Loss of RasGAP Tumor Suppressors Underlies the Aggressive Nature of Luminal B Breast Cancers

Abstract

Luminal breast cancers are typically estrogen receptor-positive and generally have the best prognosis. However, a subset of luminal tumors, namely luminal B cancers, frequently metastasize and recur. Unfortunately, the causal events that drive their progression are unknown, and therefore it is difficult to identify individuals who are likely to relapse and should receive escalated treatment. Here, we identify a bifunctional RasGAP tumor suppressor whose expression is lost in almost 50% of luminal B tumors. Moreover, we show that two RasGAP genes are concomitantly suppressed in the most aggressive luminal malignancies. Importantly, these genes cooperatively regulate two major oncogenic pathways, RAS and NF-κB, through distinct domains, and when inactivated drive the metastasis of luminal tumors in vivo Finally, although the cooperative effects on RAS drive invasion, NF-κB activation triggers epithelial-to-mesenchymal transition and is required for metastasis. Collectively, these studies reveal important mechanistic insight into the pathogenesis of luminal B tumors and provide functionally relevant prognostic biomarkers that may guide treatment decisions.

Significance: The lack of insight into mechanisms that underlie the aggressive behavior of luminal B breast cancers impairs treatment decisions and therapeutic advances. Here, we show that two RasGAP tumor suppressors are concomitantly suppressed in aggressive luminal B tumors and demonstrate that they drive metastasis by activating RAS and NF-κB. Cancer Discov; 7(2); 202-17. ©2016 AACR.See related commentary by Sears and Gray, p. 131This article is highlighted in the In This Issue feature, p. 115.

Conflict of interest statement

There are no conflicts of interest to disclose.

©2016 American Association for Cancer Research.

Figures

Figure 1.. DAB2IP is selectively lost in luminal B breast cancers and functions as a tumor suppressor by affecting multiple Ras Isoforms.

A, DAB2IP mRNA expression across the different subtypes of human breast cancer. DAB2IP is specifically low in the luminal B subtype (p< 6.69×10−9, t-test comparing LumA vs LumB). B, Tukey boxblot showing the median DAB2IP expression per subtype (p=1.5×10−14, ANOVA). C, Methylation of the CpG island in the DAB2IP promoter (chr9: 123,331,678) across subtypes. The DAB2IP promoter is specifically methylated in the Luminal B subtype (p< 0.005, t-test comparing LumA vs LumB). D, Methylation at the CpG island in the DAB2IP promoter inversely correlates with expression. E, Kaplan-Meier curve showing relapse-free survival of luminal B tumors with high or low DAB2IP expression (log rank p=0.0028). F, DAB2IP mutations in human tumor samples. Each triangle represents a nonsynonymous mutation and each diamond represents a nonsense mutation. Blue triangles indicate breast cancer mutations and red triangles indicate breast cancer mutations in the catalytic RasGAP domain. The table shows DAB2IP mutations in human breast tumors and the domain in which they occur. G, DAB2IP expression in lysates of basal (Ba, HCI-004) and luminal B (HCI-011, −013, −017, −003, −005) PDX tumors. H, DAB2IP expression in a panel of ER+ human breast cancer cell lines and normal immortalized mammary epithelial cells (MCF10A). Cell lines with minimal or no DAB2IP are indicated in bold. I, Western blot showing an increase in HRas-GTP, NRas-GTP, and pERK levels in CAMA1 cells following shRNA-mediated inactivation of DAB2IP or control shRNA. Samples were run on the same gels, but a lane was cut out as indicated by the line. Lysates were run in duplicate. DAB2IP, HRAS/HRAS-GTP and pERK were probed for on the same gel, while NRAS/NRAS-GTP were probed for on a separate gel. J, Soft agar colony formation of CAMA1 cells infected with a 3’-UTR shRNA targeting DAB2IP or nontargeting control, followed by expression of EGFP control vector, wild-type DAB2IP, or DAB2IP GAP point mutant (R413L). Data show relative number of colonies +/− SD. There was a significant increase in soft agar colony formation upon DAB2IP suppression (p=0<0.0001, t-test comparing EGFP/shCNT vs EGFP/shDAB2IP(1)). Reconstitution with wild-type DAB2IP rescued colony formation, however, the R413L GAP mutant failed to rescue (p<0.0001, t-test comparing EGFP/shCNT vs R413L/shDAB2IP(1)). K, Western blot showing a reduction of HRas-GTP, KRas-GTP, and pERK levels in MCF7 cells upon expression of DAB2IP compared to EGFP. Samples were run on the same gel, but a lane was cut out as indicated by the line. Lysates were run in duplicate. DAB2IP, HRAS/HRAS-GTP, KRAS/KRAS-GTP and pERK were probed for on the same gel, while NRAS/NRAS-GTP were probed for on a separate gel. L, Soft agar colony formation of MCF7 cells expressing DAB2IP or EGFP. Data show relative number of colonies +/− SD. There was a statistically significant decrease in soft agar colony formation upon DAB2IP-reconstitution (p=0.0002, t-test). M, Western blot showing that expression of the R413L DAB2IP-GAP mutant fails to suppress HRas-GTP, KRas-GTP, NRas-GTP, and pERK levels in MCF7 cells. Lysates were run in duplicate. DAB2IP, HRAS/HRAS-GTP, KRAS/KRAS-GTP and pERK were probed for on the same gel, while NRAS/NRAS-GTP were probed for on a separate gel. N, Soft agar colony formation of MCF7 cells expressing the R413L GAP mutant or EGFP. Data show relative number of colonies +/− SD. There was no statistically significant decrease in anchorage-independent growth upon ectopic expression of the R413L mutant. O, Xenograft tumor formation of CAMA1 cells infected with DAB2IP-targeting CRISPR guide RNA or empty control vector. Cells were injected subcutaneously into female NOD/SCID mice. Horizontal bars indicate median tumor volume and the error bars indicate +/− interquartile range. There was a statistically significant increase in tumor volume upon CRISPR-mediated DAB2IP knock-out (p=0.0043 for sgDAB2IP(1); p=0.0087 for sgDAB2IP(2), Mann-Whitney).

