SIX1 induces lymphangiogenesis and metastasis via upregulation of VEGF-C in mouse models of breast cancer
Chu-An Wang, … , J. Chuck Harrell, Heide L. Ford
Chu-An Wang, … , J. Chuck Harrell, Heide L. Ford
Published April 2, 2012
Citation Information: J Clin Invest. 2012;122(5):1895-1906. https://doi.org/10.1172/JCI59858.
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Research Article Oncology

SIX1 induces lymphangiogenesis and metastasis via upregulation of VEGF-C in mouse models of breast cancer

Abstract

An association between lymph node metastasis and poor prognosis in breast cancer was observed decades ago. However, the mechanisms by which tumor cells infiltrate the lymphatic system are not completely understood. Recently, it has been proposed that the lymphatic system has an active role in metastatic dissemination and that tumor-secreted growth factors stimulate lymphangiogenesis. We therefore investigated whether SIX1, a homeodomain-containing transcription factor previously associated in breast cancer with lymph node positivity, was involved in lymphangiogenesis and lymphatic metastasis. In a model in which human breast cancer cells were injected into immune-compromised mice, we found that SIX1 expression promoted peritumoral and intratumoral lymphangiogenesis, lymphatic invasion, and distant metastasis of breast cancer cells. SIX1 induced transcription of the prolymphangiogenic factor VEGF-C, and this was required for lymphangiogenesis and lymphatic metastasis. Using a mouse mammary carcinoma model, we found that VEGF-C was not sufficient to mediate all the metastatic effects of SIX1, indicating that SIX1 acts through additional, VEGF-C–independent pathways. Finally, we verified the clinical significance of this prometastatic SIX1/VEGF-C axis by demonstrating coexpression of SIX1 and VEGF-C in human breast cancer. These data define a critical role for SIX1 in lymphatic dissemination of breast cancer cells, providing a direct mechanistic explanation for how VEGF-C expression is upregulated in breast cancer, resulting in lymphangiogenesis and metastasis.

Authors

Chu-An Wang, Paul Jedlicka, Aaron N. Patrick, Douglas S. Micalizzi, Kimberly C. Lemmer, Erin Deitsch, Matias Casás-Selves, J. Chuck Harrell, Heide L. Ford

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Figure 4

Knockdown of Six1 in 66cl4 mammary carcinoma cells leads to a reduction in Vegf-c and lymphangiogenesis.

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Knockdown of Six1 in 66cl4 mammary carcinoma cells leads to a reduction ...
(A) Western blot analysis demonstrates increased Six1 expression in the metastatic 66cl4 cell line as compared with that in the syngeneic, nonmetastatic 67NR cell line. (B) Real-time PCR demonstrates that Vegfc mRNA is increased in 66cl4 cells as compared with that in 67NR cells. (C) 66cl4 cells contain high levels of Vegf-c in their conditioned media, as assessed by ELISA. (D) Expression of Six1 in 66cl4 cells expressing either a control (scram) or Six1 knockdown (KD1/KD2) construct. Two shRNAs were used to knockdown Six1 and are individually shown in the panel while shown as Six1 KD1 and Six1 KD2 in the following panels. (E) Vegfc mRNA is decreased in 66cl4-Six1 KD cells relative to that in 66cl4-scramble cells, as measured by real-time PCR. (F) Vegf-c protein is decreased in the conditioned media from 66cl4-Six1 KD cells relative to that in 66cl4-scramble cells, as measured by ELISA. (G) 66cl4-Six1 KD tumors express less Vegf-c, as detected by immunostaining, than 66cl4-scramble (66cl4 scramble) control tumors. Representative images are shown. Original magnification, ×200. (H) Real-time PCR demonstrates that 66cl4-Six1 KD tumors contain less Vegfc mRNA than the 66cl4-scramble control tumors. (I) 66cl4-scramble and Six1 KD tumors were stained with antibodies against Lyve-1 (red) and MECA32 (green), and representative images are shown. Original magnification, ×400. SlideBook software was used to quantify Lyve-1–positive, MECA32-negative regions in 66cl4-scramble and 66cl4-Six1 KD tumors. *P < 0.05; **P < 0.01; ***P < 0.001.