Abstract
Signaling by small guanosine triphosphatases (GTPase), Rap1/Rac1, is one of the major pathways controlling cancer cell migration and tumor metastasis. Thymosin beta-4 (Tβ4), an actin-sequestering protein, has been shown to increase migration of cancer cells. Episodes of hypoxia and re-oxygenation (H/R) are an important phenomenon in tumor microenvironment (TME). We investigated whether Tβ4 could play as an intermediary to crosstalk between Rac1- and Rap1- GTPase activation under hypoxia/reoxygenation (H/R) conditions. Inhibition of Tβ4 expression using transcription activator-like effector nucleases (TALEN) significantly decreased lung metastasis of B16F10 cells. Rac1 and Rap1 activity, as well as cancer cell migration, increased following induction of Tβ4 expression in normoxia- or H/R-experienced cells, but were barely detectable in Tβ4-depleted cells. Rap1-regulated Rac1 activity was decreased by a dominant negative Rap1 (Rap1N17), and increased by 8-(4-chloro-phenylthio)-2'-O-methyladenosine-3',5'-cyclic monophosphate (CPT), a Rap1 activator. In contrast, a Rac1-specific inhibitor, NSC23766, and dominant negative Rac1 (Rac1N17) enhanced Tβ4 expression and aberrant Rap1 activity. While NSC23766 and Rac1N17 incompletely inhibited tumor metastasis in vivo, and H/R-experienced cancer cell migration in vitro, more efficient attenuation of cancer cell migration was accomplished by simultaneous inactivation of Rap1 and Rac1 with Rap1N17 and Rac1N17, respectively. These data suggest that a combination therapy targeting both Rap1 and Rac1 activity may be an effective method of inhibiting tumor metastasis.
Highlights
In the solid tumor microenvironment (TME), the oxygen supply to tumor cells is often diminished to ~10 mmHg (1.3% O2), compared with 40–60 mmHg (5.3– 7.9% O2) in healthy cells [1, 2], due to the rapid growth of tumor cells, which outpaces the surrounding endothelial cells necessary to support blood vessel growth [3]
B16F10 cells were transfected with a transcription activator-like effector nuclease (TALEN) targeting Tβ4, effectively suppressing Tβ4 expression using Reverse transcriptase polymerase chain reaction (RT-PCR) (Figure 1A, top) and realtime PCR (Figure 1A, bottom)
Tumor metastasis was found to be reduced in mice injected with the Tβ4-transcription activator-like effector nucleases (TALEN)-transfected cells relative to control cells (Figure 1B and 1C), suggesting that tumor metastasis may be associated with Tβ4 signaling pathways
Summary
In the solid tumor microenvironment (TME), the oxygen supply to tumor cells is often diminished to ~10 mmHg (1.3% O2), compared with 40–60 mmHg (5.3– 7.9% O2) in healthy cells [1, 2], due to the rapid growth of tumor cells, which outpaces the surrounding endothelial cells necessary to support blood vessel growth [3]. This decrease in oxygen tension leads to widespread hypoxia in solid tumors, and presents significant obstacles to cancer therapy. As tumor metastasis is responsible for over 90% of cancer deaths [6], understanding the mechanisms underlying tumor metastasis is of significant concern
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