Higher metastatic efficiency of KRas G12V than KRas G13D in a colorectal cancer model

Although all KRas (protein that in humans is encoded by the KRas gene) point mutants are considered to have a similar prognostic capacity, their transformation and tumorigenic capacities vary widely. We compared the metastatic efficiency of KRas G12V (Kirsten rat sarcoma viral oncogene homolog with valine mutation at codon 12) and KRas G13D (Kirsten rat sarcoma viral oncogene homolog with aspartic mutation at codon 13) oncogenes in an orthotopic colorectal cancer (CRC) model. Following subcutaneous preconditioning, recombinant clones of the SW48 CRC cell line [Kras wild‐type (Kras WT)] expressing the KRas G12V or KRas G13D allele were micro‐injected in the mouse cecum. The percentage of animals developing lymph node metastasis was higher in KRas G12V than in KRas G13D mice. Microscopic, macroscopic, and visible lymphatic foci were 1.5‐ to 3.0‐fold larger in KRas G12V than in KRas G13D mice (P< 0.05). In the lung, only microfoci were developed in both groups. KRas G12V primary tumors had lower apoptosis (7.0 ±1.2 vs. 7.4 ± 1.0 per field, P = 0.02), higher tumor budding at the invasion front (1.2 ± 0.2 vs. 0.6 ± 0.1, P= 0.04), and a higher percentage of C‐X‐C chemokine receptor type 4 (CXCR4)‐overexpressing intravasated tumor emboli (49.8 ± 9.4% vs. 12.8 ± 4.4%, P < 0.001) than KRas G13D tumors. KRas G12V primary tumors showed Akt activation, and β5 integrin, vascular endothelial growth factor A (VEGFA), and Serpine‐1 overexpression, whereas KRas G13D tumors showed integrin β1 and angiopoietin 2 (Angpt2) overexpression. The increased cell survival, invasion, intravasation, and specific molecular regulation observed in KRas G12V tumors is consistent with the higher aggressiveness observed in patients with CRC expressing (his oncogene.—Alamo, P., Gallardo, A., Di Nicolantonio, F., Pavón, M. A., Casanova, I., Trias, M., Mangues, M. A., Lopez‐Pousa, A., Villaverde, A., Vázquez, E., Bardelli, A., Céspedes, M. V., Mangues, R. Higher metastatic efficiency of KRas G12V than KRas G13D in a colorectal cancer model. FASEB J. 29, 464‐476 (2015). www.fasebj.org

found that the prevalence of the different KRas oncoproteins varied among adenomas and among Dukes stage A, B, and C tumors (2). The RASCAL study later established the KRas G12V mutation as the most aggressive KRAS mutation, associating it with a higher risk of recurrence and death in a large cohort of patients with CRC (3)(4)(5). This finding was later confirmed in al a r g e rs e r i e s( n = 4268) (3)(4)(5). Consistently, KRas G12V mutation confers a metastatic phenotype that renders CRC tumors more aggressive; the incidence of this mutation was found to be higher in primary tumors and metastases of Dukes stage C/D than in stage A/B primary tumors. Moreover, within Dukes stage D cases, KRas G12V mutations were associated with decreased overall survival (2,6). Distinct Kras oncogene mutations have also shown different transformation capacity in 3T3 fibroblasts in vitro (2,6). In another study, 3T3 transformants that expressed KRas G12C and KRas G13D showed significant differences in their regulation of survival and anchorageindependent growth and in the induction of anoikis in culture (7). It was also shown that these 2 transformants generate different sarcoma tumor types when injected in immunosuppressed mice (8,9).
The known differences in the transformation capacity in vitro and tumorigenicity in vivo of different KRas point mutants led us to hypothesize that specific oncogenic KRAS changes could also have different metastatic capacity. The main aim of this study was to compare the metastagenic capacity of 2 SW48 human CRC recombinant clones, expressing KRas G12V or KRas G13D mutants, after their subcutaneous preconditioning followed by orthotopic implantation in the cecum.

