In the present article we show that p190B RhoGAP, a gene that is essential for mammary gland development, plays a critical role in MMTV-Neu mammary tumor progression. Haploinsufficiency for p190B increases tumor-free survival, reduces tumor penetrance, and decreases tumor multiplicity. The level of Neu transgene expression is unaltered in the p190B+/-
Neu normal mammary tissue and tumors, and thus downregulation of transgene expression is not responsible for the inhibition of tumor progression. ErbB3 expression was also similar between the two groups, suggesting that p190B deficiency does not inhibit Neu-induced tumorigenesis by altering the epidermal growth factor receptor signaling axis that promotes tumor formation in this model [23
]. Interestingly, p190B heterozygosity does not inhibit the growth rate of established tumors. Furthermore, we found in a few of the p190B+/-
Neu tumors that p190B expression levels were comparable with the levels detected in the p190B+/+
Neu tumors (data not shown).
It has been reported that GAPs and related G proteins are upregulated in MMTV-Neu tumors [19
], and our results confirm that this signaling network plays an important role in MMTV-Neu tumor progression. Based on previous studies using p190B-deficient mouse embryonic fibroblasts [12
], we anticipated that reduced p190B expression in vivo
would increase Rho GTPase activities as well as signaling through the downstream effectors ROK and PAK. GTPase assays to examine the levels of active RhoA, Rac1, and Cdc42, however, demonstrated that only Rac1 levels were altered in the heterozygous tumors, and they were in fact decreased. In addition, ROK activity - and in some cases ROK expression levels - were reduced in the p190B-deficient tumors, whereas PAK1/2 activity levels were increased. These data indicate that p190B is required for proper regulation of Rho/ROK signaling during Neu mammary tumor progression.
Rac1 and PAK activities have been shown to play important roles in mammary tumorigenesis [24
], and thus PAK1/2 activities may be elevated in the p190B-deficient mice to compensate for the decrease in Rac1 activity. It is interesting, and perhaps not surprising, that p190B deficiency does not result in persistent upregulation of Rho GTPase activity in the tumors. Rho GTPases play critical roles in essential cellular processes such as cell cycle progression, mitosis, and cell survival, and GTPase activity is tightly controlled during these processes [26
]. Downstream effectors such as PAK and ROK are also critical for these processes [27
]. A loss of p190B resulting in persistently elevated activity of several Rho GTPases is therefore likely to have a negative impact on cell survival or proliferation. Other GAPs or guanine nucleotide dissociation inhibitors may be upregulated or the guanine nucleotide exchange factor activity may be downregulated to compensate for the loss of p190B. In some tumors, we noted that p190B levels were similar to levels in wild-type tumors, suggesting compensatory mechanisms were functioning to restore p190B levels.
Taken together, these results demonstrate that p190B deficiency results in unexpected effects on the Rho signaling axis and reveal an important role for p190B in regulating these signaling pathways during mammary tumor progression. In addition, these results highlight the importance of investigating the role of these signaling pathways in tumorigenesis in the context of the complex in vivo environment.
Examination of the mammary glands from the p190B+/-
Neu tumor-burden mice indicated that there were a higher number of preneoplastic lesions. This increase probably resulted from a lack of progression, since averaging the number of preneoplastic lesions and the number of tumors showed no statistical difference in the total number of initiated lesions between the p190B+/+
Neu mice and the p190B+/-
Neu mice. These preneoplastic lesions were characteristic of the avascular phase of the angiogenic switch, as they were approximately 1 to 2 mm in diameter [22
]. Interestingly, several p190B+/-
Neu mice did not develop tumors by 18 months of age and, when their glands were examined for preneoplastic lesions, no lesions were detected (7/7 mice) (data not shown). These data suggest that p190B is important for the progression of preneoplastic lesions, but that it may also play a role in tumor initiation. Further studies in which normal mammary tissue pieces from p190B+/-
Neu mice are transplanted into wild-type stroma and are allowed to proceed through the stochastic process of tumor formation will be required to examine preneoplastic lesions in more detail.
