br ated inhibition of TUBB mRNA expression
ated inhibition of TUBB3 mRNA expression in HT-29 1132935-63-7 (Figure 5D).
Cathelicidin Inhibited Colon Cancer Metastasis via P2RX7
To validate this receptor pathway in vivo, we in-jected KN62 into the HT-29-loaded nude mice. Injection of KN62 did not significantly affect colon cancer metastasis to lungs and liver in the control vector-expressing group, but reversed the colon cancer metastasis in the cath-elicidin-expressing group (Figures 6A, 6B, 7A, and 7B). Therefore, cathelicidin inhibited colon cancer metastasis via P2RX7. Cathelicidin over-expression significantly reduced human TUBB3 mRNA expression in the lungs and liver of HT-29-loaded nude mice, which was reversed by KN62 administration (Figures 7C and 7D).
Cathelicidin Inhibited Colonic Tumor Growth via P2RX7
We also injected the HT-29 cells into the nude mice subcutaneously followed by intravenous CAMP-AAV injection and intraperitoneal KN62 injection. Consis-
tent with our previous report,16 the cathelicidin-overexpressing group had significantly reduced subcutaneous tumor diameter (Figure 7E). This reduction was partially reversed by KN62 (Figure 7E). Thus, cathelicidin-mediated inhibition of colonic tumor growth is P2RX7 dependent.
Cathelicidin Did Not Affect Colon Cancer Cell Invasion
Cathelicidin did not affect SW620 cell invasion (Figure S1A). It altered the protein secretion of MMP1, TIMP1-2, TGF-b1, and VEGF in HT-29, SW620, and SW480 cells differently (Figure S1B). We found no consistent pattern of LL-37-dependent soluble cancer mediator secretion among the three cultured colon cancer cell lines. Therefore, metalloprotease activity cannot address the anti-metastatic effect of cathelicidin.24 Also, cathelicidin overexpression did not affect the endothelial cell marker von Willebrand factor (vWF) mRNA expression in lungs and liver of the HT-29-loaded nude mice (Fig-ure S1C). LL-37 is unlikely to modulate tumoral angiogenesis in vivo.
Molecular Therapy: Oncolytics
This study demonstrates that cathelicidin inhibits colon cancer metastasis via a P2RX7-dependent pathway. Cathelicidin does not inhibit the cell viability of colon cancer cells (Figure 3A) and normal colonic fibroblasts.16 Instead, cathelicidin inhibits colonic tumor growth via indirect inhibition of TAFs.16 Both metastatic colon cancer cells and TAFs possess mesenchymal cell properties.3 The cathelicidin-dependent inhibition of colon cancer devel-opment and metastasis shares a common characteristic: the disruption of tubulin cytoskeleton in cells with mesenchymal cell properties.
Cytoskeleton, including tubulin, mediates the metastatic poten-tial of circulating tumor cells (CTCs) in the vasculature.25,26
The adherent tumor cells extravasate to form new metastatic foci in distant organs. Although the biophysics of CTCs is complex and beyond our scope of investigation, cathelicidin-
Figure 3. Cathelicidin Inhibited Cell Migration and TUBB3 Expression
(A) Cell viability of SW620 cells. (B) Cell migration of SW620 cells. (C) Green tubulin tracker staining with blue nuclear staining in human cancer SW620 cells. LL-37 reduced tubulin expression in SW620 cells. (D) TUBB1 mRNA expression in SW620 and HT-29 cells. (E) TUBB3 mRNA expression in SW620 and HT-29 cells. Results were pooled from three independent experi-ments.
mediated disruption of the cytoskeleton is associated with the specific inhibition of TUBB3 mRNA expression (Figure 3D). bIII-tubulin (TUBB3) is associated with exacerbated liver metastasis in colon cancer patients and with increased colon cancer cell migration.18,21 Overexpression of TUBB3 reversed the anti-migratory effect of cath-
elicidin because cytoskeleton mediates cell migration.18,27
Consistent with our findings, deficiency of P2RX7 augmented colon cancer tumor devel-opment and metastasis in nude mice injected with mouse colon cancer CT26 cells.28 This report suggests that P2RX7 mediates anti-cancer effects. The role of cathelicidin recep-tor in cancer development may depend on cell type and disease condition. In colon can-cer HCT116 cells, cathelicidin mediates its anti-tumoral effects via G-protein coupled receptor (GPCR)-dependent and FPRL1-inde-pendent pathways.7 Given the heterogeneity and complexity of cancer, our study cannot cover all aspects of colon cancer conditions.
This study attempts to reveal the potential cathelicidin-P2RX7 pathway in colon cancer development, which is worth further investigation.
To understand how P2RX7 modulates TUBB3 expression, we pro-filed the microRNA (miRNA) expression in SW620 cells (Fig-ure S2A). LL-37 significantly increased miR200c-3p expression in SW620 and HT-29 cells (Figures S2B and S2C). miR200c is associated with distant metastasis in colorectal cancer patients29 and is currently the only validated miRNA shown to target TUBB3.30 Inhibition of P2RX7 with shRNA did not affect basal miR200c-3p expression but abolished the LL-37-mediated increase of miR200c-3p expression (Figure S2B). Similarly, KN62 also in-hibited LL-37-induced miR200c-3p expression in HT-29 cells (Figure S2C). Pretreatment of SW620 cells with miR200c-3p inhib-itor reversed the LL-37-mediated inhibition of TUBB3 mRNA expression, cell migration, and disruption of tubulin structure