). Once AKT is phosphorylated, it releases from the plasma membrane into the cytoplasm and phosphorylates huge numbers of downstream targets. PI3K-AKT pathway signaling modulates a variety of cellular processes including apoptosis (via Bad, Mdm2), cell cycle (via p21 and p27), translation, cell metabolism and cell growth (via mTOR signaling through 4EBP and S6K) and cytoskeletal reorganization (via Tiam1 and Rac) (35).
Given the very important functions of all of the pathways regulated by K-Ras, it is unsurprising that
K-Ras plays a very important role in many cancers. It is estimated that Ras is mutated in a third of all cancers. Moreover, it is thought that Ras mutation is an early event in many cancers and may even be the defining initiating event in certain cancers such as lung adenocarcinoma. K-Ras mutations are common in some of the deadliest cancers including lung, colon and pancreatic cancer.
Mutations of K-Ras typically occur at codons 12, 13 or 69 and function to ablate the intrinsic
GTPase activity of the protein, cause insensitivity to GAPs, and thus result in constitutively active K-
Ras signaling.
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Unfortunately direct pharmacological inhibition of K-Ras, or N/H-Ras, has remained elusive. A major obstacle is the lack of any potential binding pockets given the small size of these GTPases.
Inhibitors of targets upstream of K-Ras have been successful, notably EGFR tyrosine kinase inhibitors such as erlotinib and gefitinib (6). However, these inhibitors have no effect on cancers with K-Ras mutations since K-Ras is downstream of EGFR. Inhibitors downstream of K-Ras have been less successful, except for vemurafenib and dabrafenib which inhibit mutant BRAF (BRAF
V600E), which is not commonly mutated in lung cancers but common in melanoma. Another melanoma specific target therapy includes the MEK inhibitor trametinib (36). Other MEK inhibitors in current clinical trials include selumetinib and MEK162. Several other MEK and BRAF inhibitors are in Phase I and Phase II clinical trials.
A potential downstream target K-Ras that has received relatively little attention is that of the Rho
GTPases. As will be described later in this work, Rho GTPases may be a potentially efficacious antineoplastic target and also have implications in other medical realms as vasodilators for coronary artery disease, microvascular heart disease and hypertension. Current inhibitors include the RhoA inhibitor rhosin (formally called G04), and the ROCK inhibitors fasudil and Y-27632.
Other Rho GTPase pathway inhibitors have been directed against Rac, such as NSC23766, a Rac GEF inhibitor, or the Cdc42 inhibitor ML141.
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Introduction to Rho GTPases
Rho GTPases are a family of GTPases belonging to the Ras superfamily. Just as described with Ras,
Rho GTPases are also 21 kDa signaling proteins that act as molecular signaling switches. They signal when GTP-bound by interacting with effector proteins which relay signaling and effect many cellular processes from cell morphogenesis, cell migration, cell cycle, enzyme regulation and gene expression. Most all Rho GTPases have intrinsic GTPase properties that turn off signaling by hydrolyzing GTP to GDP, thus switching the protein into an inactive state, such that it no longer interacts with effector proteins. Rho GTPases are reactivated when the “used” GDP is swapped out for a “new” GTP, as depicted in Figure 1-2 below. As with Ras GTPases, intrinsic GTP hydrolysis is slow in Rho GTPases and the process of hydrolysis is greatly accelerated by GAPs. The exchange of
GDP for GTP requires GEFs. An additional level of regulation is provided by GDP dissociation inhibitors (GDIs), which bind to GDP-bound Rho, inhibit GDP dissociation and also block GEFs from binding Rho GTPases by sequestering them away from the plasma membrane.
Figure 1-2. GTPase activity of Rho GTPases.
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Figure 1-3. Homology Relations of Rho GTPase Family Members.
Adapted from Ellenbroek, S. I. J., & Collard, J. G. (2007). Rho GTPases: functions and association with cancer. Clinical & Experimental Metastasis, 24(8), 657–72. doi:10.1007/s10585-007-9119-1
To date, twenty six Rho GTPases have been found in mammals. Based on their primary sequence homologies these Rho GTPases can be classified into six subfamilies as outlined in Figure 1-3. The most important, and well-studied, subfamilies are that of Rho, Rac and Cdc42.
Rho GTPases Structure and Function
All Rho GTPases share a common nucleotide binding domain known as the G-domain (or G-box).
This domain also catalyzes GTP hydrolysis and hence the GTPase activity of the protein. Very similar to Ras GTPases, the structure of Rho GTPases changes depending on whether the protein is
GTP or GDP bound. These changes occur at two segments known as switch I and switch II (in the human protein these correspond to residues 28–44 and 62–69 respectively). These switches are stabilized when GTP bound, especially the conformation of switch I, but the segments vary widely
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in conformation when GDP bound (37). Conformation of the switches is important because they mediate interaction with GAP, GEFs and effector proteins. Importantly, the switch I region mediates binding to GEFs containing two important domains: the DH-PH domain-containing Dbl-related
GEFs and the DHR2 domain-containing DOCK family GEFs. Specificity as to which effectors bind which GTPases is at least in part mediated by the specific orientation and residues on β-strands (B2 and B3) as well as an α-helix (A5) (37). As for GAPs, these proteins contain a RhoGAP domain which binds to the switch regions and enhances the catalytic activity of the GTPase. Over 70 RhoGAP domain containing proteins have been identified in the human genome, and most, but not all of these proteins are indeed GAPs for Rho GTPases. Interestingly, Rho GTPase GAPs are often found to be deleted in many human cancers (38).
One difference unique aspect of Rho GTPases that differentiate them from other Ras superfamily
GTPases is a 12 residue α-helical structure known as the Rho-insert, but this insert is not located in the switch regions. Another interesting aspect of Rho GTPase biology is that these proteins require a coordinated Mg2+ ion to bind guanine nucleotides. Rho function, particularly that of RhoA, can be inhibited by bacterial toxins such as C3 exoenzyme, which specifically ribosylate Rho proteins in their effector binding domain, rendering downstream signaling impossible (39).
General Considerations of Rho GTPase Function The major downstream function of Rho GTPases is regulation of cell shape and structure through modulation of actin, and to lesser extent microtubules. Different Rho family members regulate different processes. The Rho subfamily (RhoA, B & C) regulate stress fiber formation and cell contraction, Rac members regulate lamellipodia, and Cdc42 regulates filopodia. Broadly speaking,
Rho GTPases regulate cell shape, adhesion, migration, phagocytosis, cytokinesis, neurite extension and retraction, developmental morphogenesis, polarization, growth, survival and transcription.
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Figure 1-4. Cellular Functions of Rho GTPases.
Activation of Rho GTPases and Rho Regulatory Proteins As has been explained, GTPase signaling is directly modulated by three classes of proteins: the Rho
GAPs, GEFs and GDIs. Similar to the activation of Ras described previously in this chapter, the activation of Rho GTPases usually starts with activation of cell-surface receptors such as cytokine, tyrosine kinase and adhesion receptors, and most importantly GPCRs.
The first mammalian Rho GEF was identified as a transforming gene leading to diffuse B-cell- lymphoma, and was thus called Dbl. As more Rho GEFs were discovered, it was found that Dbl belongs to a large family of Rho GEFs, which comprise the majority of all Rho GEFs, and are defined
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by a Dbl homology (DH) domain followed by tandem pleckstrin homology (PH) domains. Another notably Rho GEF is LARG (Leukemia-Associated Rho GEF, also called ARHGEF12) was originally identified as part of the leukemia fusion protein with the mixed-lineage leukemia (MLL) gene as part of a common chromosomal translocation in acute myeloid leukemia. As explained earlier in this chapter, the DH domain is important for interacting with the switch 1 domain of Rho GTPases.
In many instances, Rho GEFs exhibit auto-inhibition, and there have been many instances of deletion of the auto-inhibitory region found in variety of cancers, especially leukemia. Relief of auto-inhibition and activation of Rho GTPase signaling by Rho GEFs can occur through many methods. Examples include phosphorylation of Rho GEFs by the βγ subunits of GPCR, or phosphorylation of Tiam1 by calmodulin-dependent (CaM) kinase 2 or by protein kinase C, allowing it to activate Rac1. Another method is binding of the Rho GEF to phosphatidylinositol products via the PH domain resulting in conformational changes that relieve auto-inhibition, such as occurs in the Vav1 Rho GEF. Another method of relieving auto-inhibition is through protein- protein interactions, such as when the activated Gα subunit of a GPCR binds the DH–PH portion of p115-RhoGEF, resulting in increased GEF activity.
Rho GTPases can conversely be deactivated by GAPs, such as breakpoint cluster region protein
(BCR) which is infamous as part of the fusion gene encoded by the Philadelphia chromosome in chronic myelogenous leukemia. BCR is a Rac and Cdc42 GAP, though it is unclear how Bcr is activated. These proteins all share a conserved GAP domain which is key to their GTPase activating activity. One of the best studied Rho GAPs is β2-chimerin which is a Rac GAP. β2-chimerin is activated and can associate with Rac when it is on the plasma membrane – a process which is facilitated by diacylglycerol (DAG) in response to phospholipase C activation, which is itself activated by a variety of signals such as EGFR and other GCPRs.
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Figure 1-5. Rho Subfamily of GTPases and Main Effector Pathways.
Actin filaments Rho GTPases regulate actin polymerization and assembly – as filaments, lamellipodia, filopodia, and contractile rings – and also regulate the interaction of actin with myosin.
The Rho subfamily (RhoA, B & C) regulate the actin cytoskeleton through formins, specifically the mammalian Diaphanous-related formins – mDia1 and mDia2. mDia proteins are able to elongate existing actin filaments (Figure 1-5). mDia exists in an auto-inhibited state, however, upon binding to Rho:GTP via its GTPase-binding domain (GBD) the protein is released from auto-inhibition and is able to bind to actin monomers (G-actin) via its formin homology domain (FH2) and add them to the end of actin monofilaments (F-actin), thus catalyzing polymerization of actin filaments.
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Whereas formins act to elongate actin filaments, the other major Rho subfamily effector ROCK acts to increase myosin activity. There are two isoforms of ROCK: ROCK1 and ROCK2. Both isoforms are composed of an N-terminal catalytic domain, a coiled-coil domain, and a C-terminal PH domain, which regulates the kinase activity.
ROCK is a serine/threonine kinase, and has three major targets: myosin regulatory light chain, myosin light chain phosphatase and LIM-kinase (as outlined in Figure 1-5). It activates myosin regulatory light chain and LIM-kinase, but inhibits the activity of myosin light chain phosphatase, all of which results in increased myosin activity, and thus impacts important cellular activities such as cytokinesis, cell movement, cell shape, apoptotic membrane blebbing, and smooth muscle contraction.
In the typical smooth muscle cell, intracellular influx of calcium ions results in activation of myosin light chain kinase in a calcium/calmodulin mediated process. Activation of myosin light chain kinase results in phosphorylation of myosin light chain, which enables crossbridge formation with actin. In myosin II – the major myosin isoform – this occurs at threonine 18 and serine 19, though there are many isoforms of myosin. Similarly, ROCK can directly phosphorylate myosin light chain at serine 19. These activating phosphorylations can be removed by myosin light chain phosphatase.
ROCK inhibits myosin light chain phosphatase by phosphorylating it.
LIM kinase is also a serine/threonine kinase that has two zinc finger motifs, known as LIM motifs, within its regulatory domains. The main two isoforms of LIM kinase are LIMK1 and LIMK2. ROCK, and also PAK, increase LIMK activity by phosphorylating its activation loop at threonine residues
505 and 508. The main function of LIMK is to inhibit actin depolymerization. To that end, LIMK phosphorylates cofilin at serine 3, which decreases the activity of this actin depolymerization protein. Specifically, cofilin acts to depolymerize and sever actin filaments, and phosphorylation of cofilin at serine 3 results in its translocation from the cytoplasm to the nucleus.
