Background
Ras proteins have been the subject of intense research as signalling molecules in normal and neoplastic cells [
1]. Yet, a complete understanding of their exact mode of action is still to come. Among the three
RAS genes (
H-RAS,
KRAS and
N-RAS)
KRAS is the most commonly activated in human tumours. Several lines of evidence suggest that not only the presence or absence of a
KRAS mutation but its molecular nature influences tumour cell behaviour [
2,
3]. A reduced transforming capacity of codon 13 mutation as compared with codon 12 is observed
in vitro and
in vivo, with short latency times to tumour-appearance for codon 12
KRAS overexpressing cells [
4‐
6]. Moreover, our previous results indicate that distinct mutations associate with specific metabolic phenotypes, an increased anaerobic glycolytic metabolism in cells containing codon 12
KRAS compared with cells containing codon 13 mutations. Switching to a glycolytic metabolism is a rapid adaptation to hypoxia that can be related to HIF1α expression [
7].
Perpetual blood vessel formation and remodelling (angiogenesis) is a hallmark of cancer and a prerequisite for three-dimensional tumour growth, invasion, and metastasis [
8]. Hypoxia, by inducing HIF-1α, promotes the expression of VEGF-A, the main pro-angiogenic hypoxia-induced gene [
9]. However, oncogenes are also
per se potent inductors of angiogenesis [
10]. Ras proteins are a paradigm for oncogene-dependent induction of tumour angiogenesis due to their involvement in the regulation of key pro and anti angiogenic factors [
11‐
14]. However, its cross-talk with hypoxia-dependent signals is not so clear.
To gain further insight into the metabolic potential and distinct aggressiveness of different activating KRAS mutations, we examined the expression levels of HIF-1α and VEGF-A in stable mutated 12 and 13 NIH3T3 transfectants. Our results in vivo and in vitro indicate that the distinct KRAS mutations generated different normoxic HIF-1α responses. Moreover, different VEGF-A expression patterns were observed that are independent of the HIF-1α status but dependent upon ERKs stimulation. These alterations associated with distinct tumoral angiogenic profiles.
Discussion
In the context of
KRAS-driven tumourigenesis, mutations located at codon 12 and 13 display distinct malignant potential and differentially regulate apoptosis, cell cycle [
4,
15], or metabolic profiles [
25]. Here we show that minor differences in the molecular nature of
KRAS mutations stimulate distinct intracellular signalling pathways in normoxic conditions with different impact in basal levels of HIF-1α VEGF-A production and generation of a distinct vascular network in tumours.
Upregulation of VEGF by the KRAS pathway has been previously shown [
26]. Here we show that cells expressing ASP13
KRAS mutant present higher levels of VEGF-A, the main pro-angiogenic gene induced by hypoxia, in the absence of high HIF-1α levels [
9]. In contrast, CYS12 mutants present a high glycolytic phenotype [
25] through HIF-1α-dependent induction of glycolytic enzymes including GLUT-1 glucose transporter supporting the role of HIF-1α in switching to a glycolytic metabolism [
7].
We have attempted to gain insight into the molecular mechanisms underlying the differential VEGF-A overexpression, apparently independent of HIF-1α in ASP13 clones, Our data support a direct transcriptional effect of ASP13 acting on
VEGF-A promoter. This effect is mediated by a distinct activation of Raf-ERKs pathway and AP2/Sp1 elements within the proximal
VEGF-A promoter. Of note it is independent of hypoxia-dependent elements and of PI3K activity. Extracellular signals that induce VEGF-A through this proximal region include, among others, growth factors such as EGF, insulin and PDGF in fibroblasts [
18], prostaglandin E2 in human muscle cells [
27], M-CSF in monocytes [
28] and lysophosphatidic acid (LPA) in ovarian cancer cells [
29]. All of them affect promoter activity through modulation of at least Sp1 transcriptional activity. Noteworthy, Sp1 is also regulated by different signalling pathways including ERKs, PKA and PI3K-Akt [
18,
30]. We have not detected changes in total Sp1 protein levels between ASP13 and CYS12 mutants, but other mechanisms with impact in the activity of this transcription factor could be implicated, such as acetylation, sumoylation, glycosylation or phosphorylation [
24].