SW48 KRas recombinant clones
The SW48 recombinants expressing heterozygous KRasVal12 (KRas G12V) or KRasAsp13 (KRas G13D) oncogenic mutation were generated by homologous recombination using an adeno-associated-virus (AAV), as previously described (10). The mutations are knocked-in in a heterozygous fashion, and the mutant alleles are therefore expressed under the gene's own promoter. Clones obtained were sequenced to verify the presence of the corresponding KRas point mutation.

Generation of the SW48 metastatic CRC models
We used 5-week-old Swiss female nu/nu mice weighing 18 to 20 g (Charles River Laboratories, L-Arbresle, France) for all in vivo experiments. Mice were housed in a sterile environment with bedding, water, and g-ray-sterilized food ad libitum. Experiments were approved by the Animal Ethics Committee at Hospital de la Santa Creu i Sant Pau.
In a preliminary study, we injected the SW48 recombinants expressing KRas G12V or KRas G13D directly into cecum. Due to the low metastatic rate, we used subcutaneous preconditioning before orthotopic injection. This procedure was developed for CRC models and increases the metastatic rate without changing the pattern of metastases (11). This approach provides a sufficient rate of metastasis to compare metastatic development and regulation between KRas G12V-and KRas G13D-derived models.
Briefly ,5m i c ew e r es u b c u t a n e o u s l yi n j e c t e dw i t h23 10 7 control SW48 cells or 2 3 10 7 recombinant cells (KRas G12V or KRas G13D) in DMEM in 2 flanks. Tumors were excised when they reached a volume of 700 mm 3 and disaggregated. A cell suspension (2 3 10 6 cells resuspended in 50 ml) derived from subcutaneous tumors was injected directly into the mouse cecum wall in control (KRas WT, n = 9) and KRas G12V (n =11)andKRasG13D(n =7) mice using the orthotopic cell microinjection procedure (12). Mice were followed once a week and killed when they lost 10% of their body weight or showed signs of pain or illness.

Necropsy and histopathological analysis of primary tumor and metastases
At death, complete necropsy of each animal was performed. We recorded the presence and size of the primary tumor and any visible metastatic foci. Local tumor and the organs with expected metastases (lymph nodes, liver, and lung) were removed, collected, and processed for the histopathological analysis and molecular studies as described previously (12).
Histopathology of primary tumor and all targeted metastatic organs was analyzed by 2 independent observers in samples stained with hematoxylin and eosin (H&E) using 4-203 magnification. We counted the number and area of micro-and macroscopic tumor foci in the affected organs using CellD software, (v3.3), (Olympus). Foci with a diameter of 1 mm or larger and occupying an area . 750,000 mm 2 (13) were considered macroscopic. All smaller foci were considered microscopic.
In primary tumors, we recorded the degree of differentiation and cell morphology, the percentage of tumor necrotic area, the apoptotic and mitotic rate, and the tumor invasion. The apoptotic rates were calculated by counting the number of apoptotic figures in 10 randomly selected 4003 field sections stained with H&E. The primary tumor invasive capacity was analyzed as previously described (11). Briefly, after anti-A1/A3 keratin staining, we counted the number of keratin-positive single epithelial tumor cells as well as tumor cell clusters containing 10 or fewer cells (tumor budding) at the primary tumor front. We recorded the number of keratin positive cells or clusters in 3 different tumor fields (4003 magnification) for each group.

Molecular analysis of primary tumors and metastatic foci
Molecular analysis was performed using immunohistochemistry (IHC) on formalin-fixed paraffin-embedded tumor tissue. IHC staining was performed using the Dako Autostainer automated Link48 (Dako, Carpinteria, CA, USA) and standard procedures. Samples were incubated with the corresponding primary antibody using the following dilutions: integrins b1, b2, b3, b4, b5, a1, a2, a3, a4, a5, a6, and av BD Transduction Laboratories, Franklin Lakes, NJ, USA), which were followed by incubation with mouse or rabbit secondary antibodies (EndVision; Dako). We next incubated the preparation with 3, 3'-diaminobenzidine substrate (Dako) for 5 min, a n dc o n t r a s t e dt h i sw i t hh e m a t oxylin. Immunohistochemical slides were evaluated by 2 independent observers who quantified the percentage of stained cells in relation to the total number of tumor cells and their staining intensity (between 0 and 3, where 3 represents the maximum intensity). Finally, the multiplication of both values represented the expression of the protein in each sample.
