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Tran et al. J Cancer Metastasis Treat 2020;6:47 I http://dx.doi.org/10.20517/2394-4722.2020.104 Page 9 of 11

AKT activation. Previous studies of various tumor cells have reported important functions of ROR2 in
migration and invasion [25-29] , which are essential metastasis initiation steps in the pre-circulatory processes.
The lung metastasis model used in this study was designed to focus on the post-circulatory processes of
metastasis, allowing us to discover another important function of ROR2 in the lung metastatic process.

Previous study using the same lung metastasis model revealed that CYGB function is crucial for LM8
[16]
extravasation ability, and Cygb was previously identified as a LEF1-regulated gene . Although Ror2 is one
of the Wnt signaling-related genes, the ROR2 expression level did not correlate with the LEF1 expression
level [Supplementary Figure 4A]. In contrast, Cygb mRNA levels were significantly reduced in LM8-L
and H/Ror2-KO [Supplementary Figure 4B], suggesting that ROR2 signaling may be an alternative CYGB
regulator independent of LEF-1 signaling in LM8. In fact, overexpression of ROR2 in LM8-L resulted in
significantly increased endothelial transmigration abilities while knocking out of Ror2 in LM8-H resulted
in significantly decreased endothelial transmigration abilities [Supplementary Figure 4C]. These results
are similar to those observed in the previous study using CYGB-overexpressing LM8-L and Cygb-KO
[16]
LM8-H . Together with our previous study of the LEF1-CYGB axis, this study of ROR2-CYGB
axis provided new insight into the mechanism of Wnt-signaling in OS lung metastasis. Although the
Wnt5a-ROR2 axis has been reported to induce the migration of human OS cells [25,28,30,31] , we found that
recombinant Wnt5a did not promote transmigration of LM8-H [Supplementary Figure 5], suggesting that
Wnt5a-ROR2 signaling was not involved in the post-circulatory process of lung metastasis in LM8 sublines.

We demonstrated that the anoikis resistance regulated by ROR2 was dependent on AKT activation and that
AKT inhibition reduced ROR2-induced anoikis resistance. Since Ror2 expression level did not affect LM8
cell proliferation under adhesion conditions [Figure 1D], the increased viability of LM8 sublines with high
ROR2 expression is not due to increased proliferation. The AKT inhibitor used here, MK2206, is currently
in phase II clinical trials for recurrent and advanced endometrial cancer. In addition to inhibiting tumor
[32]
growth in neuroblastoma and colorectal cancer , MK2206 also impairs the proliferation of human OS
[33]
cells such as U2OS and HOS . MK2206 was found to reduce LM8-H viability only under low adhesion
conditions, and no cell death was observed with MK2206 treatment under adhesion conditions. These
results indicate that MK2206 might be an ideal drug to specifically kill OS cells that have lost anchorage
dependency, such as cells that have acquired the ability to invade and metastasize.

In conclusion, our findings suggested that the ROR2-AKT axis enhanced OS lung metastasis through
anoikis resistance and that MK2206 enhances the induction of apoptosis in OS cells under low adhesion
conditions. These discoveries may shed light on specific stages of the metastatic process and provide clues
for the development of effective OS treatment.

DECLARATIONS
Acknowledgments
We thank Sakiko Yonezawa and Ngoc Thi Hong Hoang for technical advice of Western blotting, qPCR
and RT-PCR. We also thank the Open Research Facilities for Life Science and Technology, Tokyo Institute
of Technology for technical assistance. We would like to thank Editage (www.editage.com) for English
language editing.

Authors’ contributions
Designed overall projects: Tran DTP, Kuchimaru T, Kizaka-Kondoh S
Performed experiments: Tran DTP, Kuchimaru T
Analyzed and interpreted data: Tran DTP, Kuchimaru T, Pongsuchart M, Nguyen TK, Co Soriano JC,
Kadonosono T, Kizaka-Kondoh S
Wrote the manuscript: Tran DTP, Kizaka-Kondoh S