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Lastly, and briefly, I will describe the mode of action of Rac and Cdc42. Rac and Cdc42 form lamellipodia and filopodia, respectively (40). Both Rac and Cdc42 regulate the Arp2/3 complex, which serves to create new “daughter branches” of actin filaments off of existing “mother” filaments at characteristic 70-degree branches (41). The Arp2/3 complex – named as an acronym of Actin-
Related Protein – consists of the highly conserved and important Arp2 and Arp3 proteins with
ATPase activity, along with five scaffolding proteins named ARPCs 1 through 5. Rac and Cdc42 regulation of Arp2/3 occurs through intermediary proteins. In the case of Cdc42, GTP-bound Cdc42 is enabled to bind Wiskott-Aldrich syndrome proteins (WASP), more specifically N-WASP, which disinhibits the protein allowing binding and activation of the Arp2/3 complex. Rac functions in a similar manner by binding WAVE (WASP-family verprolin-homologous protein) (41).
Microtubules Rho GTPases also control microtubules, which are important for a variety of cellular functions such as cell polarity, organelle localization, centrosome dynamics and formation of the mitotic spindle.
The relationship between Rho GTPases and microtubules is less clear than with the actin cytoskeleton. Briefly, it is clear that Rho GTPases are critical to cell motility as is the microtubule cytoskeleton. It has been suggested that Rac GTPases are important for focal adhesion initiation whereas Rho subfamily GTPases promote focal adhesion maturation. As an example, integrins accumulate at focal adhesions at the leading edge of a cell, where APC plays an important role, and in conjunction with CLIP and EB1, interacts with the Rac/Cdc42 effector protein IQGAP1 (42).
Cell shape and movement independent affects Rho GTPases have been shown to be involved directly in gene expression. The best studied example of this is via SRF (serum response factor) which binds to serum response elements and upregulates transcription of many genes important for cell cycle progression, growth and differentiation.
However SRF-mediated transcription requires the binding partners such as MLK (megakaryoblastic
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leukemia 1, also known as MAL). MLK is normally sequestered by binding to G-actin in the cytosol.
Once Rho GTPases induce F-actin formation, MLK translocates to the nucleus to interact with SRF and induce transcription. Additionally, Rho GTPases play an important role in cell cycle progression. Specifically, RhoA and Rac1 promote transition through the G1-S checkpoint by upregulating cyclin D1 levels, and negatively regulating the cell cycle inhibitors p21cip1 and p27kip1.
The mechanisms are unknown, but many studies have reproduced these cell cycle control effects and some have shown them to be ROCK-dependent (43).
The Intersection of Rho GTPases and Cancer
Pro-neoplastic aspects of Rho A Signaling Elevated levels of Rho GTPase proteins are found in a wide variety of cancers. Specifically, elevated levels of RhoA protein have been found in breast cancer, lung cancer, colon cancer, esophageal and neck squamous cell carcinomas, gastric cancer, hepatocellular carcinoma, ovarian cancer, testicular germ cell cancer, and bladder cancer (44–51). More important than protein levels, some reports have demonstrated increased Rho GTPase activity in breast, colon and lung cancers (44,45).
Despite consistent findings of either increased Rho GTPase protein levels or activity, only recently have there been reports of functionally relevant mutations in Rho GTPases. Two reports utilizing whole genome sequencing of over 120 melanoma samples found a frequent and previously undescribed Rac1 mutation. These two reports show that ~10% of sun-exposed melanomas have a recurrent Rac1(P29S) mutation that apparently increases both GTP bound states and increased binding to effector proteins (52,53). Two other reports also utilizing whole genome sequencing found recurrent RhoA mutations in ~15-25% of gastric carcinomas (54,55). The mutations appear
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to be gain-of-function mutations in the switch regions related to effector and GEF binding, but the functional significance of the mutations remains unclear.
Additionally, mutations have been described in signal proteins downstream of RhoA and RhoC.
Again, utilizing whole genome sequencing of lung adenocarcinomas a group reported duplications of exons in ROCK that presumably results in a gain-of-function (11). Evidence supporting the importance of RhoA signaling through ROCK was recently established in a basic science study investigating K-Ras driven lung cancer in mice (56). In this report it was shown that tumors almost completely regressed when mice were treated with a combination of fasudil (a ROCK inhibitor) and bortezomib (a proteasome inhibitor used to treat melanoma).
The first evidence that Rho GTPases could promote cancer came from work using fibroblasts. Since
Ras was known to transform cells efficiently it was hypothesized that similar GTPases such as the
Rho family may also be involved in transformation. The first studies found that constitutively active
RhoA and Rac were modestly efficient at transforming cells (57–62). Interestingly, it was found that the efficiency of transformation could be increased synergistically when combined with oncogenic
Raf mutants (also only modestly efficient at transformation) (60). The converse was also found; specifically that dominant negative RhoA or Rac could block Ras transformation (60,61). These early studies laid the groundwork for the next 20 years of research into the role of Rho GTPases in cancer.
An important study implicating Rho involvement in Ras-driven tumorigenesis was performed in
Drosophila. In this powerful study, a forward genetic screen was utilized to find genes that cooperate with Ras to drive eye hyperplasia/tumorigenesis (63). Specifically, the authors used random translocation of a strong eye specific promoter system. Interestingly, the majority of positive hits in their screen belonged to Rho GTPase pathway including Rho1, Rac1 and RhoGEF2,
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which all enhanced Ras-driven eye hyperplasia in a JNK pathway dependent manner. The authors also showed that dominant negative Rho and Rac blocked tumorigenesis.
Another piece of evidence is the RhoA and Ras connection to SRF, a transcription factor that binds to serum response elements and induces the expression of genes important for cell cycle progression, growth, division and differentiation, such as c-fos and jun (64,65). SRF is regulated by
MAPK pathways such as the K-Ras and JNK pathways. To this effect, SRF works in combination with other transcription factors such as MLK (megakaryoblastic leukemia 1, also known as MAL) and
TCF-containing transcription factors such as Ets-1. MLK remains in the cytoplasm when bound to G- actin. However, upon serum stimulation, Rho activity increases directly resulting in F-actin polymerization and stress fiber formation; thus decreasing G-actin levels. With reduced binding to
G-actin, MLK is able to translocate to the nucleus and interact with SRF (66).
More recently other studies have implicated RhoA directly in lung cancer using murine models of lung cancer including the RhoA-ROCK study alluded to earlier, as well as another study of murine
KRas-driven, INK4A/ARF-deficient lung adenocarcinomas , which found the RhoA-FAK axis to be vital to tumorigenesis (56,67)
RhoB and RhoC in Cancer In contrast to findings suggestive of increased Rho GTPase activity in many cancers, it has actually been found that RhoB is deleted in many cancers including lung (68–70). These findings are consistent with the finding that RhoB is an inhibitory isoform to the effects of RhoA and RhoC (71).
Accordingly, RhoB deletion was shown to accelerate chemically induced skin tumors (72).
RhoC has long been implicated in cell movement and metastasis. The first mouse model to address the role of RhoC in metastasis in vivo was that of Hakem and colleagues after they produced a constitutively RhoC-null mouse that, surprisingly, had no baseline abnormal phenotype (73). To assess the effect of RhoC on metastasis the authors used the MMTV-PyVT transgenic mouse [mouse
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mammary tumor virus (MMTV) driven Polyoma Virus middle T antigen (PyVT)], which develops mammary tumors that metastasize to the lung with high penetrance. Using this model, the authors showed that RhoC-null mice had dramatically fewer metastases to the lung. Moreover, they confirmed their results in vitro, by assaying mammary tumor cells in transwell Matrigel invasion chambers (73).
Rac and Cdc42 Signaling in Cancer Both Rac and Cdc42 have been implicated in cancers as well. One important study demonstrated that Rac1 and Cdc42 directly bind to and activate p110β, a subunit of PI3K (74). P13K is well known to be important in many cancers, and in the case of this study it was shown that deletion of the RBD of p110β – the “Rho Binding Domain” through which Rac1 and Cdc42 bind – blocked bleomycin induced lung injury. These reports were further strengthened by evidence utilizing Raf,
MEK, P13K, mTOR, IGF1R and ROCK inhibitors in both K-Ras-driven mouse models of lung adenocarcinoma and lung cancer cell lines (75,76).
Several studies have shown the importance of Rac1 in lung cancers specifically (44,77). More specifically, the Rac1b splice variant of Rac1 has emerged as a variant that is upregulated in several cancers such as lung, breast, colon and thyroid cancers (45,78,79). Rac1b is an important splice variant of Rac1 that contains a 19 amino acid insert adjacent to the switch II domain. The effect of this insert appears to be increased Rac1b activity with decreased GTPase ability, but with differential binding to certain effectors as compared to Rac1 (80). These differences may contribute to increased ROS generation (81). Also, there is evidence that in more than one human cancer, that
Rac1b cooperates specifically with BRAF V600E (79,82).
Yet another important link between Rho GTPases and cancer is through the Rac and Cdc42 effector
PAK (p21-activated kinase), which has been shown to phosphorylate important cell signaling proteins such as Bcl-2, Raf1 and is part of the MAPK, JNK and NF-kappa-B pathways. There are
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multiple isoforms of PAK and unsurprisingly PAKs have been found to upregulated in several human cancers (83).
Other forms of cross-talk between Ras and Rho GTPases have been documented via GEFs. For instance it has been shown that the Rac GEF Tiam1 is also a GEF for H- and K-Ras, though interestingly both its upregulation and deletion have been implicated in initiation and metastasis, respectively (84). Also, the Rho GEF H1, a RhoA GEF, is a critical part of a positive feedback loop in the Ras pathway via direct interaction with KSR-1 (85).
Possible anti-neoplastic aspects of Rho GTPases Until very recently, the consensus in the scientific literature appeared to be that Rho GTPases are uniformly pro-neoplastic (except for RhoB). However, very recently data has emerged to challenge this notion. A recent study of KRas-induced hepatic adenoma formation in Zebra fish found that constitutively active RhoA (RhoAG14V) resulted in smaller adenomas and increased survival, and that dominant negative RhoA (RhoAT19N) resulted in larger adenomas and decreased survival (86).
These results are exactly the opposite of studies done in transforming fibroblasts with KRas and
RhoA. The authors found that increased neoplasia resulting from dominant negative RhoA was in part due to increased AKT and S6 signaling, and upregulation of cyclin D1. This finding is in line with two in vitro studies which found RhoA negatively regulated AKT phosphorylation and decreased cyclin D1 levels in endothelial cells and KRas-driven adrenocortical cancer cell lines
(87,88). Another recent study of a murine model of colon cancer utilizing mutant APC, found that simultaneous expression of dominant negative RhoA (RhoAT19N) resulted in more adenomas, larger adenomas and decreased survival (89).
Another compelling finding has been recent whole exome sequencing of angioimmunoblastic and peripheral T cell lymphomas, which found that 68% of these T cell lymphomas had the same recurrent RhoAG17V mutation (90). The authors demonstrated that not only was this mutated RhoA
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protein inactive and unable to bind GTP, but that it was able to inhibit wildtype RhoA signaling as well.
The vast majority of the literature supports the notion that RhoA and Rho GTPases in general are pro-neoplastic. More recently however, there have been both animal models of cancer and genetic studies of human cancers that support the counter-idea that Rho GTPases can also have anti- neoplastic properties. These conflicting results likely mean that the role of Rho GTPases in cancer is not entirely straight-forward and is dependent on the cancer-type and specific drivers of a particular cancer.
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Chapter 2 – Loss of RhoA Exacerbates, Rather than
Dampens, Oncogenic K-Ras Induced Lung
Adenoma Formation in Mice.
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Title Page Zandvakili et al.
Loss of RhoA exacerbates, rather than dampens, oncogenic K-Ras induced lung
adenoma formation in mice.
