In the present study we show that production of angiogenic factors and EC activation is primarily controlled by breast epithelial cell polarity and tissue architecture. In conventional (2D) tissue culture, non-malignant and malignant breast epithelial cells do not differentially secrete VEGF or recruit ECs. However, once provided with 3D lrECM, non-malignant cells organize into quiescent, polarized acini, produce low levels of VEGF and limit migration of co-cultured ECs. Disorganized, malignant epithelial cells, on the other hand, produce more VEGF and significantly increase recruitment of ECs, mimicking the stromal angiogenic response of malignant breast tumors in vivo
. Significantly, phenotypically reverting malignant epithelial cells to polarized acini via reintroduction of the HoxD10 tumor suppressor or by treatment with various signaling inhibitors(20
) reduces VEGF and reduces migration of co-cultured ECs to levels observed with non-malignant cells. Hence, differential expression of VEGF by non-malignant and malignant breast epithelial cells is evident only when cells undergo characteristic morphological changes in 3D lrECM. Morever, reduced VEGF transcription in tumor cells re-polarized by HoxD10 is unlikely a direct effect as HoxD10 did not influence VEGF production when cells were unable to re-organize into polarized structures in 2D cultures, although it remains possible that accompanying morphological changes in 3D unmask binding sites within the VEGF promoter. Still, these findings indicate that activation of the angiogenic switch is not simply due to genetic changes within the tumor cells, but rather linked to how cells sense their architecture and interact with their microenvironment.
Importantly, we also show that in tumor cells, which characteristically exhibit increased growth and metabolic demand compared to non-malignant cells, restoring normal tissue architecture is essential for attenuating their angiogenic potential. Indeed, a ten-fold increase in proliferation of non-malignant cells was not sufficient to explain resistance to chemotherapeutic agents(17
) and is also not sufficient to activate VEGF expression when growing cells maintain a polarized architecture.
Many studies have established that increased proliferation, metabolic demand and hypoxia in tumor cells stabilizes HIF1α and drives VEGF expression (3
). Blocking HIF1α expression/activity reduces VEGF expression, and HIf1α null mice display defective angiogenesis and fail to support tumor growth (8
). However, our results show that neither differences in HIF1α expression or activity underly reduced VEGF in reverted, polarized tumor cells and implicate other pathways and/or transcriptional mediators in regulating the angiogenic switch in this system.
In a mouse model of pancreatic cancer, hypoxia-independent activation of the angiogenic switch occurs via increased MMP-9 activity, which liberates bioactive VEGF trapped within the tumor matrix (37
). Despite the fact that MMP-9 is highly expressed in disorganized T4-2 cells and reduced in reverted T4-2 (A. Beliveau, and MJ Bissell submitted), elevated secretion of VEGF is reflected by increased VEGF mRNA. Whether reduced VEGF transcription by re-polarized breast tumor cells increases inhibitory factors (38
) and/or attenuates other positive inducers of VEGF requires further investigation.
Previous studies also demonstrated that PI3K signaling induces VEGF (13
), and our current study suggests that the Rac1 branch of this pathway directly drives VEGF expression. We reported PI3K inhibition phenotypically reverts tumorigenic breast epithelial cells and is accompanied by down regulation of both the Akt and Rac1 effector pathways (22
). However, while attenuation of Akt reduces proliferation, it did not restore a polarized phenotype. Instead, suppression of Rac1 activity was necessary for re-establishing an organized polarized phenotype, and selective re-activation of Rac1 disrupts polarity without re-initiating growth when Akt remained blocked (22
). Moreover, whereas activation of Rac1 in non-malignant breast epithelial cells normally induces apoptosis, in breast tumor epithelial cells lacking scribble
, Rac activation promotes tumorigenesis and loss of cell polarity (40
). In the present study, we demonstrate that re-activation of Rac1 and disruption of polarity restores VEGF expression and induces EC migration despite the fact that growth remains suppressed via low pAkt levels (latter data not shown).
Active Rac1 directly binds and phoshphorylates STAT3 (41
), which in turn forms Sp1/STAT3 complexes that bind the VEGF promoter (42
) and the SP1 site within the -86- -66 region of the VEGF promoter is linked to HIF1α-independent transcription of VEGF (10
). Phosphorylated STAT3 can also bind to HIF1α and prevent its degradation (44
). Although we observed an increase in phospho-STAT3 in Rac1-overexpressing cells, reversion of malignant cells by HoxD10 did not reduce phospho-STAT3 (data not shown) and HIF1α protein levels were similar in both tumorigenic and HoxD10-reverted tumor cells. Thus binding of STAT3 to either SP1 or HIF and VEGF expression may be context dependent as previously suggested by investigations of pathways impinging upon VEGF expression (39
), and our current study also emphasizes the critical role tissue polarity in mediating VEGF expression in breast tumor cells.
It is noteworthy that inhibiting EGF receptors represses VEGF in both a HIF-dependent and HIF-independent manner, with the latter attributed to reduced phosphorylation and binding of Sp1 to the VEGF promoter (43
). A number of EGFR inhibitors, including those used in this study, not only reduce VEGF expression and inhibit breast tumor angiogenesis in vivo
, but also phenotypically revert tumor cells to a polarized morphology in culture (19
). Finally, mice lacking LKB1, a tumor suppressor which directs cell polarity and when mutated, produces epithelial cancers, show markedly increased VEGF (47
The present study addresses breast tumor epithelial organization and VEGF production, but it is important to note that other components of the tumor microenvironment, including fibroblasts and macrophages, are also rich sources of angiogenic factors. In DCIS where newly formed capillaries appear immediately adjacent to the basal surface of the breast epithelium (5
), the BM is largely intact but a majority of epithelial cells have lost their characteristic polarized morphology and display attenuated expression of the HoxD10 tumor suppressor (3
). As macrophage recruitment typically occurs in later stages of malignant progression accompanied by BM degradation (49
), it is likely that the loss of epithelial cell polarity marks an early and critical step in activation of the angiogenic switch.