ELISA assays were performed to determine VEGFA in primary tumor samples extracts following the manufacturer'sr e c o m m e ndations (Human VEGFA Platinum ELISA, ref. BMS277/2CE for VEGFA; eBioscience, San Diego, CA, USA).

Statistical analysis
Fisher's exact test was used to analyze possible significant differences between groups in primary tumor or metastatic rates. The Mann-Whitney test was used to compare tumor size, the number of apoptotic or mitotic figures, single tumor cells, and tumor clusters or metastatic foci between groups. Differences in survival between groups were evaluated using Kaplan-Meier curves and the log-rank test. All quantitative values were expressed as mean 6 SE, and the statistical tests were performed using SPSS, version 11.0 (IBM, Armonk, NY, USA). Differences between groups were considered significant at P , 0.05.
Related to metastatic dissemination, all 3 groups developed lymphatic and lung metastases ( Fig. 1,Table1),whereas no liver metastases were recorded in any group. The number of mice developing lymph node metastases was also higher in KRas G12V (73%) and KRas G13D (29%) than in KRas WT (11%) mice. Moreover, there were significantly more lymph node metastatic foci in KRas G12V (n = 26) or KRas G13D (n =4 1 )t h a ni nK R a sW T mice (P , 0.05). Similarly, there were more lung metastases in KRas G12V (n = 34) and KRas G13D (n = 27) than in KRas WT (n = 10) mice (Table 1) KRas G12V showed higher tumor cell survival, invasion, and CXCR4 expressing intravasated tumor emboli than KRas G13D We analyzed the number of apoptotic and mitotic cells in H&E-stained sections derived from KRas G12V and KRas G13D primary tumors. KRas G12V primary tumors displayed significantly (P = 0.02) fewer apoptotic figures per field (7.0 6 1.2) than KRas G13D tumors (7.4 6 1.0; Fig. 2A, B). Analysis of the mitotic rate showed a trend toward more mitotic figures in KRas G12V (4.9 6 0.5) than in KRas G13D (2.8 6 0.4) primary tumors, but it did not reach statistical significance (not shown). In contrast to the observations in primary tumors, the mitotic and apoptotic parameters in KRas G12V and KRas G13D metastatic foci displayed no significant differences between groups (not shown).
We also analyzed the invasion front in the primary t u m o r s . Based on the established relationship between the induction of epithelial-mesenchymal transition (EMT) in tumor cells and the acquisition of an increased invasive and metastatic capacities (14), we studied the expression of Snail-1, as a molecular marker of EMT and the expression of E-cadherin (down-regulated during EMT) and b-catenin (up-regulated during EMT) in the primary tumors, especially at their invasion front, of both groups. We found no differences in Snail-1, E cadherin, and b-catenin expression between KRas G12V and KRas G13D groups (data not shown). To assess differences in intravasation capacity and CXCR4 expression, we counted the number of tumor emboli inside blood vessels in the tissues adjacent to the primary tumors (Fig. 2) and performed CXCR4 immunostaining. We observed no significant differences in the number of intravasated tumor emboli in the submucosal and pericolic layers of the cecum between groups. The percentage of tumor cells with CXCR4 membrane expression in intravasated tumor emboli of KRas G12V mice (49.8 6 9.4%) was significantly (P , 0.001) higher than in KRas G13D (12.8 6 4.4%) intravasated tumor emboli (Fig. 2E, F, H).