Authors: Inuk Zandvakili1,2,3, Ashley Kuenzi Davis1, Guodong Hu1 and Yi Zheng1,2,3
Affiliations:
1Experimental Hematology and Cancer Biology, Cincinnati Children’s Hospital Medical Center,
Cincinnati Ohio, USA
2Molecular and Developmental Biology Graduate Program, Cincinnati Children’s Hospital Medical
Center, Cincinnati Ohio, USA
3Medical-Scientist Training Program, College of Medicine, The University of Cincinnati, Cincinnati
Ohio, USA
Contact:
Inuk Zandvakili: [email protected]
Ashley Kuenzi Davis: [email protected]
Guodong Hu: [email protected]
Yi Zheng (corresponding author): [email protected]; 3333 Burnet Avenue, Cincinnati, OH 45229,
USA. Phone: 513-636-0595; Fax: 513-636-3768
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Abstract Zandvakili et al.
Abstract
Numerous cellular studies have indicated that RhoA signaling is required for oncogenic Ras- induced transformation, suggesting that RhoA is a useful target in Ras induced neoplasia. However, to date very limited data exist to genetically attribute RhoA function to Ras-mediated tumorigenesis in mammalian models. In order to assess whether RhoA is required for K-Ras-induced lung cancer initiation, we utilized the K-RasG12D Lox-Stop-Lox murine lung cancer model in combination with a conditional RhoAflox/flox and RhoC-/- knockout mouse models. Deletion of the floxed Rhoa gene and expression of K-RasG12D was achieved by either CCSP-Cre or adenoviral Cre, resulting in simultaneous expression of K-RasG12D and deletion of RhoA from the murine lung. We found that deletion of RhoA, RhoC or both did not adversely affect normal lung development. Moreover, we found that deletion of either RhoA or RhoC alone did not suppress K-RasG12D induced lung adenoma initiation. Rather, deletion of RhoA alone exacerbated lung adenoma formation, whereas dual deletion of RhoA and RhoC together significantly reduced K-RasG12D induced adenoma formation.
Deletion of RhoA appears to induce a compensatory mechanism that exacerbates adenoma formation. The compensatory mechanism is at least partly mediated by RhoC. This study suggests that targeting of RhoA alone may allow for compensation and a paradoxical exacerbation of neoplasia, while simultaneous targeting of both RhoA and RhoC is likely to lead to more favorable outcomes.
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Introduction Zandvakili et al.
Introduction
In the United States, lung cancer kills more people each year than breast, prostate and colon cancer combined (1). Lung adenocarcinoma is the most common subtype of lung cancer and often harbors activating mutations of K-Ras (11). K-Ras is a founding member of the Ras GTPase superfamily and is a key signal transduction protein that integrates extracellular stimuli and promotes cell proliferation and survival. Activating mutations of K-Ras disrupt the GTPase activity of the protein, increasing levels of GTP-bound K-Ras, which results in continuous signaling. Despite being among the very first oncogenes discovered, direct pharmacological inhibition of K-Ras has remained elusive (91). An alternative strategy to inhibiting K-Ras directly is to target its downstream signaling pathways. While the RAF-MEK-ERK signaling pathway is the most important transducer of K-Ras signaling, several other downstream signaling axes including the PI3K-AKT-mTOR pathway, Ral GTPases, and the Rho family of GTPases have each been implicated as required elements for Ras induced transformation.
The mammalian Rho GTPase family includes over 20 members, of which RhoA, Rac1 and Cdc42 are among the best characterized. These Rho GTPases regulate the cell cycle and actin cytoskeleton and are thus critical regulators of processes such as cell shape, adhesion, migration, polarity and proliferation (40). Given these essential functions of Rho GTPases and the availability of pre-clinical and clinical inhibitors of Rho GTPase signaling, they pose an important topic in cancer research
(92–95). Importantly, Rho GTPases have been shown to be critical for Ras-induced transformation of fibroblasts and epithelial cells (59–62,96). Over a decade ago, several classic studies in the
GTPase field demonstrated that blocking RhoA signaling could suppress Ras-induced transformation, and conversely that constitutively active RhoA could cooperate synergistically with
Raf to promote cell transformation (59,60,62). Additionally, studies have shown that RhoA plays an important permissive role in cell cycle progression through the G1-S phase: namely, increased RhoA activity inhibits p21Waf1/Cip1 and results in increased amounts of cyclin D1 and p27Kip1 (97–99).
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Introduction Zandvakili et al.
Indeed, blockage of RhoA signaling is thought to induce INK4 activity, in turn halting the cell cycle
(99). Thus, the plurality of evidence shows RhoA is a positive regulator of the cell cycle.
Subsequently, RhoA has been found to be either overexpressed or hyperactive in a variety of cancers, and RhoA activity is correlated with negative outcomes in gastric, hepatocellular, esophageal squamous cell, breast and lung carcinomas (44–47,51,100).
More recently, greater attention has been paid to the role of Rac1 in tumorigenesis due to the availability of murine genetic models. These studies have confirmed a positive role of Rac1 in tumor initiation. Rac1 was found to be required for K-Ras-induced lung adenoma formation in mice and a
Rac1 splicing variant with increased activity, Rac1b, appears to promote tumorigenesis in this context (77,78). Two other recent studies have indicated that pharmacological inhibition of the downstream mediators of RhoA signaling, ROCK and FAK, are promising therapeutic targets
(56,67). However, to date no mammalian genetic studies have directly assessed whether RhoA is required for oncogenic Ras-mediated tumorigenesis.
In the present work, we have sought to investigate the role of RhoA and the closely related Rho
GTPase, RhoC, in K-Ras-induced tumorigenesis using a well-established inducible K-RasG12D knock- in mouse model of lung adenoma induction in combination with our RhoA conditional knockout,
RhoAflox/flox, and RhoC-/- mouse models. Our investigation of these genetic studies produced surprising results that individually RhoA and RhoC are dispensable for the oncogenic K-Ras induced lung tumorigenesis and loss of RhoA alone exacerbates, rather than suppresses, tumor initiating activity. Our study implies that pharmacological targeting of multiple Rho family members, e.g.
RhoA and RhoC in this context, is required to prevent tumorigenesis.
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Results Zandvakili et al.
Results
We first sought to determine whether mice would be viable without RhoA expression in their bronchiolar epithelium. We crossed a RhoA conditional mouse (RhoAflox/flox) with mice harboring the Club Cell Secretory Promoter Cre (CCSP-Cre), as outlined in Table 1. This promoter is expressed mainly in Club cells, eponymously referred to as Clara cells, which are a major component of the bronchial epithelium (101).
Mice harboring CCSP-Cre RhoAflox/flox transgenes were born at the expected Mendelian ratio, did not differ in weight during weaning or adulthood and lived as long as Cre-negative littermates. These mice did not display any signs of overt respiratory distress. Their lungs were normal appearing both grossly and microscopically (Fig. 1A&B). We wondered whether the Cre expression would be altered by RhoA deletion so we crossed these mice to the double-reporter “mTmG” mouse. These mice constitutively express tdTomato, which switches to eGFP with the expression of Cre- recombinase (102). We found Cre expression in a patchy distribution within bronchiolar epithelium, which was similar between RhoAcKO and RhoWT backgrounds (Fig. 1C).
We next assessed for the penetrance of RhoA deletion. Using immunohistochemistry, we found
RhoA was deleted from the bronchioles in a patchy pattern similar to the distribution of eGFP in the reporter mice (Fig. 1D). Recombination of the RhoAflox/flox allele was also evident in RhoAcKO via PCR with RhoA deletion specific primers (S1 Fig.). Moreover, we did not find any qualitative differences in the expression pattern of Club cells or Type II alveolar cells as assessed by CCSP and SPC staining, respectively (Fig. 1E&F). Thus, deletion of RhoA from Club cells by the CCSP-Cre resulted in viable mice with no signs of respiratory dysfunction. There were no overt differences between RhoAcKO mice and control mice with regards to lung architecture or the amount and distribution of major lung cell types.
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Results Zandvakili et al.
Next we sought to determine whether RhoA is required for oncogenic K-Ras-induced tumor initiation in vivo. We bred the Lox-STOP-Lox K-RasG12D (LSL-K-RasG12D) transgene into the CCSP-
Cre;RhoAflox/flox mice. At least two different constructs of CCSP-Cre (also referred to as CC10-Cre) have been successfully used to induce lung tumors in mice (103–106). It was also previously shown that CCSP-Cre LSL-K-RasG12D mice develop a strong inflammatory component to their disease with abundant infiltration of alveolar macrophages and neutrophils (103,104). Consistent with previous works, we found the CCSP-Cre;LSL-K-RasG12D;RhoAflox/flox mice to have grossly abnormal lungs with abundant infiltration and adenoma formation (Fig. 2A&B). These mice also displayed prominent hyperplasia of the bronchiolar epithelium and atypical adenomatous hyperplasia (AAH) (107). We did not find any differences in the disease pathology of RhoAcKO mice in comparison to RhoWT mice and both groups formed adenomas. Only a proportion of the adenomas in either group stained positively for pERKThr202/Tyr204, in agreement with previous reports (Fig. 2C) (67,108).
Immunohistochemical staining of the lungs of these mice demonstrated positive RhoA staining for all adenomas in the RhoWT group, whereas a fraction of adenomas stained positively in the RhoAcKO group (Fig. 2D&E). Although only ~15% of the adenomas observed were RhoA-null in the RhoAcKO group, we found numerous RhoA-null hyperplastic growths, including AAH (S2A Fig.).
Given that most adenomas evaded RhoA-deletion by CCSP-Cre, our results imply that there is selection pressure for the maintenance of RhoA signaling during K-RasG12D-induced transformation.
Our results are similar to the finding that Rac1 is required for K-Ras-induced adenoma formation by
Kissil et al. (77). In their findings, Kissil et al. utilized a similar approach with Rac1flox/flox transgenic mice and found the Rac1 locus was never recombined, and always remained undeleted in adenomas. Our findings differ in that we do find RhoA-null adenomas, thus demonstrating that
RhoA signaling is not essential for oncogenic K-Ras-induced adenoma formation.
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We wondered if other closely related Rho GTPases may be redundant for RhoA function, and decided to investigate RhoC as a candidate since RhoC shares a very high degree of sequence homology with RhoA and is also thought to be pro-tumorigenic (109). We did not investigate the third Rho subfamily member, RhoB, because gene targeting and other studies have shown it plays an opposite function to RhoA (68–71). As previously reported, constitutively RhoC null mice, RhoC-
/- mice, are viable and do not demonstrate any overt phenotype (73). Similar to RhoA deletion alone,
CCSP-Cre;RhoAflox/flox;RhoC-/- double-knockout (DKO) mice were born at the expected Mendelian ratio, did not display any signs of overt respiratory distress and their lungs were grossly normal
(Fig. 3A&B). When crossed with the mTmG reporter line we found no differences in Cre expression between RhoWT, RhoC-/- or DKO mice (Fig. 3C). We found RhoA expression, as assessed by immunohistochemistry, to be similar between RhoC-/- and RhoWT mice (Fig. 3D). RhoA expression in double-knockout mice was similar to that of RhoAcKO mice (Fig. 3D). Specifically, we found RhoA deletion from the bronchiolar epithelium in DKO mice to be in a patchy distribution (Fig. 3D).
Effective RhoA-locus recombination was also evident by PCR (S1 Fig.). We did not find any qualitative differences in the expression pattern of Club cells or Type II alveolar cells in any of these mice as assessed by CCSP and SPC staining, respectively (Fig. 3E&F). Thus, dual RhoA and RhoC deletion from a large proportion of the bronchiolar epithelium does not have any substantial effect on normal lung development or histology.