The possibility that p190B was important for the progression of avascular preneoplastic lesions to vascularized lesions prompted us to examine angiogenesis in the p190B+/-Neu mice. To characterize the potential for a vascular defect in these mice we utilized the Superarray qRT-PCR angiogenesis platform, which allowed us to examine the expression of 87 angiogenesis genes, both positive and negative regulators, including vascular endothelial growth factor, matrix metalloproteinase-2 and matrix metalloproteinase-9, hypoxia inducible factor 1 alpha, fibroblast growth factor genes, TBS-1, and transforming growth factor beta genes. Only TBS-1 was significantly increased within the adult virgin mammary glands of the p190B+/-Neu mice, and quantification of immunostaining for TBS-1 confirmed this increase.
Previous studies have demonstrated that TBS-1 is a component of the extracellular matrix that functions as a negative regulator of tumor vasculature, and TBS-1 expression dramatically effects mammary tumorigenesis. For example, overexpression of TBS-1 inhibited angiogenesis in transgenic mice overexpressing activated Neu, resulting in delayed tumor onset and reduced tumor penetrance [30
]. Conversely, TBS-1 deficiency in the transgenic mice overexpressing activated Neu increased angiogenesis, enhanced growth, and reduced tumor latency [30
TBS-1 has previously been linked to GTPase signaling. Ras/Rho signaling has been shown to repress TBS-1 through effects on phosphatidyl inositol-3 kinase [31
], and Ras/Rho-mediated suppression of TBS-1 has been suggested as necessary to promote angiogenesis [32
]. Disruption of Rho/ROK signaling may therefore account for the elevated TBS-1 expression that was detected in the p190B+/-
Neu mice. Increased expression of this potent angiogenic inhibitor may be responsible, in part, for the dramatic inhibition of tumorigenesis that occurred in the p190B+/-
Consistent with the increased TBS-1 expression in p190B+/-
Neu mammary glands, p190B haploinsufficiency also resulted in decreased angiogenesis in the tumors. This persistent vascular defect may be responsible for the decreased number of metastatic nodules detected in the tumor-bearing p190B heterozygous mice. Interestingly, an increased vascular density has been correlated with poor breast cancer prognosis, and TBS-1 expression was inversely correlated with malignant progression of mammary and lung carcinomas as well as melanoma [33
Our transplantation experiments showed that the reduction in angiogenesis was probably due to defects in the stroma or vasculature because transplantation of p190B+/-
Neu tumor pieces into wild-type stroma restored angiogenesis to the levels detected in wild-type tumors. Conversely, wild-type tumors pieces failed to grow in the p190B-deficient stroma, suggesting that loss of p190B. These data are consistent with recent studies in which siRNA knockdown of p190B inhibited capillary tube formation in Matrigel by human umbilical vein endothelial cells [35
]. Interestingly, these transplantation experiments revealed a decreased time to tumor burden in the p190B+/-
Neu transplant tumors compared with the p190B+/+
Neu tumors. The mechanism responsible for this difference remains to be elucidated. One potential explanation, however, may be that the cells which initiated the p190B+/-
Neu tumors had already acquired significant genetic changes in order to develop the initial tumor in the absence of one allele of p190B, which in the presence of wild-type stroma impart a growth advantage. For example, PAK1/2 activity is elevated in the heterozygous tumors, and PAK1 hyperactivation promotes mammary tumor formation in part by activating p38MAPK signaling and proliferation [25
]. Elevated PAK activity may therefore allow the tumor cells to respond more robustly to proliferative cues present in the wild-type stroma. Further studies, including microarray analysis of the original and transplanted tumors, as well as transplantation experiments in which p190B+/-
Neu mammary epithelium is introduced into wild-type stroma and is allowed to go through the stochastic process of tumor formation, will be required to fully understand the contribution of p190B in the epithelium to tumor initiation, growth, and progression.