KRas G12V promotes a higher growth rate in lymph node metastases than KRas G13D mice We analyzed the number of lymphatic and lung metastases, determined their size, and classified them into micro-, macro, and visible metastases. The percentage of mice with lymphatic metastasis was significantly higher in KRas G12V (73%, 8/11) than in KRas G13D (23%, 2/7) mice ( Fig. 1, Table 1). Moreover, in KRas G12V mice, the mean area of lymphatic microfoci (30.3 6 8.3 3 10 4 mm 2 )w a ss i g n i ficantly (P = 0.02) larger than in KRas G13D mice (12.1 6 2.8 3 10 4 mm 2 ). The mean area of the lymphatic macrofoci in KRas G12V mice (193.2 6 28.6 3 10 4 mm 2 ) was also larger than in KRas G13D mice (158.5 3 10 4 mm 2 ). Similarly, the mean area of visible lymphatic metastases in KRas G12V mice (7523 6 1937 3 10 4 mm 2 )w a ss i g n i ficantly (P = 0.05) larger than in KRas G13D mice (2622 6 400 3 10 4 mm 2 ;F i g .1 ,T a b l e1 ) . Thus, as compared with KRas GD13, the expression of the KRas G12V oncogene increased lymph node colonization and increased metastatic foci growth in lymph nodes by promoting the transition from micro-to macrometastases and from macro-to visible metastases. There were no significant differences in the number of mice affected with lung metastasis between KRas G12V and KRas G13D groups. The analysis of metastatic size showed only micrometastasis in KRas G12V or KRas G13D mice. The total number of lung metastases in KRas G12V mice (n =3 4 ) w a sh i g h e rt h a ni nK R a sG 1 3 Dm i c e( n =2 7 ) .T h em e a n size of the lung microfoci was significantly (P , 0.001) larger in KRas G13D (11.9 6 3.8 3 10 4 mm 2 )t h a ni n KRas G12V mice (6.4 6 1.5 3 10 4 mm 2 ; Table 1, Fig. 1). No hepatic metastases were observed in SW48-derived KRas WT, KRas G12V, or KRas G13D mice.

KRas G12V and KRas G13D induced different molecular changes in primary tumors
The differences in metastatic dissemination between the KRas G12V and KRas G13D groups triggered the analysis of the expression and/or activation of proteins involved in signaling downstream of KRas (PI3K and MAPK pathways) as well as regulators of survival, adhesion, invasion, and metastatic dissemination to unveil some of the molecular changes that could underlie the observed differences in metastatic dissemination.
KRas G12V primary tumors showed a modestly (P = 0.05) higher activation of PI3K pathway than KRas G13D tumors (Fig. 3) as measured by the level of phospho-AKT. Nevertheless, in lymphatic and lung metastasis, there were no significant differences in AKT activation between the KRas G12V and KRas G13D groups. The activation of the Erk pathway, as measured by p-MAPK, showed a trend toward higher activation in KRas G12V vs. KRas G13D tumors (data not shown). No significant differences were recorded in the activation of the Erk pathway in lymph node or lung metastasis between KRas G12V and KRas G13D groups (data not shown).
To analyze whether the KRas G12V and KRas G13D oncogenes differentially regulated adhesion, we evaluated the expression of a (1, 2, 3, 4, 5, 6, and n) and b (1-6) integrins by IHC. Of the 13 evaluated integrins, only b5 and b1 showed a differential expression pattern between groups. The level of b1 integrin expression was significantly (P = 0.039) higher in KRas G13D primary tumors than in KRas G12V tumors (Fig. 4A, B). In contrast, the expression of b5 integrin was higher in the KRas G12V primary tumor than in KRas G13D tumors (P = 0.037, Fig. 4G, H). In addition, the level of b5orb1 integrin expression was low in both lymph node and lung metastases in the KRas G12V and KRas G13D groups. We did not therefore detect significant differences in the expression of these integrins in metastases between the groups (Fig. 4C-F, I-L).
We did not observe expression of CXCR4 in the bulk of primary tumors or their invasive front in KRas G12V and KRas G13D groups. However, CXCR4 was expressed in a subset of cells in metastatic foci involving the lymph nodes, or lung metastasis. The percentage of tumor cells that overexpressed CXCR4 in their membrane in the metastases affecting the lymph nodes in KRas G12V mice was significantly (P = 0.009) higher than that in KRas G13D mice (Fig. 5). There were no significant differences between groups in CXCR4 expression in lung metastases. We also evaluated the expression of Serpine-1, a regulator of adhesion and invasion. The expression of Serpine-1, as measured by IHC, was significantly (P =0.033) higher in KRas G12V than in KRas G13D primary tumors. These differences in expression were not maintained at the metastatic sites. Thus, no significant differences in Serpine-1 expression in lymphatic metastases were found between KRas G12V and KRas G13D groups. In contrast, the expression of Serpine-1 in lung metastases was significantly (P , 0.001) higher in KRas G13D than in KRas G12V mice (Fig. 5).