We further assessed whether double deletion of RhoA and RhoC would affect K-RasG12D induced adenoma formation. We crossed RhoAflox/flox;RhoC-/- mice with the CCSP-Cre;LSL-K-RasG12D line and observed adenoma formation in both RhoC-/- and DKO mice (Fig. 4A&B). It was previously shown that RhoC-/- mouse embryonic fibroblasts are able to form Ras-induced colonies in agar, and that
RhoC-/- mice can form MMTV-PyMT induced breast tumors (73), but to the best of our knowledge this is the first time RhoC has been shown to be dispensable for Ras-induced tumor formation in vivo. We found that these adenomas stained positively for pERKThr202/Tyr204 by
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immunohistochemistry (Fig. 4C). Additionally, immunohistochemistry for RhoA showed that almost all adenomas in the RhoC-/- and DKO stained positively for RhoA (Fig. 4D&E). These results raise the possibility that there is a strong selective disadvantage for RhoA deletion during adenoma formation in the absence of RhoC. While a RhoA deletion band is present by PCR (S1 Fig.), it may be derived from heterozygous RhoA lox recombination or non-adenoma tissue. Deletion of either
RhoA or RhoC did not result in differences in Ki67 or cleaved-caspase 3 staining of adenomas suggesting that RhoA and RhoC status do not affect cell proliferation or survival (S2B and S2C Fig.)
Importantly, we found that “RhoA-null” tumors in DKO mice maintained pMLCSer20 staining, suggesting compensatory activity for RhoA loss (S3 Fig.). Taken together, these results indicate that while RhoA and RhoC are each dispensable for K-RasG12D mediated tumorigenesis, together they contribute to adenoma formation.
To eliminate potential developmental effects of K-RasG12D and RhoA deletion during mouse development, as well as to achieve more robust RhoA deletion, we next used adenoviral mediated induction of lung adenomas to yield a sporadic tumor model (110,111). Six- to eight-week-old mice were administered intratracheal Cre-expressing adenovirus (Adeno-Cre) in order to simultaneously induce oncogenic K-Ras and delete the floxed Rhoa gene. Twelve weeks post- adenoviral induction mice were euthanized and their lungs harvested. Hematoxylin and eosin
(H&E) staining demonstrated adenoma formation in RhoWT, RhoAcKO, RhoC-/- and DKO mice (Fig.
5A). As before, only a proportion of the adenomas stained positively for pERKThr202/Tyr204 in each group (Fig. 5B). Immunohistochemical staining revealed positive RhoA staining in all adenomas from the RhoWT, RhoC-/- and DKO groups (Fig. 5C), whereas only a subset of adenomas from the
RhoAcKO group expressed RhoA (Fig. 5C&D). Therefore, similar to the CCSP-Cre model, RhoA-null adenomas were able to form by Adeno-Cre induction in K-RasG12D;RhoAflox/flox mice, indicating RhoA alone is not essential for K-RasG12D mediated tumorigenesis.
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We quantified the tumor burden of the Adeno-Cre mice via serial sectioning of their lungs.
Surprisingly, we found that there were many more adenomas in the RhoAcKO group as compared to the control, RhoWT group (Fig. 5E), and in addition to quantity, many adenomas in the RhoAcKO group are larger in size than those in controls (Fig. 5F). Interestingly, analysis of the RhoA status of the larger adenomas within the RhoAcKO group revealed that the relatively larger adenomas
(>10,000 μm2) were ones in which RhoA was not fully deleted. Quantification of the number and size of tumors revealed similar numbers and sizes of tumors between RhoWT and RhoC-/- mice (Fig.
5E&F). However, DKO mice had fewer adenomas and these tumors were smaller than those formed in either RhoWT or RhoC-/- mice (Fig. 5E&F). Interestingly, we found noticeably increased amounts of hyperplastic lesions in the RhoAcKO group (S4 Fig.). Similar to the CCSP-Cre model, increased hyperplasia did not translate to differences in Ki67 or cleaved-caspase 3 staining of adenomas regardless of RhoA status (S4B and S4C Fig.).
Due to the heterogeneity of RhoA-positive and RhoA-null adenomas in the RhoAcKO mouse group, we subdivided tumors based on their RhoA deletion status in our analysis. The RhoA expression status of each adenoma was quantified by immunohistochemistry. We found that all adenomas in the RhoWT, RhoC-/- and DKO groups expressed RhoA in the Adeno-Cre induction model (Fig. 5G). In particular, every adenoma found in the DKO group retained RhoA and escaped recombination of the
RhoAflox/flox allele (Fig. 5G) whereas a portion of the tumors in the RhoAcKO group was completely depleted of RhoA, similar to that of the CCSP-Cre model (Fig. 5D&G). However, the sum of the RhoA- null tumors in the RhoAcKO group did not account for the overall increased numbers of tumors in this group (Fig. 5G). This data suggests that there is a strong selection advantage to RhoA expression in the absence of RhoC during K-RasG12D-driven adenoma formation.
Finally, we microdissected Adeno-Cre induced adenomas from mice with different Rho backgrounds and assessed downstream signaling via Western blot (Fig. 5D). Both RhoA and RhoC
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Results Zandvakili et al.
are known to bind to and signal via ROCK and regulate phosphorylation of MLC (112,113). We found that RhoA-null adenomas maintained or upregulated pMLCSer19 signaling, suggesting likely compensation by RhoC or other related Rho GTPases. The upregulated levels of pMLCSer19 in
RhoAcKO mice correlate with the increased tumorigenesis in these mice. Moreover, we found that pMLCSer19 levels are not increased in DKO mice, correlating with the observation that these mice have both smaller and fewer tumors than RhoWT, RhoAcKO or RhoC-/- background mice. These observations are consistent with the interpretation that there is a selection pressure to maintain downstream pMLC signaling when RhoA signaling is lost.
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Discussion Zandvakili et al.
Discussion
Several previous studies of the relationship between RhoA and Ras signaling in cell transformation prompted our current investigation of whether RhoA or RhoC are required for K-Ras-induced tumor formation (60,61,96). The main readouts of the earlier studies were anchorage-independent growth in soft agar, cell proliferation, decreased requirement of serum for growth, and focus formation in the Petri dish. These studies addressed two important questions: whether increased
RhoA signaling could lead to transformation of cells and whether abrogation of RhoA signaling could prevent Ras-induced transformation. To answer the first question, several studies reported that over expression of wild type RhoA, or constitutively active RhoA or Rac1, can weakly transform cells, suggesting an oncogenic role of active RhoA (57–62). Transformation of cells expressing active Raf were synergistically enhanced with constitutively active RhoA (60). Answering the second question, it was found that dominant negative forms of RhoA can block Ras-induced transformation (60,61). However, these classical cell biology studies were limited by the nature of the approaches – dominant-negative or constitutively active mutant over expression – by the models – mostly fibroblasts and tumor cell lines – and by the in vitro nature of the readouts.
Nevertheless, these works laid out the conventional rationale that RhoA is a proto-oncogene product and RhoA targeting may be of therapeutic value (109).
Our investigation focuses on addressing the role of RhoA in K-Ras-driven tumorigenesis in vivo in established mouse models of lung cancer. First, it appears that neither RhoA nor RhoC is required for normal lung developmental or histology in mice. Second, our data shows K-RasG12D-induced adenomas can form in the absence of RhoA, indicating that RhoA is not required for K-RasG12D- induced tumorigenesis. Third, the closely related RhoA homolog, RhoC, which has been implicated as an important pro-tumor metastasis molecule, is also dispensable for K-RasG12D-induced tumorigenesis. Fourth, we found an increase in the numbers of adenomas in the RhoAcKO sporadic lung tumor model in which RhoA is deleted by adenoviral expression of Cre. Fifth, an attempt at
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Discussion Zandvakili et al.
combined deletion of RhoA and RhoC resulted in reduced adenoma formation and retention of
RhoA in all adenomas. These results indicate that there is a selective advantage to retain RhoA in the absence of RhoC, and there is likely a redundant and/or compensatory role of RhoC in RhoA signaling downstream of oncogenic K-Ras in adenoma formation. Consistent with this interpretation, the phosphorylation status of MLC, downstream of RhoA and RhoC signaling, was not decreased in either RhoA or RhoC-null adenomas, nor in adenomas from DKO mice, in which
RhoA is retained.
Our counterintuitive findings are in line with several recent studies that have begun to challenge the conventional paradigm that RhoA is a simple proto-oncogene. Two whole-exome sequencing studies of human lymphomas found recurrent Gly17Val mutations in RhoA which confer a dominant negative or loss of function phenotype (90,114). Another very recent study using two different mouse models of colon cancer found that mice carrying a dominant negative RhoA transgene (RhoAT19N) had increased tumor burden (89). Furthermore, a study of K-Ras-driven liver tumorigenesis in Zebrafish has found that an active form of RhoA (RhoAG14V) hindered tumor formation whereas dominant-negative RhoA (RhoAT19N) accelerated tumorigenesis (86). While the pro-oncogenic mechanism of RhoA function loss remains unclear, our work suggests that other Rho family members such as RhoC may compensate for RhoA loss, or possibly even “overcompensate”, resulting in a paradoxical increase in tumorigenesis.
A limitation of our mouse model in this study is that RhoA is deleted at the same time as K-RasG12D is expressed, and thus our work does not address whether RhoA plays a role in tumor maintenance or progression. It is possible that once oncogenic K-Ras induced tumors have developed, they become addicted to permissive signaling from wild-type RhoA. In this case, RhoA signaling would be required for tumor maintenance. Indeed, an examination of human cancer cell lines treated with
RhoA-specific siRNA or the Rho inhibitor Rhosin shows that they are dependent on RhoA function
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Discussion Zandvakili et al.
for proliferation and progression (data not shown). These findings pose an intriguing possibility that targeted inhibition of RhoA activity alone is useful for suppressing advanced tumors in certain instances, but may exacerbate preneoplastic lesions. Another caution interpreting our results is that both mouse cancer models are accompanied by significant inflammatory responses which could potentially be modulated by RhoA status, which in turn may be promoting the inflammatory component (i.e. effects through tumor microenvironment).
The interplay between RhoA and RhoC signaling is in some ways similar to that of B-Raf and c-Raf in oncogenic K-Ras-driven adenoma initiation. Recent studies have found that c-Raf, but not B-Raf, is required for K-Ras-driven adenoma formation, despite historical evidence in mouse embryonic tissues and fibroblasts suggesting the opposite, that B-Raf but not c-Raf is required for ERK phosphorylation (115–119). In fact, similar to our findings, pharmacologic targeting of B-Raf results in increased activity of c-Raf and a paradoxical increase in ERK phosphorylation and tumorigenesis in K-Ras-mutant cancers (117,120–122); though this is not the case with genetic deletion of B-Raf.
Interestingly, Blasco et al. also found that whole organism deletion of both B-Raf and c-Raf resulted in no obvious phenotype in mice, also similar to our finding of overtly healthy mice with dual deletion of RhoA and RhoC from the bronchiolar epithelium (115).
Our study of the relationship between RhoA, RhoC and K-Ras in murine lung adenoma formation sheds light on the broader subject of cellular signal transduction networks, and how signal redundancy and compensation in these networks can result in counterintuitive results when the network is modified. These insights are important considering the increased interest and emphasis on targeted therapeutics against individual pathways. The robustness of signal transduction networks paired with the evolutionary power of clonal selection in cancer suggests the best antineoplastic strategies may need to incorporate multiple drug targets with an anticipation of how cancer cells will adapt to and evade targeted therapies. Our study suggests that simultaneous
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targeting of both RhoA and RhoC signaling is critical, as targeting of RhoA alone may worsen clinical outcomes. Further study confirming our proposed compensatory mechanism is warranted, as is a deeper understanding of the overlapping and differential functions of RhoA, RhoC and other related
Rho GTPases in cancer biology. Last, our study suggests that targeting ROCK signaling, which is a shared downstream mediator of both RhoA and RhoC, may be sufficient to block K-Ras-driven cancer as has been previously suggested (56,123).
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Materials and Methods Zandvakili et al.
