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 5

Six1 knockdown decreases distant metastasis, and the Six family member, Six2, likely compensates for loss of Six1.

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Six1 knockdown decreases distant metastasis, and the Six family member, ...
(A) Diagrammatic representation of the metastasis experiment performed in A–C, in which tumors were allowed to grow for the same amount of time before sacrifice. 66cl4-scramble or 66cl4-Six1 KD1/2 cells were injected into the fourth mammary fat pad of Balb/c mice, and metastases were measured weekly. (B) Representative bioluminescent imaging of Balb/c mice 40 days after injection of 66cl4-scramble or 66cl4-Six1 KD1/2 cells into the fourth mammary fat pad. Quantitation of distant luminescent signal, likely in lungs (yellow boxed region), in 66cl4-scramble and Six1 KD groups. p/s, photons per second. (C) Clonogenic assays were used to detect metastases in lungs of animals bearing 66cl4-scramble and 66cl4-Six1 KD tumors. Representative images from 66cl4-scramble and 66cl4-Six1 KD are shown. (D) Diagrammatic representation of the metastasis experiment performed in D–G, in which tumors were allowed to grow to the same size before sacrifice. 66cl4-scramble or 66cl4-Six1 KD1/2 cells were injected into the fourth mammary fat pad of Balb/c mice, and metastases were measured when tumor sizes were comparable. (E) Quantitation of bioluminescent imaging (photons per second) emanating from the region of lungs in Balb/c mice injected with 66cl4-scramble or 66cl4-Six1 KD1 and KD2 when tumors reached comparable sizes (day 46 for 66cl4-scramble and day 54 for 66cl4-Six1 KD1 and KD2). (F) Six1 expression was measured by real-time PCR in parental Six1 KD2 cells (in culture), Six1 KD2 cells retrieved from lungs of 3 individual mice, and 66cl4-scramble control cells retrieved from lung of 1 animal. The numbers over the bars represent Six2 expression fold change over the value of Scram. (G) Six2 expression was measured by real-time PCR in the above-mentioned cells. *P < 0.05; **P < 0.01.