In primary tumors, the expression of VEGFA measured by ELISA showed significantly higher (P = 0.025) levels in KRas G12V than in KRas G13D. We also used IHC to detect VEGFA expression in metastatic foci. We observed no significant differences in lymphatic metastatic foci between groups. In contrast, lung metastases in the KRas G13D group showed a significantly (P , 0.001) higher level of VEGFA expression in KRas G13D than in KRas G12V lung metastases (Fig. 6). Angpt2 is involved in angiogenesis and metastatic spread. The analysis of its expression by IHC showed that Angpt2 was significantly (P = 0.022) higher in KRas G13D than in KRas G12V primary tumors. The observed differences in Angpt2 expression were also maintained in metastases, because KRas G13D displayed a significantly higher level of this protein in metastatic foci involving lymph nodes (P = 0.048) and the lung (P , 0.001) than in the KRas G12V group. The level of Angpt2 expression in lymph node metastases that developed in the KRas G13D group was significantly higher (P = 0.045) than that observed in KRas G13D primary tumors (Fig. 6). Subcutaneous SW48 KRas G12V or SW48 KRas G13D tumors used to generate the metastatic orthotopic model  mostly exhibited protein expression levels similar to those found in the derived primary tumors (data not shown). These expression profiles differed from those that we were able to obtain from cell recombinants cultured in vitro. Therefore, most molecular changes appear to be induced during their subcutaneous passage.

KRas G12V enhances metastases to lymph nodes, an indication of its higher aggressiveness
Our aim was to evaluate whether there were differences in metastatic dissemination between SW48-derived mouse CRC models expressing the KRas G12V or the KRas G13D oncogene. We observed that the KRas G12V mutation increased the percentage of mice with lymph node metastases and the area of lymphatic microfoci, macrofoci, and visible lymph node metastases, as compared with KRas G13D. Therefore, the KRas G12V oncogene increased both the colonization of the lymph nodes and the growth rate of the metastatic foci at this site, promoting the transition from microfoci to large metastases. Our observation of the significantly higher capacity for KRas G12V expressing tumor cells to develop lymphatic metastases suggests higher tumor aggressiveness for this mutation. This argument is consistent with the presence of lymph node metastasis as the strongest predictor of poor p r o g n o s i sinpatientswithCRC (15).Moreover,thehigher aggressiveness observed in KRas G12V mice is consistent with the shorter overall survival observed in patients with CRC with KRas G12V mutation compared with other Kras mutations in the RASCAL study (3). In human CRC tumors, of 12 different point mutations found at KRas codon 12 or 13, only the KRas G12V mutation conveyed an increased risk of recurrence and death (3,4). In another study, an analysis restricted only to Dukes stage D patients showed that KRas G12V decreased overall survival as compared with other KRas mutations (6). In our study, we did not detect any difference between KRas G12V and KRas G13D mice regarding the percentage of animals with lung metastases. KRas G13D seems to stimulate the growth of the lung metastasis more than KRas G12V, but it does not appear to be able to induce the transition from micro-to macrometastases, because a l lm e t a s t a s e sr e m a i n e dm i c r o s c o p i cb o t hi nK R a sG 1 3 D and KRas G12V mice. In liver, we did not observe any metastatic dissemination, because the SW48 CRC cell line does not show an intrinsic ability to metastasize to the liver. We used the SW48 CRC cells because they bear the Kras wild type as well as because they have previously been reported to be amenable to genetic engineering by homologous recombination.