Materials and Methods
Animal work
RhoAflox/flox mice generated as previously described (124). RhoCΔ2-3, LSL-K-Ras, CCSP-Cre and mTmG mice have been previously characterized (73,102,103,125). Mice were bred and housed in a specific pathogen-free vivarium at Cincinnati Children’s Hospital Medical Center and protocols were approved by the Institutional Animal Use and Care Committee of the Cincinnati Children’s Hospital
Research Foundation (IACUC2013-0069), in compliance with the Association for Assessment and
Accreditation of Laboratory Animal Care and the Office of Laboratory Animal Welfare. All in vivo experiments were performed using age-matched mice. Genotyping primers can be found in the supplementary information (S1 Table). Adeno-Cre virus (Ad5-CMV-Cre) was obtained from the
University of Iowa Gene Transfer Vector Core. Administration of Adeno-Cre virus was performed as previously described using a dose of 108 PFU per mouse (110). Mice were anesthetized with isoflurane and pain control was achieved with buprenorphine.
Western blotting
Western blotting was performed using standard protocols, with important changes or steps described herein. Briefly, lysis was performed using radioimmunoprecipitation assay buffer (RIPA buffer) supplemented with protease inhibitors cocktail (cOmplete Protease Inhibitor Cocktail
Tablets, Roche) and phosphatase inhibitor cocktail consisting of sodium fluoride, sodium orthovanadate, sodium pyrophosphate, ß-glycerophosphate and sodium molybdate (Simple Stop 1
Phosphatase Inhibitor Cocktail, Gold Biotechnology). Transfer was performed using the Trans-Blot
Turbo Transfer System (Bio-Rad). Visualization was performed using fluorescently conjugated secondary antibodies and an infrared imaging System (LiCor Odyssey CLx).
Immunohistochemistry
Briefly, tissues were fixed using 3.7% paraformaldehyde in PBS overnight and embedding to paraffin. Secondary antibodies and horse-radish peroxidase (HRP) used consisted of either Signal
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Stain Boost (Cell Signaling Technology) or anti-rabbit donkey secondary (GE Healthcare) and
Vectastain Elite ABC Kit (Vector Labs) avidin-biotin complex. The developing reagent was DAB (3,
3’-diaminobenzidine) HRP substrate (Vector Labs). Counterstaining was performed using Gill’s hematoxylin or nuclear fast red. Quantification of stainings were made by serially sectioning lungs in 250μm step increments and counting a sample of evenly spaced sections for the number of tumors. Tumor size was expressed as tumor area as measured in ImageJ.
Primary antibodies
Primary antibodies for immunoblotting or immunohistochemistry are as follows: RhoA (67B9)
Rabbit mAb (1:400 dilution, Cell Signaling Technology [CST] #2117), Myosin Light Chain 2 (D18E2)
Rabbit mAb (CST #8505), Phospho-Myosin Light Chain 2 (Ser19) Mouse mAb (1:150 dilution, CST
#3675), Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) Rabbit mAb (CST #4370),
Phospho-MEK1/2 (Ser221) (166F8) Rabbit mAb (CST #2338), GAPDH (D16H11) Rabbit mAb (CST
#5174), Anti-Myosin Light Chain (phospho S20) antibody (Abcam ab2480), Ki67 Rabbit mAb, (790-
4286 Ventana), cleaved-caspase 3 Rabbit Ab (orb137850 Biorbyt), N-Terminal Pro SPC Rabbit Ab
(Seven Hills Bioreagents WRAB-9337), and CCSP Rabbit Ab (Seven Hills Bioreagents WRAB-3950).
All antibodies were used at the manufacturers recommended dilutions unless otherwise stated.
Data analysis and presentation
Data was gathered and analyzed in either ImageJ 1.47 (NIH), GraphPad Prism (GraphPad) or the
GNU Image Manipulation Program 2.8 (GIMP). Analysis in GraphPad Prism comprised of unpaired two-way student T-tests or ANOVA.
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Acknowledgments Zandvakili et al.
Acknowledgments
Thanks to Drs Jeffery A Whitsett, Anna Perl and Lee Grimes for the transgenic mice they provided us. Many thanks to Drs Timothy D. LeCras, Vladimir V. Kalinichenko, John C. Morris, James C.
Mulloy, Leesa L. Sampson and Kathryn A. Wikenheiser-Brokamp for their insightful comments and guidance. Additionally, we would like to acknowledge support from the University of Cincinnati
Medical Scientist Training Program and the Molecular and Developmental Biology Graduate
Program at Cincinnati Children’s Hospital Medical Center.
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References Zandvakili et al.
Bibliography
A compete bibliography can be found at the end of this dissertation work.
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Figure Legends
Fig1. CCSP-promoter driven deletion of RhoA does not affect normal lung development.
RhoWT and RhoAflox/flox mice were mated with CCSP-Cre mice on a K-RasWT background.
(A & B) Lungs by H&E (bars represent 1mm and 200μm for panels A & B respectively).
(C) Mice were crossed to a tdTomato to eGFP reporter line to assess for difference is Cre-expression pattern (bars represent 100μm). Green represents eGFP and Cre activity. Red presents tdTomato and the absence of Cre activity. Blue represents DAPI staining.
(D) Immunohistochemistry for RhoA (brown) with hematoxylin counterstain (bars represent
25μm).
(E) Immunohistochemistry for CCSP (black), counterstained with nuclear fast red (bars represent
100μm).
(F) Immunohistochemistry for SPC (black), counterstained with nuclear fast red (bars represent
50μm).
Fig2. RhoA is not essential for CCSP-promoter driven, K-RasG12D-induced, lung adenoma formation in mice.
RhoWT and RhoAflox/flox mice were mated with CCSP-Cre mice on a K-RasG12D background.
(A & B) K-RasG12D lungs by H&E (bars represent 1mm and 200μm for panels A & B respectively).
(C) Immunohistochemistry for pERKThr202/Tyr204 (brown) with hematoxylin counterstain (bars represent 100μm).
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References Zandvakili et al.
(D) Immunohistochemistry for RhoA (brown) with hematoxylin counterstain (bars represent
100μm).
(E) Quantification of the RhoA-status of adenomas as assessed by immunohistochemistry. Greater than 30 tumors were counted from four mice per group.
Fig3. Deletion of RhoA and RhoC together does not impair normal lung development.
RhoWT, RhoC-/- and DKO mice were mated with CCSP-Cre mice on a K-RasWT background.
(A & B) Lungs by H&E (bars represent 1mm and 200μm for panels A & B respectively).
(C) Mice were crossed to a tdTomato to eGFP reporter line to assess for difference is Cre-expression pattern (bars represent 100μm). Green represents eGFP and Cre activity. Red presents tdTomato and the absence of Cre activity. Blue represents DAPI staining.
(D) Immunohistochemistry for RhoA (brown) with hematoxylin counterstain (bars represent
25μm).
(E) Immunohistochemistry for CCSP (black), counterstained with nuclear fast red (bars represent
100μm).
(F) Immunohistochemistry for SPC (black), counterstained with nuclear fast red (bars represent
50μm).
Fig4. Neither RhoA nor RhoC is required for K-RasG12D-induced adenoma formation in a
CCSP-Cre model.
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RhoWT, RhoC-/- and DKO mice were mated with CCSP-Cre mice on a K-RasG12D background.
(A & B) K-RasG12D lungs by H&E (bars represent 1mm and 200μm for panels A & B respectively).
(C) Immunohistochemistry for pERKThr202/Tyr204 (brown) with hematoxylin counterstain (bars represent 100μm).
(D) Immunohistochemistry for RhoA (brown) with hematoxylin counterstain (bars represent
100μm).
(E) Quantification of the RhoA-status of adenomas as assessed by immunohistochemistry. Greater than 30 tumors were counted from four mice per group.
Fig5. RhoA and RhoC are important in combination, but not individually, for K-RasG12D induced sporadic lung adenoma formation.
Mice from different Rho backgrounds were administered Adeno-Cre virus endotracheally. Lungs were harvested after 12 weeks.
(A) H&E of adenomas (bars represent 100μm).
(B) Immunohistochemistry for pERKThr202/Tyr204 (brown) with hematoxylin counterstain (bars represent 100μm).
(C) Immunohistochemistry for RhoA (brown) with hematoxylin counterstain (bars represent
100μm).
(D) Western blots of microdissected tumors (N = normal lung, T = tumor).
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(E) Quantification of tumor burden. Means represent the quantification of four separate, evenly spaced and similarly sized lung sections, from four mice for each group (n = 4, * = p ≤ 0.001). Data representative of two separate experiments.
(F) Quantification of tumor sizes. Data points represent the area of individual tumors expressed in
μm2. Red bars represent the mean tumor area.
(G) Proportion of RhoA-positive and RhoA-null tumors. Displayed as RhoA staining by the level of staining intensity (levels are as follows: no staining; normal = same intensity staining as adjacent normal alveoli; high = greater than adjacent normal alveoli; very high = much greater intensity staining than adjacent normal alveoli).
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Supporting Information Captions
S1 Figure. PCR analysis of mouse genotypes and lox deletion bands.
DNA was isolated from mouse lungs of different Rho background mice mated to either CCSP-Cre K-
RasWT (K-WT) or CCSP-Cre LSL-K-RasG12D mice (K-Ras). Pairs of K-RasWT and LSL-K-RasG12D mice for each Rho background are shown. Deletion bands for LSL-K-RasG12D mice demonstrate expression of
K-RasG12D (deletion band is the larger band at 315bp). Deletion bands for RhoA demonstrate recombination of the RhoAflox/flox locus (deletion band at 667bp). Yellow arrows point to the 500bp band of a 100bp ladder.
S2 Figure. CCSP-promoter driven hyperplastic growths and adenomas.
(A) RhoA immunohistochemistry (brown) of hyperplastic lesions with hematoxylin counterstain of
CCSP-Cre LSL-K-RasG12D mice from different Rho backgrounds (bars represents 100μm).
(B) Percentage of Ki67 positive cells in CCSP-Cre LSL-K-RasG12D driven adenomas (n = 4, p >0.05).
(C) Representative image of cleaved-caspase 3 (CC3) staining of CCSP-Cre LSL-K-RasG12D driven adenomas. Positive control is injured renal tubules (20X). Bars represent 100μm.
S3 Figure. Downstream K-Ras and RhoA signaling status in CCSP-promotor driven adenomas.
Immunohistochemistry of serially sectioned adenomas from CCSP-Cre LSL-K-RasG12D mice. Rows represent serial sections of the same adenoma from their respective Rho backgrounds. Columns are
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References Zandvakili et al. of RhoA, pMLCSer20, pMEKSer221 and pERKThr202/Tyr204 stainings respectively with hematoxylin counterstain (bars represents 100μm).
S4 Figure. Adeno-Cre induced hyperplastic growths and adenomas.
(A) RhoA immunohistochemistry (brown) with hematoxylin counterstain of Adeno-Cre induced
LSL-K-RasG12D mice from different Rho backgrounds (bars represents 100μm).
(B) Percentage of Ki67 positive cells in Adeno-Cre LSL-K-RasG12D driven adenomas (n = 4, p >0.05).
(C) Representative image of cleaved-caspase 3 (CC3) staining of Adeno-Cre LSL-K-RasG12D driven adenomas. Positive control is injured renal tubules (20X). Bars represent 100μm.
S5 Figure. Original Western Blots
S1 Table: Genotyping and deletion primers
Table of genotyping and deletion primers used for PCR. Specific PCR protocols can be found in the references for transgenic mice under Materials and Methods.
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Tables
Table 2-1. Breeding Schematic for Transgenic Mice
Transgenes Shorthand RhoWT RhoAcKO RhoC-/- Double Knockout (DKO) RhoA genotype RhoA+/+ RhoAflox/flox RhoA+/+ RhoAflox/flox RhoC genotype RhoC+/+ RhoC+/+ RhoCΔ2-3 RhoCΔ2-3 Additional Transgenes: K-Ras knock-in LSL-K-RasG12D Cre-recombinase Adeno-Cre or CCSP-Cre
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Figures: Figure 2-1. CCSP-promoter driven deletion of RhoA does not affect normal lung development.