Figure 2.. Concomitant loss of DAB2IP and RASAL2 occur in the most aggressive luminal B tumors and specifically enhance invasion and EMT

A, RASAL2 and DAB2IP protein expression in a panel of ER+ human breast cancer cell lines and normal immortalized mammary epithelial cells (MCF10A). The blots marked by an asterisk are a duplicate from Figure 1H for comparison. B, DAB2IP and RASAL2 expression in lysates of basal (Ba, HCI-004) and luminal B (HCI-011, −013, −017, −003, −005) PDX tumors. The blots marked by an asterisk are a duplicate from Figure 1G for comparison. C, Pie chart of human luminal B tumors. 16% of luminal B tumors have low levels of RASAL2 (red), 24% have low levels of DAB2IP (blue), and 22% have low levels of both, RASAL2 and DAB2IP (black). D, Kaplan-Meier curve showing relapse-free survival of luminal B tumors with high RASAL2 and DAB2IP expression or low RASAL2 and DAB2IP expression (log rank p=3.1e-08). E, Representative IHC images of human luminal B tumors with low (top) and high (bottom) DAB2IP and RASAL2 expression. The images marked by the red asterisk are images of the same section of the same tumor. The scale bars correspond to 100μm. F, Xenograft tumor formation of CAMA1 cells infected with nontargeting control vectors, DAB2IP-targeting CRISPR guide RNAs (p=0.0043 for sgDAB2IP(1); p=0.0087 for sgDAB2IP(2), Mann-Whitney), RASAL2-targeting shRNAs (p=0.0016 for shRASAL2(1); p=0.0022 for shRASAL2(2), Mann-Whitney), or both sgDAB2IP(1)/shRASAL2(1)-targeting constructs (p=0.008, Mann-Whitney). Cells were injected subcutaneously into female NOD/SCID mice. Horizontal bars indicate median tumor volume and the error bars indicated +/− interquartile range. The data indicated in grey is a duplicate from Figure 1O. There was no statistically significant increase in tumor volume upon combined inactivation of DAB2IP and RASAL2. G, Transwell invasion of MCF10A cells infected with control shRNA, an shRNA targeting RASAL2 (R(1)) and/or a sgRNA targeting DAB2IP (D(2)). Invasion was measured after 24 hours and reported as average +/− SD. H, Transwell invasion of MCF10A cells infected with control shRNA, an shRNA targeting RASAL2 (R(1)) and/or an siRNA targeting DAB2IP (D). Invasion was measured after 24 hours and representative images are shown. I, Transwell invasion of CAMA1 cells infected with control shRNA, an shRNA targeting RASAL2 (R(1)) and/or an siRNA targeting DAB2IP (D). Invasion was measured after 24 hours and reported as average +/− SD. J, Transwell invasion of MCF10A cells infected with control shRNA or 3’-UTR shRNAs targeting RASAL2 and DAB2IP, followed by expression of EGFP/LacZ control vectors or wild-type DAB2IP/RASAL2. Invasion was measured after 24 hours and reported as average +/− SD. K, Western blot showing the expression of molecular markers of an EMT, E-cadherin, N-cadherin, and Slug in MCF10A cells infected with an shRNA targeting RASAL2 and/or an siRNA targeting DAB2IP. Lysates were run in duplicate. DAB2IP and E-cadherin were probed for on the same gel with the loading control Tubulin (1), while RASAL2, N-cadherin and Slug were probed for separately with the loading control Tubulin (2). L, Real-time PCR quantification of E-cadherin, N-cadherin, Slug, Fibronectin, Vimentin, Snail, and ZEB1 in MCF10A cells in response to RASAL2 and/or DAB2IP suppression. Data show average relative amount of mRNA +/− SD.