KRas G12V alters protein regulation and induces higher tumor cell survival, invasion, and intravasation
KRas G12V and KRas G13D tumors presented differences in protein expressions involved in invasion, cell survival, and intravasation processes. KRas G12V enhanced cell survival, invasion, and intravasation and overexpressed CXCR4, b5 integrin, VEGFA, and Serpine-1 and overactivated Akt in primary tumors. These alterations may underlie the increased metastatic growth in the lymph nodes found in KRas G12V mice, as compared with KRas G13D. In agreement, the different ras mutants show markedly different transformation capacities (16,17) and have been associated with different interaction with the downstream effectors (18). In our model, KRas G12V induced activation of the AKT pathway and showed f e w e ra p o p t o t i cm a r k e r si nc o m p a r i s o nw i t hK R a s G13D. It has been shown that the Ras Val12 oncogene activates the PI3K/Akt pathway (19,20), whereas tumors use PKB/Akt pathway activation to inhibit apoptosis, leading to higher migratory, invasive, and metastatic capacities (21,22). Consistently, we previously showed that the KRas G13D oncogene displays lower transformation capacity in fibroblasts, associated with Akt inactivation and increased apoptosis in vitro (7) and in vivo (8). Consequently, the KRas G13D oncogene yielded more indolent sarcomas than fibroblasts transformed with the KRas Cysteine 12, the expression of which was associated with Akt activation and less apoptosis. The activation of the Akt pathway has also been observed in stage II patients with CRC where AKT activation predicts tumor recurrence (23). We also observed that KRas G12V enhanced budding in primary tumors associated with enhanced lymph node metastases. In agreement with this finding, in patients with CRC, tumor budding at the invasive front is associated with lymph node metastases and poor prognosis (24)(25)(26)(27)(28).
The increased invasion observed in KRAS mutant tumors could be caused by several distinct molecular mechanisms. We discarded EMT as responsible for the enhanced metastases observed in KRas G12V because we did not observe differences in single cell count or in Snail-1, E-cadherin, or b-catenin expression between KRas G12V and KRas G13D tumors at the invasive front, characteristic of EMT (14). Nevertheless, in CRC models, several reports support higher migratory and metastatic capacity for tumors that overexpress b5 integrin together with Akt activation (observed in our KRas G12V tumors) compared with tumors with b1 expression and Akt inactivation (observed in KRas G13D tumors). Similarly, migration of the SW480 CRC cell line (which expresses KRas G12V) in type I collagen is dependent on avb5 expression and Akt activation (29). In contrast, down-regulation of avb5 integrin in this model leads to a2b1-dependent and Aktindependent migration, supporting a cross-regulation between b5 and b1 associated with differential Akt regulation (29). Moreover, avb5-dependent migration on vitronectin has been associated with enhanced liver metastases in the LM-LM6 CRC model, whereas Akt inactivation blocked this migration (30). Consistently, the KRas G12V oncogene mutation induces b5 expression and blocks integrin b1 expression in colon epithelial cells (31).
Serpine-1 is also overexpressed in KRas G12V tumors and may contribute to their enhanced invasiveness and metastases. Thus, in patients with CRC, Serpine-1 overexpression in primary tumors is associated with lymph node metastasis (32), whereas high levels of Serpine-1 in plasma is a marker of poor prognosis (33). In addition, cross-talk between b5 and b1, like that described in CRC models, has been reported in fibroblasts, in which integrin b5 degradation leading to b1 up-regulation is controlled by urokinase-type plasminogen activator system (uPA/uPAR) system activation (34). This system is inactivated by serpine-1. This suggests that Serpine-1, a protein overexpressed together with b5 integrin in KRas G12V tumors, may regulate this crosstalk in our CRC model, when serpine-1 covalently binds to and inactivates the uPA/uPAR system.
In KRas G12V mice, we observed an increased percentage of CXCR4-overexpressing cells in intravasated tumor emboli in the submucosal and pericolic layers of the cecum. We also found more CXCR4 cells in lymph node metastases. These findings suggest the CXCR4 receptor plays a role in intravasation, enhancing lymph node metastasesinourmodel.Thissuggestionisinagreement with the association between intravasated tumor emboli (35) and lymphovascular invasion (36) with lymph node metastasis and poor prognosis in patients with CRC. Similarly, CXCR4 overexpression in primary tumors increased the risk of recurrence and poor survival (37) in patients with CRC. Moreover, expression of CXCR4 in the HT29 CRC model favors tumor cell extravasation (38), whereas CXCR4 expression promotes the growth of CT-26 CRC micrometastases in the colonized organ (39). In addition, CXCR4 + HT29 CRC cells are capable of establishing a paracrine signaling with stromal cell-derived factor 1 (SDF1a) secreting lymph node stromal cells (40), which may drive their dissemination toward this metastatic site. Similarly, CXCR4-expressing tumor cells are able to migrate through hypoxic SDF1a, secreting endothelial cells (transendothelial migration), and invade blood vessels in a breast cancer model (41).