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Figure 2-2. RhoA is not essential for CCSP-promoter driven, K-RasG12D-induced, lung adenoma formation in mice.
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Figure 2-3. Deletion of RhoA and RhoC together does not impair normal lung development.
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Figure 2-4. Neither RhoA nor RhoC is required for K-RasG12D-induced adenoma formation in a CCSP-Cre model.
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Figure 2-5. RhoA and RhoC are important in combination, but not individually, for K-RasG12D- induced sporadic lung adenoma formation.
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Supplemental Figures:
Figure S 2-1. PCR analysis of mouse genotypes and lox deletion bands.
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Figure S 2-2. CCSP-promoter driven hyperplastic growths and adenomas.
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Figure S 2-3. Downstream K-Ras and RhoA signaling status in CCSP-promotor driven adenomas.
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Figure S 2-4. Adeno-Cre induced hyperplastic growths and adenomas.
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Figure S 2-5. Original Western Blots.
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Chapter 3 – RhoA is required for Malignant
Phenotypes of Human Lung Adenocarcinoma Cell
Lines
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Introduction
Rho GTPases have been demonstrated to regulate or affect cell cycle progression, growth and proliferation, cell polarity, differentiation, vesicle trafficking, invasion and metastasis. These cellular behaviors have direct implications on carcinogenesis and cancer progression: self- sufficiency of growth signals, insensitivity to anti-growth signals, evasion of apoptosis, angiogenesis, tissue invasion and metastasis, among the other cancer “hallmarks” (126). As has been described earlier in this dissertation, Rho GTPases are highly involved in almost every malignant phenotype of cancers. Clearly Rho GTPases play a direct role in cell migration, invasion and metastasis (127).
Results
In order to assess the requirement of RhoA in a disease specific context, we decided to study three
Ras-driven human lung adenocarcinoma cell lines: A549 , H441 and H1299 cells harboring K-
RasG12S, K-RasG12V and N-RasQ61K mutations, respectively (128). We performed short hairpin RNA
(shRNA) mediated knockdown of RhoA and found efficient reduction of the target gene protein as compared to non-targeting (NT) control shRNA (Fig. 1). Knockdown as most efficient with shRNAs
1 & 3, with only partial knockdown obtained with shRNA 2. All assays of shRNA knockdown cells were done in accordance with a strict timeline outlined in the Methods section. We did not observe any consistent compensatory increases in RhoC protein levels in response to RhoA, except for some modest increases in RhoC levels with shRNA 3, most notably in H1299 cells (Fig. 3-1).
However, downstream RhoA signaling remained intact as assessed by the ratio of phosphorylated myosin light chain to total myosin light chain (pMLCSer19 to MLC, also known as MYL9) suggesting compensatory activity of RhoC during RhoA knockdown (Fig. 3-1). In instances where RhoC protein
79
levels are increased – presumably in response to RhoA knockdown – there are appeared to be increased pMLCSer19 signal (Fig. 3-1). Other downstream markers such as pERKThr202/Tyr204
(MAPK1/3), pAKTSer473 (AKT1/2/3) and pS6Ser240/244 (RPS6) levels remained mostly unchanged with RhoA knockdown.
Functionally, RhoA-knockdown cells demonstrated decreased proliferation in contrast to control shRNA (Fig. 3-2). Moreover, RhoA-knockdown cells demonstrated a profound decrease in ability to form anchorage-independent colonies in soft agar (Fig. 3-3). Taken together, these results are consistent with previous reports using Ras-transformed mouse fibroblasts (60,61). Moreover, they are also consistent with Rac1 knockdown experiments in K-Ras-induced fibroblasts (60,62). Thus, we conclude that RhoA signaling is important for human lung adenocarcinoma tumor maintenance as evident by decreased proliferation and anchorage-independent growth. Moreover, we find that downstream pMLCSer19 signaling is maintained after RhoA loss, and may be as a result of compensatory RhoC signaling.
Discussion
As has been described previously in this manuscript, there have been only a few studies specifically investigating the requirement of RhoA for Ras-induced transformation of cells (60,61,96), or whether increased Rho GTPase expression or activity could weakly transform cells (57–62).
Moreover, a few of these studies demonstrated the converse, that is that dominant negative forms of RhoA can block Ras-induced transformation (60,61).
However, for RhoA to be an appropriate therapeutic option, perturbation of RhoA signaling must hinder the malignant phenotype of cancer cells rather than premalignant cells. To that end, there have been surprisingly few studies that have directly targeted RhoA or RhoC, though there are a
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few RNA interference studies of Rho GTPase signaling. An siRNA silencing study by Vega et al. demonstrated that knockdown of either RhoA or RhoC resulted in impaired migration of PC3 prostate cancer cells (129). They also found that RhoC knockdown in MDA-MB-231 breast cancer cells resulted in decreased cell invasion, but that RhoA knockdown actually increased invasion, similar to a study by Sequeira et al. in PC3 cells (130). However, siRNA knockdown studies by Pille et al. in MDA-MB-231 cells demonstrated decreased invasion with both RhoA than RhoC (131).
Other reports of RhoA and RhoC knockdown in gastric cancer cell lines demonstrated decreased proliferation, invasion, metastasis and xenograft formation (99,132–134).
There have been other studies abolishing downstream RhoA signaling by various methods such as knockdown or pharmacologic inhibition. Specifically, abrogation of ROCK signaling has been shown to extend the lifespan of murine models of leukemia, hepatocellular, breast and lung carcinomas
(135,136). Studies of other downstream proteins such as various formins have yielded similar results with regards to inhibiting cancer cell motility and progression (137). Inhibition of ROCK signaling by inhibitors such as fasudil or Y-27632 has yielded interesting results, and has gained popularity as a method of creating immortalized human epithelial cells as well as conditionally reprogrammed cells when combined with fibroblast feeder cells (138). Conditionally reprogrammed cells have characteristics similar to those of induced pluripotent stem cells.
Overall, our results demonstrate that RhoA is at least partially required for cell growth and anchorage-independent growth – as well as transwell migration and invasion (data not shown) – which is consistent with studies of RhoA, RhoC and downstream mediators such as ROCK and the formins. There have even been reports of inhibition of pancreatic and breast cancer cell xenograft growth in mice when inhibiting RhoA and ROCK respectively (131,139). Therefore, although there is a lack of definitive understanding of exactly how inhibition of the Rho pathway abrogates
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malignant phenotypes of cancer cells, there is promising evidence that inhibition of RhoA and RhoC will yield promising antineoplastic effects.
Methods
Cell culture work
A549, H441 and H1299 were cultured in accordance with American Type Culture Collection (ATCC) recommendations. Short hairpin RNA (shRNA) knockdown experiments were performed using constructs obtained from the MISSION RNAi Consortium library (Sigma Aldrich), on the base vector pLKO.1-puro. For human cells the following constructs were used: TRCN0000047708,
TRCN0000047709, TRCN0000047710 and control shRNA SHC002. Transduction was performed using lentiviral mediated transduction with polybrene (Millipore) and selection using puromycin
(Sigma). Transduced cells were passaged no more than once before use in any assay.
Assays
Proliferation studies were conducted in 96-well format using CellTiter 96 AQueous One Solution
Cell Proliferation “MTS” Assay (Promega, G3580) and performed in either triplicate or quadruplicate recordings using the absorbance at 490nm wavelength. Transwell migration and invasion assays were performed using 8μm polycarbonate membrane cell culture inserts from
Corning Transwell and Corning BioCoat Matrigel Invasion Chambers (Corning).
Western blotting
Western blotting was performed using standard protocols, with important changes or steps described herein. Briefly, lysis was performed using radioimmunoprecipitation assay buffer (RIPA buffer) supplemented with protease inhibitors cocktail (cOmplete Protease Inhibitor Cocktail 82
Tablets, Roche) and phosphatase inhibitor cocktail consisting of sodium fluoride, sodium orthovanadate, sodium pyrophosphate, ß-glycerophosphate and sodium molybdate (Simple Stop 1
Phosphatase Inhibitor Cocktail, Gold Biotechnology). Transfer was performed using the Trans-Blot
Turbo Transfer System (Bio-Rad). Visualization was performed using either fluorescently conjugated secondary antibodies and an infrared imaging System (LiCor Odyssey CLx), or horseradish peroxidase conjugated secondary antibodies and chemiluminescence (GE Healthcare and SuperSignal West Pico Chemiluminescent Substrate Thermo Scientific, respectively).
Primary antibodies
Primary antibodies for immunoblotting are as follows: RhoA (67B9) Rabbit mAb (Cell Signaling
Technology [CST] #2117), RhoC (D40E4) Rabbit mAb (CST #3430), Myosin Light Chain 2 (D18E2)
Rabbit mAb (CST #8505), Phospho-Myosin Light Chain 2 (Ser19) Ab (CST #3671), Phospho-Myosin
Light Chain 2 (Ser19) Mouse mAb (CST #3675), p44/42 MAPK (Erk1/2) (L34F12) Mouse mAb (CST
#4696), Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) Rabbit mAb (CST #4370),
S6 Ribosomal Protein (54D2) Mouse mAb (CST #2317), Phospho-S6 Ribosomal Protein
(Ser240/244) (D68F8) Rabbit mAb (CST #5364), Akt (pan) (40D4) Mouse mAb (CST #2920),
Phospho-Akt (Ser473) (D9E) Rabbit mAb (CST #4060), GAPDH (D16H11) Rabbit mAb (CST
#5174).
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Figure 3-1. Rho and Ras GTPase Downstream Signaling after RhoA Knockdown
Short hairpin RNA mediated knockdowns of RhoA were performed in A549, H441 and H1299 cells.
Three shRNAs, numbered 1, 2 and 3 plus a non-targeting control (NT) shRNA were used.
Analysis of downstream Rho and K-Ras signaling was performed via Western blot. All cells were harvested between 7-10 days post infection with lentiviral mediated transduction of shRNA.
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Figure 3-2. Cell Proliferation after RhoA Knockdown
Short hairpin RNA mediated knockdowns of RhoA were performed in A549, H441 and H1299 cells.
Three shRNAs, numbered 1, 2 and 3 plus a non-targeting control (NT) shRNA were used.
Cells were selected for using puromycin supplemented culture medium. Proliferation was measured using an MTS assay and performed in triplicate (* = p ≤ 0.05).
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Figure 3-3. Anchorage-independent Growth after RhoA Knockdown.
Short hairpin RNA mediated knockdowns of RhoA were performed in A549, H441 and H1299 cells.
Three shRNAs, numbered 1, 2 and 3 plus a non-targeting control (NT) shRNA were used.
Anchorage independent growth was assayed via soft agar colony formation. Five-thousand cells were plated and growth medium was supplemented with puromycin. Colonies were counted after three weeks of incubation (* = p ≤ 0.001).
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Chapter 4 – Summaries and Perspectives
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Summary of Findings
This dissertation work focuses on addressing the role of RhoA in K-Ras-driven tumorigenesis. This work investigates two in vivo models of lung cancer and multiple lung cancer cell lines. Beginning with the in vivo models, this work first establishes that, broadly speaking, neither RhoA nor RhoC is required in the lung epithelium of mice for normal lung function. More specifically, neither RhoA nor RhoC alone, or in combination, is required in the CCSP-Cre positive lung epithelial population.
The expression of CCSP-Cre is largely limited to the Club cell population, but also expressed in a subset of alveolar cells (103).
