Activation of tumor cell integrin alphavbeta3 controls angiogenesis and metastatic growth in the brain

Abstract

The incidence of brain metastasis is rising and poses a severe clinical problem, as we lack effective therapies and knowledge of mechanisms that control metastatic growth in the brain. Here we demonstrate a crucial role for high-affinity tumor cell integrin alpha(v)beta(3) in brain metastatic growth and recruitment of blood vessels. Although alpha(v)beta(3) is frequently up-regulated in primary brain tumors and metastatic lesions of brain homing cancers, we show that it is the alpha(v)beta(3) activation state that is critical for brain lesion growth. Activated, but not non-activated, tumor cell alpha(v)beta(3) supports efficient brain metastatic growth through continuous up-regulation of vascular endothelial growth factor (VEGF) protein under normoxic conditions. In metastatic brain lesions carrying activated alpha(v)beta(3), VEGF expression is controlled at the post-transcriptional level and involves phosphorylation and inhibition of translational respressor 4E-binding protein (4E-BP1). In contrast, tumor cells with non-activated alpha(v)beta(3) depend on hypoxia for VEGF induction, resulting in reduced angiogenesis, tumor cell apoptosis, and inefficient intracranial growth. Importantly, the microenvironment critically influences the effects that activated tumor cell alpha(v)beta(3) exerts on tumor cell growth. Although it strongly promoted intracranial growth, the activation state of the receptor did not influence tumor growth in the mammary fat pad as a primary site. Thus, we identified a mechanism by which metastatic cells thrive in the brain microenvironment and use the high-affinity form of an adhesion receptor to grow and secure host support for proliferation. Targeting this molecular mechanism could prove valuable for the inhibition of brain metastasis.

Fig. 1.

Impact of tumor cell integrin α_vβ₃ expression and activation on lesion growth in the brain and mammary fat pad (MFP). (A) Analysis of integrin α_vβ₃ expression by flow cytometry. Parent, MDA-MB-435 transduced with control shRNA ScrB; β₃kd, MDA-MB-435 transduced with β₃ shRNA; WT, MDA-MB-435/β₃kd reconstituted with wild-type β₃ (non-activated α_vβ₃); D723R, MDA-MB-435/β₃kd reconstituted with β₃D723R (activated α_vβ₃). (B) Lesion growth in the brain (days 0–21) or MFP (days 0–34) based on bioluminescence signal of F-luc–tagged tumor cells by non-invasive in vivo imaging. (C) Lesion growth in the brain (days 0–21) or MFP (days 0–36) of MDA-MB 435/β₃- cells selected from parental cells with saporin-anti-β₃ antibody and reconstituted by transfection with β₃WT of β₃D723R based on in vivo imaging (brain) or caliper measurement (MFP). Each dot represents one animal and red lines the mean values per group; group sizes in (B): brain: parent and β₃kd, n = 6; β₃WT and β₃D723R, n = 11; MFP: parent, n = 10; β₃kd, n = 8; β₃ and β₃D723R, n = 6; (C), all groups, n = 7. P values obtained by a two-tailed Student's t test with unequal variance.

Fig. 2.

Tumor cell integrin α_vβ₃ activation enhances proliferation in the brain. (A) Non-invasive bioluminescence imaging 21 days after intracranial implantation of 1 × 10⁴ β₃WT- (Left) or β₃D723R- (Right) expressing MDA-MB-435 tumor cells. (B) Growth of β₃WT and β₃D723R-expressing tumor cells in the brain. Each dot represents one animal; horizontal lines represent mean values per group. (C and D) Intracranial proliferation of β₃WT- and β₃D723R-expressing cells in brain lesions by in vivo BrdU uptake; n = 5 per group, P values obtained by a two-tailed Student's t test with unequal variance. (E) Apoptosis in β₃WT and β₃D723R brain lesions by TUNEL staining (brown).

Fig. 3.

Tumor cell integrin α_vβ₃ activation increases angiogenesis and reduces hypoxia in brain lesions. (A) Quantification of the total vessel area in β₃WT and β₃D723R brain lesions 21 days after tumor cell implantion. Vessels were visualized by CD34 staining. (B) Immunohistochemistry of tumor vessels (brown) and hypoxic regions (gray-blue) in brain lesions on day 21. Borders between tumor area and brain parenchyma are outlined showing representative small (≤1.5 mm) and large (≥ 1.5 mm) β₃WT lesions. All β₃D723R lesions were >2 mm. (C) (Left) In vivo signal of β₃WT (black) and β₃D723R (red) brain lesions on day 21, and on day 29 for β₃WT (gray) to obtain larger lesions for this group. Only lesions with a diameter ≥1.5 mm (≈5 × 10⁷ bioluminescence photons in vivo) were included in the quantification of hypoxic tumor regions (marked with a bracket as large tumors). (Right) Percentage of hypoxic area in brain lesions >1.5 mm in diameter; n = 5 per group; P values based on two-tailed Student's t test with unequal variance. (D) Large β₃WT lesions (> 1.5 mm) show increased apoptosis (Left; TUNEL staining, brown; nuclei, green) within hypoxic regions (Right; Hypoxyprobe-1 staining, gray-blue; nuclei, green) in adjacent sections. (E) Formation of the VEGF:VEGFR2 complex on tumor vessels in β₃D723R brain lesions (Gv39M staining, brown).

Fig. 4.

Tumor cell integrin α_vβ₃ activation increases VEGF expression in the brain post-transcriptionally by 4E-BP phosphorylation. (A) VEGF protein expression in normoxic (upper panels) versus hypoxic areas (lower panels) of β₃WT and β₃D723R brain lesions by immunohistochemistry. Hypoxic regions defined by positive staining for Hypoxyprobe-1 (Left) and HIF-1α (Middle). β₃WT and β₃D723R lesions express VEGF in hypoxic regions. In normoxic regions, only β₃D723R lesions express VEGF (Right; enlarged on far right). (B) VEGF protein levels in microdissected β₃WT and β₃D723R brain lesions 21 days after tumor cell implantation (immunoprecipitation [IP], VEGF; Western blot [WB], VEGF). (C) VEGF mRNA levels in microdissected β₃WT and β₃D723R brain lesions on day 21 post-implantation (RT-PCR). Lesions from three different mice per group. (D) Phospho-4E-BP1 signal in microdissected β₃WT and β₃D723R brain lesions 21 days after tumor cell implantation (Western blot with anti-phospho-4E-BP1 and anti-tubulin antibodies). (E) Down-regulation of 4E-BP1 in β₃WT cells by shRNA results in a strong increase of VEGF protein level under normoxia in vitro. VEGF was immunoprecipitated from cell culture supernatants before Western blot analysis. (F) Detection of mTOR and Akt activation levels with phospho-specific antibodies in microdissected β₃WT and β₃D723R brain lesions 21 days after tumor cell implantation.

Fig. 5.

Tumor cell integrin α_vβ₃ activation does not prevent hypoxia in mammary fat pad tumors. Hypoxia (Hypoxyprobe-1 in gray-blue, Upper), apoptosis/necrosis (TUNEL staining in brown, Middle) and blood vessel denstity (CD34 in brown, Lower) in MFP tumors 34 days after implanting 1 × 10⁴ β₃WT- or β₃D723R-expressing tumor cells. Quantifications are included (n = 5 per group). P values are based on two-tailed Student's t test with unequal variance. Note: All 34-day old MFP tumors were smaller than 21-day-old β₃D723R brain lesions.