Figure 3.. DAB2IP and RASAL2 cooperatively regulate metastasis in vivo

A, Representative bioluminescence images of luciferase-expressing MDA-MB-361 cells that were reconstituted with LacZ/LacZ, LacZ/RASAL2, DAB2IP/LacZ or DAB2IP/RASAL2 and injected intracardially into NOD/SCID mice. B, Quantification of the number of mice with metastatic lesions as detected by bioluminescence imaging two months post injection of MDA-MB-361 cells. Expression of RASAL2 and DAB2IP together significantly suppressed metastasis formation compared to expression of RASAL2 alone (p=0.043, Fisher Exact), DAB2IP alone (p= 0.002, Fisher Exact), or LacZ control (p=6×10−5, Fisher Exact). C, Total Flux [p/s] (e+07) per mouse as determined by bioluminescence imaging four months post inter-cardiac injection. Only expression of RASAL2 and DAB2IP together significantly suppressed bioluminescence signal compared to the LacZ control (p=0.004, Mann-Whitney). D, Western blot confirming ectopic expression of DAB2IP and RASAL2. Lysates were run in duplicate. DAB2IP and RASAL2 were probed for on the same gel with the loading control Tubulin (1), while the tags, V5 and HA, were probed for separately with the loading control Tubulin (2). E, Fold growth at day three of MDA-MB-361 cells expressing LacZ/LacZ, LacZ/RASAL2, DAB2IP/LacZ or DAB2IP/RASAL2 +/− SD. F, Western blot confirming RASAL2 and DAB2IP suppression in McNeu cells, a luminal mouse cancer cell line that was infected with shRNAs targeting RASAL2 and DAB2IP or non-targeting controls. G, Representative H&E images of lungs, with McNeu metastases marked by red circles. McNeu cells were infected with shRNAs targeting RASAL2 and DAB2IP or non-targeting controls and were injected into tail veins of syngeneic mice. Lungs were harvested three weeks post injection and the number of metastases per mouse was quantified. Scale bars correspond to 500μm. H, Suppression of RASAL2 and DAB2IP significantly enhanced metastasis formation (p=0.0019, Mann-Whitney). Horizontal bars indicate the median number of metastatic lesions per mouse.

Figure 4.. RASAL2 and DAB2IP loss cooperates to activate the Ras and the NFκB pathways