Second, our data shows K-RasG12D-induced adenomas can form in the absence of RhoA, indicating that RhoA is not an absolute requirement for K-RasG12D-induced tumorigenesis. Third, we have demonstrated that the closely related RhoA homolog, RhoC, which has been implicated as a pro- metastatic, is also dispensable for K-RasG12D-induced tumorigenesis. Fourth, we found an increase in the numbers of adenomas in the RhoAcKO sporadic lung cancer model in which RhoA is deleted by adenoviral expression of Cre. Fifth, an attempt at combined deletion of RhoA and RhoC in the sporadic lung cancer model resulted in reduced adenoma formation. Sixth, we found that RhoA was efficiently deleted in hyperplastic lesions in both the tissue-specific promoter driven and sporadic lung, but that there was strong selection pressure against RhoA deletion in the initiation of adenomas in the RhoAcKO and DKO background mice. Taken together, these results indicate that there is a selective advantage to retain RhoA in the absence of RhoC, and there is likely a redundant and compensatory role of RhoC in RhoA signaling downstream of oncogenic K-Ras in adenoma formation. Consistent with this interpretation, seventh, we find that the phosphorylation status of
MLC, downstream of RhoA and RhoC signaling, was not decreased in either RhoA or RhoC-null adenomas, nor in adenomas from DKO mice, in which RhoA is retained.
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Eighth, our investigation into lung cancer cell lines, namely those driven by K-Ras and derived from lung adenocarcinomas – A549 and H441 cells – indicate that RhoA plays a very important role in proliferation and anchorage-independent growth – processes that together can be called tumor maintenance. Again, similar to the in vivo results, we found that downstream MLC signaling remained intact as indicative by pMLCSer19 levels in Western blots.
Limitations of this Study and Alternative Approaches
One clear limitation of this study is the Cre-recombinase approaches used. Briefly, the problems afflicting the CCSP-Cre and Adeno-Cre methods are as follows: One problem that affects the CCSP-
Cre model is the spatiotemporal expression of the CCSP promoter. A second problem that complicates both the Adeno-Cre and CCSP-Cre methods is efficient recombination of the RhoAflox/flox locus. A third problem that complicates both methods is the temporal relationship between expression of KRasG12D and deletion of RhoA. Though understanding whether RhoA is required at the time of KRas-induced adenoma initiation, a more clinically relevant question would be whether
RhoA is required for the maintenance of an already established adenoma, or better yet, deletion of
RhoA from an adenocarcinoma.
Beginning with the first problem, the spatiotemporal expression of the Cre, and specifically the spatial aspect, we find the problem lies in the predominant cell in which Cre-recombinase is expressed: that is the Club cell. Recent evidence suggests that the cell of origin of adenomas in mice is most likely Type II cells and not Club cells. In fact, CC10 may be expressed in a subset of Type II cells which give rise to adenomas, or alternatively evidence also suggests that some Club cells may be undergoing differentiation into Type II cells, which then give rise to adenomas (140,141). The notion that Type II cells are likely the source of adenoma formation is supported by our data showing that there are scant cells within the alveolar regions that express CCSP-Cre as seen by eGFP expression in the mTmG model as shown in Chapter 2, Figure 2-1 and Figure 2-3. The 89
aforementioned scant cells in the alveoli are likely Type II cells as based on morphology.
Unfortunately our attempts at co-immunofluorescence of SPC in the mTmG mice to prove the origin of these cells were unsuccessful. A method to address the cell of origin problem would be to use
SPC-Cre which is restricted in expression to Type II cells. Although the Adeno-Cre virus theoretically obviates this problem, another way to further dissect the cell of origin would have been to use Adeno-Cre virus that is restricted in expression to either the SPC or CC10 expressing cell types. Virus such as Ad5-SPC-Cre and Ad5-CC10-Cre is commercially available and as has been utilized successfully in this manner (142).
The next aspect of the spatiotemporal limitation of CCSP-Cre is the temporal restriction. CCSP-Cre is expressed perinatally (~E18.5) which may cause some confounding developmental aberrations.
The temporal aspect of Cre-recombinase expression is two-fold. First, and as already discussed,
CCSP-Cre may have developmental ramifications, but mostly the problem with this timing is that it is not ideal for modeling a disease that afflicts older adults rather than newborns. The second aspect of the temporal expression of Cre relates to the timing of oncogenic KRas activation and
RhoA deletion. An approach which could possibly circumvent these problems would be through the use of a reverse tetracycline-controlled transactivator (rtTA, or Tet-on system). The use of rtTA to control Cre expression would address both of these problems since doxycycline could be given to adult mice avoiding developmental effects, and secondly doxycycline could be given in multiple doses, which could address the final and third limitation discussed in the following paragraph.
The third problem described in the introduction to this section relates to the deletion efficiency of
RhoA. In some ways the incomplete deletion of RhoA from adenomas has yielded insight by suggesting that there is selection pressure against RhoA deletion. However, this phenomenon has also confounded interpretation of results because there may be unexplored reasons as to why RhoA is not efficiently deleted which support a conclusion other than our interpretation. Deletion
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efficiency could also be address with a reverse tetracycline-controlled transactivator system.
Though not validated, it would seem reasonable that continuous administration of doxycycline may result in improved deletion efficiency of RhoA from adenomas. If this were true, then simple quantification of the number and size of adenomas formed in the presence of clean and clear RhoA deletion would provide unambiguous data as to how RhoA deletion effects KRas-induced adenoma initiation. Another approach that could be taken would be administration of a short period of low dose of doxycycline to these mice which would activate oncogenic KRas, and as our data shows, likely not result in efficient RhoA deletion. These tumors could then be allowed to continue to incubate and grow within the mice for a period of time. A subset of mice could then be sacrificed, their lungs harvested and tumor burden quantified to serve as a baseline level of tumor burden.
Next, the remaining subset of mice could then be subjected to a high dose continuous doxycycline to promote deletion of RhoA from formed adenomas. The tumor burden of this second group of mice could be compared to the tumor burden of the baseline group of mice to answer the question: Does deletion of RhoA from formed adenomas cause regression of those adenomas? However, one problem with this method would be that new adenomas and hyperplasia would be induced with repeated exposure to doxycycline, as least with the use of a tissue-specific promotor system such as
SPC or CCSP promotor (e.g. SPC-rtTA/tetO-Cre). A way to circumvent this last problem would be the delivery of rtTA/tetO-Cre to the lung parenchyma via virus. This can actually be accomplished as there is also commercially available Adeno-rtTA/tetO-Cre. With these last few alternative methods we approach the limits of feasibility as each layer of complexity adds more variables and requirements. For instance dosing of virus, dosing of doxycycline, timing of each exposure, and timing of incubation periods each require investigation. Each step requires validation and experimentation to learn how modifying variables affects experimental results and interpretation of those results. Some of the alternative approaches suggested in this section would require multiple levels of experimental optimization and validation. 91
Another limitation of this study was lack of tumor quantification in the CCSP-Cre mouse model. The abundance of hyperplasia and inflammation made quantification of adenomas difficult in this model. An approach by H&E staining was unsuccessful as was the use of staining for CCSP or SPC
(data not shown). Despite our technical inability to achieve CCSP/CC10 staining, this approach has been successfully utilized by others to positively identify adenomas (103). Another approach we sought was staining for Ki67 which was also unsuccessful at helping with quantification (data not shown). We also attempted to use mortality as a surrogate for tumor burden, but found that regardless of Rho status the mice all died at the same rate (data not shown). However, upon reflection we could have utilized several other methods. One method which has been previously used in conjunction with a CCSP-Cre LSL-KRasG12D model is quantifying tumor burden by lung weight normalized to total body weight (105). Another method we could have used would be to stain for other tumor markers that differentiate inflammatory cells from epithelial cells such as E- cadherin, TTF-1, beta-catenin, and cytokeratins 5/6 and 7. Another alternative approach would be to stain inflammatory cells rather than adenomas to differentiate inflammation from tumor (e.g.
CD45 staining). Ultimately, we used the Adeno-Cre model to circumvent the problem of quantification, but accurate quantification in both models would have yielded more reliable interpretation of the data.
Lastly, a major limitation of this study is the use of a pre-metastatic mouse model. Activation of oncogenic KRas alone in the lung results in adenomas and not adenocarcinomas in mice (111). To propagate adenocarcinomas and cause metastases requires additional hits such as inactivation of p53, Lkb1 (Stk11), the CDKN2A locus (p16INK4a and p14Arf) or PTEN. Of course this would have added power to our current study, but again may have not been feasible in a realistic timeframe as the number of transgenes required to produce such a mouse along with RhoAflox/flox and RhoC-/- loci would be impressive, and possible lethal. An important way in which a metastatic model would
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have added to this study would be in comparison of RhoC+/+ to RhoC-/- mice, since RhoC has been long implicated in metastasis. Moreover, this would be important data to gather with regards to a pre-clinical model to establish evidence for the use of a Rho GTPase inhibitor in cancer patients.
The reasoning being that it is often metastasis and not the primary cancer that causes morbidity and mortality. Therefore a drug that could inhibit metastasis would have profound clinical utility.
Deciphering the Paradoxical increase in Adenoma Formation and Growth in
RhoAflox/flox background mice
A defining piece of this dissertation work is that loss of RhoA alone exacerbates K-Ras-induced lung adenoma formation in mice. Given the accumulation of years of evidence that RhoA is an important downstream mediator of K-Ras signaling, how could this result be? As outlined in the discussion section of Chapter 2, the main hypothesis we put forth is that of increased RhoC activity. There are two key pieces of evidence that suggest compensation by RhoC. The first piece of evidence stems from investigating the deletion efficiency of RhoA from newly sprouting adenomas. Whereas RhoA deletion occurs at similar frequencies in the adenomas of RhoC+/+ mice in both the Adeno-Cre and
CCSP-Cre models, there is no RhoA deletion in RhoC-/- background mice in the Adeno-Cre model and at very low levels in the CCSP-Cre model. The second piece of evidence is that downstream Rho
GTPase signaling is maintained in adenomas regardless of the RhoA or RhoC gene status of murine adenomas. This is evident by Western blot analysis of multiple tumors demonstrating intact pMLCSer19 status (only one blot shown, but results have been reproducible). Lastly, this same pattern of intact Rho GTPase signaling is also seen when RhoA is knocked-down by shRNA in human lung cancer cell lines as outlined in Chapter 3.
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Unfortunately, we were unable to assay RhoA and RhoC activity directly. Ideally, a pulldown of active RhoC would directly demonstrate whether RhoC activity levels increased in response to
RhoA deletion. This can be accomplished by precipitation of GTP-bound RhoC with rhotekin Rho binding domain, and then blotting with RhoC specific antibody (that is, antibody that does not react with RhoA, which exists and can be bought commercially). However, microdissection of murine adenomas followed by RhoC pulldown, though not impossible, is technically challenging. Therefore, we assayed downstream signaling that arises from RhoA and RhoC activity, namely phosphorylation of MLC by ROCK.
Another distinct possibility is upregulation of another pathway in response to RhoA deletion – that is something other than RhoC. We did explore upregulation of the PI3K-AKT-mTOR pathway, but did not find compelling evidence of increased signaling in this pathway leading to increased tumorigenesis and adenoma growth (data gathered for in vivo studies but only in vitro studies are shown). There are of course practically endless other pathways that could be hypothesized and tested. For instance, Rac or Ras activity could easily be increased by dysregulation of GEF activity resulting from RhoA deletion (i.e. Vav2 or Ras-GRF1).
For the Adeno-Cre experiments, an important consideration is that although there was increased adenoma formation and growth in RhoAcKO mice, the vast majority of the tumors still expressed
RhoA. This is a confusing result since there should be no effect in RhoAflox/flox mice if RhoA is not actually deleted – how can there be more adenomas and hyperplasia in RhoAcKO mice versus RhoWT mice if both have largely RhoA-intact tumors? It should be noted that the RhoAflox/flox and RhoC-/- mice are both on a C57BL/6 genetic background, the CCSP-Cre on a FVB/B6 hybrid background, and that all mice were backcrossed. Therefore mouse background should not be a factor here.