In agreement with enhanced VEGFA tumor expression and lymph node metastases observed in KRas G12V mice, VEGFA and CXCR4 are associated with lymph node metastasis and poor prognosis in patients with CRC (42)(43)(44). Similarly, in the CT26 CRC model, VEGFA induces angiogenesis and promotes vascular permeability, leading to metastatic spread (45,46). Finally, and in contrast with the reports on KRas G12Vdisregulated proteins, no publications on the prognostic value in patients with CRC have been reported for the proteins overexpressed in KRas G13D tumors (integrin b1 or Angpt2).

Differential protein regulation in KRas G12V and KRas G13D mice between primary tumors and metastases
Whereas protein expression in primary tumors may contribute to determine the mechanism of invasion and intravasation, such expression in metastases may indicate the pathway used for metastatic foci growth at the metastatic site. In our model, KRas G13D tumors overexpressed Angpt2 and developed larger lung micrometastasis than with KRas G12V. In this regard, Angpt2 expression in the primary tumor may have promoted colonization of the lung, because it enhances cancer cell extravasation by loosening the endothelial cell junctions, leading to increased metastasis in the lung (47,48). In contrast, the high levels of Angpt2 observed in lung microfoci in KRas G13D mice may have determined the inability of these tumor cells to promote vascularization. It has been observed that high levels of Angpt2 induce microvessel regression and inhibit tumor growth in the HT29 subcutaneous CRC model (49).
The oncogene expressed in primary tumors (KRas G12V or KRas G13D) may determine the pattern of protein expression. Interestingly, the pattern of protein expression we found in orthotopic primary tumors was already present in the subcutaneous tumors used for KRas G12V and KRas G13D preconditioning. Thus, both orthotopic and subcutaneous tumors that expressed KRas G12V showed higher Akt activation and integrin b5 overexpression than KRas G13D. However, the environment in the site where the tumor grows may also contribute to regulate protein expression in tumor cells. In contrast with our observations in primary tumors, the number of mitotic and apoptotic figures in KRas G12V and KRas G13D metastatic foci did not differ significantly between groups. Therefore, different metastatic organs appear to regulate apoptosis in a different way. We also observed that the pattern of protein expression in metastases differed greatly from the corresponding pattern in the primary tumor. This suggests that that the tumor environment also contributes to determining this pattern. In support of this suggestion, we observed that VEGFA or Serpine-1 was overexpressed in KRas G12V but not in KRas G13D primary tumors. In lung metastases, these 2 proteins were overexpressed in KRas G13D compared with KRas G12V mice.

Clinical implications
In summary, this is the first report to describe a different metastatic capacity and a pattern of protein expression for distinct KRas point mutations in an orthotopic mouse CRC model, as well as an association between metastatic capacity and CXCR4-overexpressing tumor emboli. Our results highlight the need to consider the different KRas mutants as separate entities.
These results are consistent with the higher aggressiveness of KRas G12V mutation observed in patients with CRC, as compared with any other KRas mutation, and support the notion that different KRas mutants may associate with different prognosis. The different regulation of cell survival and Akt-p between KRas G12V and KRas G13D in our model may also indicate different responses between KRas G12V and KRas G13D to antitumor drugs. This is in agreement with the reported lack of Akt activation by KRas G13D (7,8) and with the higher sensitivity of KRas G13D CRC cells, as compared with KRas G12V to the epidermal growth factor receptor (EGFR) inhibitor cetuximab in vitro and longer overall survival in KRas G13D patients compared with other KRas mutations when being treated with the EGFR inhibitor cetuximab (50). However, more recent clinical data are contradictory (51). Future studies should explore other KRas mutations with lower transforming capacity in relation to their ability to engage different pathway effectors, their propensity to metastasize, as well as their impact on anticancer treatments.