SIX1 induces lymphangiogenesis and metastasis via upregulation of VEGF-C in mouse models of breast cancer
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
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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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 2

SIX1 transcriptionally activates VEGFC.

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SIX1 transcriptionally activates VEGFC. (A) Microarray analysis demo...
(A) Microarray analysis demonstrates that SIX1 overexpression leads to increased VEGFC mRNA in both MCF7-SIX1 cells and MCF7-SIX1 tumors. (B) Quantitation of VEGFC gene expression in MCF7-Ctrl and MCF7-SIX1 using real-time PCR. (C) SIX1 induces VEGFC promoter activity. VEGFC promoter-luciferase reporter constructs were transiently transfected into MCF7 cells, along with increasing amounts of SIX1 and a constant amount of the SIX1 cofactor, EYA2. Luciferase activity was analyzed after 48 hours. (D) ChIP was performed to detect SIX1 presence on the VEGFC promoter in MCF7 cells transfected with SIX1 and EYA2. Protein-DNA complexes were precipitated with a SIX1-specific antibody as well as a control rabbit IgG antibody, after which real-time PCR was performed with primers that flank 5 predicted SIX1 binding sites within the VEGFC promoter (red circles denote the TGATAC binding sites; green triangles denote the ATCCTGA binding sites) as well as 1 upstream region with no predicted SIX1 binding site as a negative control. Dashed line indicates the background non-specific binding of SIX1 and x axis units are base pairs upstream of transcription start site. (E) Functional VEGF-C secreted by MCF7-Ctrl and MCF7-SIX1 cells was measured by ELISA and Western blot analysis (3 clonal isolates of MCF7-Ctrl cells [lanes 1–3] and 3 clonal isolates of MCF7-SIX1 cells [lanes 4–6]) in conditioned medium after serum starvation for 48 hours. (F) MCF7-SIX1 tumors express higher levels of VEGF-C in vivo compared with the MCF7-Ctrl tumors. Immunostaining was used to detect VEGF-C and VEGF-D. Original magnification, ×400. *P < 0.05; **P < 0.01.