There are two possibilities that come to mind. The first is that there is a selective advantage for
RhoA heterozygosity. This possibility is interesting since it implies there is selective pressure
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against total RhoA deletion, however, there may be advantages to modestly decreased RhoA signaling – as discussed in the previous paragraph, it may be that decreasing RhoA levels slightly frees GEFs to interact with other GTPases such as RhoC to result in increased cell proliferation. The notion that RhoA heterozygosity imparts selective advantage for tumor growth is supported by data we collected on the RhoA staining intensity of adenomas. Reviewing Figure 5 in Chapter 2, it is clear that there are many more adenomas that have decreased levels of RhoA in the RhoAcKO group as compared to any of the other Rho backgrounds.
Another possibility as to why the RhoAcKO group has more adenomas despite having largely RhoA intact adenomas is that there is a cell non-autonomous effect of RhoA deletion. That is, it may be that deletion of RhoA from surrounding tissues causes increased adenoma initiation and growth. It is known that Rho GTPases regulate secretion of factors such as matrix metalloproteases, and it could be that secretion of such factors accounts for the increased tumorigenesis.
In summary, this dissertation work puts forth several facts. First there is increased adenoma initiation and growth in the RhoAcKO background mice, despite little actual RhoA deletion. Second, there is a strong selection advantage against RhoA deletion in DKO mice, and there are fewer adenomas in these mice. Thirdly, there are decreased RhoA levels in the adenomas of RhoAcKO mice and there is preserved pMLCSer19 signaling regardless of the RhoA or RhoC status of any adenoma.
The best explanation that marries these facts is that there is selection advantage to moderately decreased RhoA signaling, but strong selection advantage against total RhoA signaling loss. The rationale for this could be that slightly decreased RhoA signaling allows for increased signaling through more highly pro-neoplastic pathways such RhoC or Rac. Due to technical challenge we were unable to directly test this rationale which would support our main conclusion.
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A Case for Rho GTPase inhibition: Reconciling cell line and in vivo findings
An obvious and outstanding question of this dissertation work is reconciling the in vivo and the in vitro results. On the one hand the in vitro results are as expected: there is decreased proliferation and anchorage-independent growth. However this result is neither surprising nor groundbreaking as there have been hundreds of genes shown to be important for in vitro assays of malignancy that have then fallen flat when pharmaceutical inhibitors to those gene products are tested in vivo. On the other hand, the in vivo data is unexpected, counterintuitive and the opposite as would be expected from the results of numerous studies of Rho GTPases.
The in vitro and in vivo results however are not irreconcilable. As explained earlier in this chapter, the in vivo results do suggest RhoA signaling is not required for adenoma initiation and growth but that the level of signaling may be what is important and that too much or too little signaling is not advantageous. Why then do the cell lines seem exquisitely sensitive to RhoA knockdown? There are a few reasons as to why this may be the case. First off, it may be simply because RhoA signaling is important in the context of 2-dimensional growth on a plastic Petri dish. Alternatively, it may be that cell lines have a highly optimized signaling transduction network that is geared towards rapid growth, and because of this are reliant on these signaling pathways. In this scenario it would be easy to comprehend that cell signaling transduction networks in these cells are inflexible and sensitive to even minor perturbations. This would be in contrast to less malignant murine adenoma cells that demonstrate resilience due to their more malleable signaling networks capable of undergoing selection pressures. Lastly, another possibility that is rather unlikely would be a fundamental difference between K-Ras driven murine cells and K-Ras driven human. Though, this last scenario is highly unlikely. Therefore, we have assayed if RhoA signaling is required for KRas- driven lung cancer under two different contexts: adenoma initiation in our in vivo model, and adenocarcinoma maintenance in our in vitro model. These experiments have yielded different
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results due to differences in experimental context, and thus we have obtained a better, more nuanced, understanding of RhoA signaling biology in this regarded.
Parallels to the Paradoxical Activation of the MAPK Pathway through B-Raf inhibition
As paradoxical as it seems to have decreased RhoA signaling being pro-neoplastic in certain contexts and then antineoplastic in other contexts, this is not an entirely unheard-of phenomenon.
A prominent example that is in many ways similar to our findings is that of the adverse effects of B-
Raf inhibitors. Clinical trials of the B-Raf inhibitors vemurafenib and dabrafenib yielded the unexpected result that patients often developed cutaneous squamous cell carcinomas and keratoacanthomas, and that these lesions commonly harbored K-Ras mutations (143,144).
Examination of these adverse drug responses and a look back at some clues in the literature has led to the consensus that in certain contexts B-Raf inhbitors actually led to increased MAPK pathway signaling. The first clues came in 1999 when Hall-Jackson et al. demonstrated that a competitive
ATP substrate inhibitor of Raf resulted in increased MEK phosphorylation. Despite the fact that the inhibitor was effective against both purified B-Raf and c-Raf in vitro they found >100 fold increase in c-Raf activity and thus increased MEK phosphorylation in vivo (145). The authors speculated that feedback loops exists such that Raf inhibits its own activity via a MEK dependent manner and these feedback loops were being perturbed by their inhibitors.
A few year later in 2002 reports emerged that B-Raf mutations were common in many cancers, especially melanomas. Next in 2004, Wan et al. examined the cellular and molecular biology of various B-Raf mutations that had been newly discovered in human cancers, and were perplexed to find some B-Raf mutations found in cancers encoded kinase-dead versions of the protein (146).
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They found that most of the mutations – which occur in P loop or the “activation segment” – result in promotion of the active conformation of B-Raf and thus increased B-Raf signaling. This is the case of the very common B-Raf V600E mutation, which is found in ~55% of melanomas (147). What was perplexing was that some mutations resulted in kinase-dead versions of B-Raf, that could not phosphorylate MEK in vitro, yet when these same B-Raf mutants were introduced into cells they caused increased phosphorylation of ERK. Further investigation showed that the increased MAPK signaling resulting from these kinase-dead versions of B-Raf occur in a c-Raf dependent manner.
Thus, the authors speculated that the most likely explanation was that kinase-dead B-Raf catalyzed activation of c-Raf by promoting the active conformation of c-Raf.
Supporting the hypothesis that certain kinase-dead B-Raf mutants can catalyze activation of c-Raf is the notion that Raf activates through dimerization. In their article Wan et al. also produced a crystal structure of B-Raf and a sorafenib and found the protein crystalized as dimers (146). Preliminary evidence suggestion dimerization had been previously reported and later confirmed by careful mutational studies (121,146,148). Currently, the consensus in the literature is that under physiologic states active Raf isoforms act as a scaffold to promote the active conformation of un- activated Raf molecules. In the case of Raf inhibitors, this process is mimicked when the inhibitor binds the active site to block kinase activity, but at the same time shifts the conformation of other
Raf proteins into the active state.
The case study of kinase-dead B-Raf sheds light into how a B-Raf inhibitor could result in increased
MAPK pathway signaling. Current B-Raf inhibitors function by competitively binding the ATP pocket of B-Raf to block kinase activity. In doing so, and in order to fit within the ATP pocket, these inhibitors promote the active conformation of the protein – as when ATP is occupies the pocket – but because these inhibitors are not hydrolysable, they block any kinase activity. However, once B-
Raf assumes an active conformation – whether from physiologic signaling, mutation or due to
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binding of inhibitor – it is able to catalyze formation of the active conformation in c-Raf. Therefore, despite blocking B-Raf kinase activity, these inhibitors can promote c-Raf signaling.
It is not completely clear as to why this process is hyper-activated in Ras-mutant cells. One proposal with good basic science evidence is that in Ras-mutant cells, constant signaling from Ras to B-Raf results in B-Raf autophosphorylation and inactivation, as a natural negative feedback blocking excess MAPK pathway signaling. However, current B-Raf inhibitors remove this negative feedback by priming B-Raf to conform to the active conformation and catalyze c-Raf dependent signaling.
The seemingly paradoxical effects of B-Raf inhibitors and the interplay between B-Raf and c-Raf can shed light on the findings of this dissertation. In many ways the surprising and complex findings of
B-Raf and c-Raf dynamics parallel our finds with regards to RhoA and RhoC, especially our finding that RhoA inhibition can increase adenoma initiation in some contexts, decrease adenoma formation when RhoC is also absent, and also be vital for the malignant phenotypes of advanced cancers. This is very similar to B-Raf inhibitor induced lysis of advanced cancers such as malignant melanoma, yet simultaneously inducing new cancerous skin lesions, often in a KRas-dependent manner. Other similarities include the fact that both sets of signaling molecules work downstream of K-Ras, each signaling pathway has highly homologous but distinct isoforms, and both bind nucleotides to assume active conformations. Unlike Raf proteins, Rho GTPases have no kinase activity and there is no evidence of dimerization or signal amplification through catalysis. However, as has been described in this dissertation, Rho GTPases do engage in complex signaling cascades involving interactions with many effectors, GAPs, GEFs and GDIs. It may be through these complex interactions that inhibiting RhoA results in increased RhoC signaling and ultimately increased adenoma initiation.
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The lessons learned from B-Raf inhibitors and this dissertation highlight the complex nature of signal transduction networks and caution that simply inhibiting one part of a pathway can counterintuitively result in increased signaling overall. Thus, a strategy of multiple pharmacological inhibitors or promiscuous inhibitors that bind multiple protein isoforms may at times be a better strategy when it comes to therapies for human disease.
Final Comments and Future Directions
This dissertation work has certainly yielded interesting and wholly unexpected findings. There remain many questions as to why exactly decreased RhoA signaling in some contexts appears to yield pro-neoplastic effects but then yields antineoplastic effects in other contexts (i.e. when RhoC is also absent). Though this work is not definitive, it would seem to suggest that any pharmacological inhibitors used in either pre-clinical or clinical trials would work best if they inhibited both RhoA and RhoC signaling simultaneously. The current ROCK inhibitor, fasudil, which is approved for use in pulmonary hypertension in Japan – and may be used as a coronary artery vasodilator in the USA – would fit this description. Another pre-clinical inhibitor, known as rhosin
(or G04) developed by our lab, also fits this description. Using rational drug design, rhosin was developed to bind between the two important “switch” regions of RhoA and inhibits its interaction with GEFs such as LARG. Not only does this inhibitor decrease phosphorylation of myosin and have antineoplastic effects in vitro, but importantly it inhibits both RhoB and RhoC as well (93,94).
Future directions expanding on this thesis work would include direct measurement of RhoA and
RhoC activity in the context of K-Ras driven tumorigenesis. Measurements could be made using
“rho binding domain” pulldown from rhotekin protein expressed as a glutathione S-transferase fusion protein as described earlier. Other methods may include GTP-based Enzyme-Linked
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Immunosorbent Assay “G-LISA” or through the use of fluorescence resonance energy transfer
(FRET) microscopy which has been used by many groups to measure Rho GTPase activity.
Another limitation of this study is the crude nature of the lung cancer model. The murine models we used have several advantages including simplicity and reproducibility and are thus excellent for studying cell signaling. However, this also limits how well they recapitulate human disease. For instance, our murine lung cancer model only develops adenomas and not adenocarcinomas. An interesting and enlightening experiment would to inhibit RhoA and RhoC signaling in primary human lung adenocarcinoma xenografts. As part of this thesis work I was able to grow several human lung adenocarcinomas as xenografts as proof of principle. Inhibition could be obtained through intra-tumoral injection of shRNA, siRNA or through the use of pharmacological inhibitors such as fasudil or rhosin.
This dissertation specifically studied RhoA and RhoC signaling requirements in the context of KRas- driven cancers. However, our results may have implications outside of this context, and Rho GTPase signaling is certainly important in non Ras-driven cancers, and even in other diseases processes.
Rho GTPases haven been studied and found to be important targets in a wide variety of cancers from colon cancers to breast cancers and leukemias (149–151). Rho GTPases are also important for neuronal development and neurite formation and may be targets in neurological diseases. As mentioned, fasudil has been used to treat pulmonary hypertension and explored in the context of refractory angina (152–154). Rho GTPases may even be good targets for hypertension in general.
As with any scientific inquiry, there remains much to be understood and many benefits to society to be realized only by further study.
101
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~End of Dissertation~
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