A, Quantification of KRas- and HRas-GTP levels in MCF10A cells upon shRNA-mediated suppression of RASAL2 (R(1)) and/or DAB2IP (D(1)). The Ras-GTP/Ras ratio of each sample was calculated and normalized to the non-targeting shRNA control. B, Western blot showing downstream Ras pathway activation (pAKT and pERK) following RASAL2 and/or DAB2IP knock down in MCF10A cells. C, Transwell invasion of MCF10A cells infected with shRNAs targeting RASAL2 and DAB2IP. Invasion was measured after 24 hours in the presence of vehicle (DMSO), MEK inhibitor (PD-0325901 at 500nM) and PI3K inhibitor (GDC-0941 at 750nM). The number of cells that invaded was normalized to the non-targeting shRNA control and reported as average +/− SD. D, Transwell invasion of MCF10A cells infected with control shRNA or 3’-UTR shRNAs targeting RASAL2 and DAB2IP, followed by expression of EGFP and LacZ control vectors, wild-type DAB2IP and RASAL2, or the GAP mutants (DAB2IP413L and RASAL2K417E). Invasion was measured after 24 hours and reported as average +/− SD. The data indicated in grey is a duplicate from Figure 2J for comparison. E, Western blot showing the expression of molecular markers of an EMT in MCF10A cells. Cells were infected with non-targeting shRNAs or shRNAs targeting RASAL2 and DAB2IP. Addition of MEK or PI3K inhibitors had no effect on the induction of an EMT. Lysates were run in duplicate. DAB2IP, RASAL2, pAKT and pERK were probed for on the same gel with the loading control Tubulin (1), while E-cadherin, N-cadherin and Slug were probed for separately with the loading control Tubulin (2). F, Schematic of DAB2IP’s functional domains. The RasGAP domain allows DAB2IP to negatively regulate Ras while the Period-like (Per-like) domain allows DAB2IP to negatively regulate NF-κB through a direct interaction with TRAF2. G, NF-κB activity reported as relative light units +/− SD. MCF10A cells were infected with non-targeting control shRNAs (CNT) or shRNAs targeting RASAL2 (R(1)) and DAB2IP (D(1)) alone or together and NF-κB activity was measured using a reporter assay. While suppression of DAB2IP alone significantly induced NF-κB activity compared to the control (p=0.0025, t-test), suppression of both RASAL2 and DAB2IP even further induced NF-κB activity (p=0.04, t-test shCNT/shDAB2IP vs shRASAL2/shDAB2IP). H, NF-κB activity reported as relative light units +/− SD. MCF10A cells were infected with non-targeting control shRNAs or shRNAs targeting RASAL2/DAB2IP. Subsequently cells were infected with control cDNA (EGFP and LacZ), wild-type RASAL2/DAB2IP cDNA, GAP point mutant cDNA (DAB2IPR413L/RASAL2K417E) and DAB2IP double mutant/RASAL2 GAP point mutant cDNA (DAB2IPR413L/S728A/RASAL2K417E). Data was reported as average +/− SD. While expression of wild-type RASAL2/DAB2IP and expression of the GAP mutants rescued NF-κB activation, the DAB2IP double mutant failed to suppress NF-κB activity.

Figure 5.. NF-κB activation is required for EMT and metastasis

A, NF-κB activity, reported as relative light units +/− SD, from MCF10A cells infected with shRNAs targeting RASAL2 and DAB2IP and co-infected with either vector control or the IKBα−SR. Co-infection with the IKBα−SR completely ablates NF-κB activity. B, Transwell invasion of MCF10A cells infected with control shRNAs or shRNAs targeting RASAL2 and DAB2IP. Invasion was measured after 24 hours and reported as average +/− SD Suppression of NF-κB activity (co-infection of the IKBα−SR) had no effect on invasion. C, Western blot showing the expression of the EMT markers E-cadherin and N-cadherin in MCF10A cells that were infected with control shRNAs or shRNAs targeting RASAL2 and/or DAB2IP in the absence or presence of the IKBα−SR. Lysates were run in duplicate. RASAL2, N-cadherin and Slug were probed for on the same gel with the loading control GAPDH (1), while DAB2IP and E-cadherin were probed for separately with the loading control GAPDH (2). D, Real-time PCR quantification of Fibronectin, Vimentin, Slug, Snail, and ZEB1 in MCF10A cells in response to RASAL2 and DAB2IP suppression with and without the IKBα−SR. Data show average relative amount of mRNA +/− SD. E, Representative H&E images of lungs, with McNeu metastases marked by red circles. McNeu cells were infected with non-targeting control shRNAs or shRNAs targeting RASAL2 and DAB2IP with and without the IKBα−SR. Cells were injected into tail veins of syngeneic mice, lungs were harvested three weeks post injection and the number of metastases per mouse was quantified. Scale bars correspond to 500μm. F, Co-infection of the IKBα−SR significantly suppressed metastasis formation (p=0.037, Mann-Whitney). Horizontal bars indicate the median number of metastatic lesions per mouse. Note: The data indicated in grey is a duplicate from Figure 3G.

Figure 6.. DAB2IP and RASAL2 Cooperate to Drive Distinct Aspects of Metastasis Through Ras and NF-κB

Cartoon depicting the mechanism by which RASAL2 and DAB2IP regulate invasion, EMT, and metastasis in breast cancer. DAB2IP and RASAL2 both possess catalytic RasGAP domains. Accordingly, loss of RASAL2 and DAB2IP together potently activate all three major Ras isoforms and downstream effectors (although when all Ras isoforms are expressed they appear to exert more potent effects on K- and H-Ras). Loss of RASAL2 and DAB2IP also potently activates NF-κB. NF-κB activation requires the loss of DAB2IP, which directly affects the NF-κB pathway through its period-like domain, however NF-κB activity is further enhanced by Ras pathway activation. Our studies further suggest that while Ras activation drives invasion, NF-κB is required for EMT and metastasis. We hypothesize that it is the combined and potent activation of these two important signaling pathways that underlies the aggressive and metastatic nature of luminal B breast cancers that have lost both DAB2IP and RASAL2.