In our xenograft model, ASP13 xenografts consistently develop angiogenic sprouts of large diameter, invested by mural cells. These structures seem to be sufficient to support the increased utilization of the oxidative pentose phosphate pathway observed in the more benign ASP13 tumours [
25]. While development of these complex vascular structures may account for the initial delay observed in tumour growth, we speculate that they are able to support the very rapid growth occurring later [
4]. Nonetheless, the presence of significant tumour necrosis and less Carbonic anhydrase IX to hypoxic adaptation, observed in established ASP13 tumours may depict the relative insufficiency of this vascular tree [
8]. In contrast, histological analysis reveals that the more aggressive CYS12 tumours educe a dense endothelial-lined microvascular network that allows an early, steady and sustained tumour growth. This vascular strategy appears to be effective for these tumour cells that are more resistant to hypoxia, do not proliferate fast [
31] and have relatively low energetic requirements associated with an increased anaerobic glycolysis.
The vascular pattern observed in ASP13 xenografts is in line with previous observations linking high VEGF-A levels with an increased diameter of newly forming vessels [
32,
33]. The prominent stimulation of DNA synthesis in primary HUVECs by whole ASP13 conditioned medium, and in a less conspicuous manner by CYS12 supernatants, propose significant paracrine effects of tumour cell-derived VEGF-A in neovascularization [
34]. Also, ASP13 tumours vessels are covered with α-Sma(+)/Desmin(+) cells [
35] further highlighting the contribution of VEGF-A to vessel maturation and tumour growth.
The retarded growth of ASP13 tumours harbouring elevated VEGF-A levels is consistent with reports challenging the concept that VEGF is just a positive angiogenic regulator. While angiopoietin2 levels did not show differences between transfectants, we cannot exclude a role of other angiogenic factors in differences observed between ASP13 and CYS12 tumoral vessels [
36].
The impact of the genetic background of tumour cells on the angiogenic phenotype is relevant since they may have consequences regarding efficacy of specific antiangiogenic strategies. An evolving tumour with an ever-changing genetic background likely educes a dynamic vascular strategy that may escape to specific antiangiogenic treatment such as those targeting VEGFRs or its ligand [
37]. This is of importance now that more antiangiogenic drugs are being introduced to the clinical setting and there is a need for biomarkers that help in the selection of patients to be treated.
KRAS mutations are used as negative predictors of antiEGFR therapies in colorectal cancer [
38]. The role of
KRAS mutation as a predictive marker of bevacizumab-based treatment has been also explored. Indeed, better response rates to bevacizumab can be observed in
KRAS wt colorectal tumors when compared to
KRAS mutant [
39]. Of note, some authors have explored a potential differential behaviour of codon 13 mutant tumors with no conclusive results [
40]. It will of interest to explore in the adequate clinical setting whether our experimental observations correlate with clinical outcome in other tumor types such as colorectal cancer.
Acknowledgements
This study was supported by grants from SAF 2009–07319, SAF2012-3363, FIS 01/1264 to Gabriel Capellà, FIS 03/0290 to Maria Antonia Arbós and from the Ministerio de Ciencia y Tecnología (SAF2010-20859), the Spanish Ministry of Health (RTICC RD2006-0092) and Generalitat de Catalunya (2009SGR283) to Francesc Viñals. The research team belongs to the Network of Cooperative Research on Cancer RD06/0020/0150 RD12/0036/0031 funded by the Instituto Carlos III, Ministerio de Sanidad y Consumo of Spain, F05-01 from the Fundació Gastroenterologia Dr. Francisco Vilardell, and Acción Transversal contra el Cáncer and Fundación Científica de la AECC.
Role of the funding source
Funding sponsors had no involvement in any scientific area to this work.
Competing interest
None of the authors have any financial or non-financial competing interests in relation to this paper.
Authors’ contributions
AF: study concept and design; acquisition of data; analysis and interpretation of data; drafting of the manuscript; critical revision of the manuscript. MAA: study concept and design; acquisition of data; analysis and interpretation of data; drafting of the manuscript; critical revision of the manuscript; MTQ: acquisition of data; analysis and interpretation of data; critical revision of the manuscript; FV: study concept and design; acquisition, analysis and interpretation of data; drafting of the manuscript; critical revision of the manuscript; obtained funding; study supervision; JRG: material support; obtained funding; critical revision of the manuscript; GC: study concept and design; analysis and interpretation of data; drafting of the manuscript; critical revision of the manuscript; obtained funding; study supervision. All authors read and approved the final manuscript.