HOXC9 Induces Phenotypic Switching between Proliferation and Invasion in Breast Cancer Cells

Abstract

HOX genes encode a family of transcriptional regulators that are involved in pattern formation and organogenesis during embryo development. In addition, these genes play important roles in adult tissues and some of the dysregulated HOX genes are associated with cancer development and metastasis. Like many other HOX genes, HOXC9 is aberrantly expressed in certain breast cancer cell lines and tissues; however, its specific functions in breast cancer progression were not investigated. In the present study, we demonstrated that HOXC9 overexpression in breast cancer cell lines such as MDA-MB-231 and MCF7 increased the invasiveness but reduced the proliferation of cells, resembling a phenotype switch from a proliferative to an invasive state. Furthermore, the reciprocal result was detected in MCF7 and BT474 cells when the expression level of HOXC9 was reduced with siRNA. The clinical impact of HOXC9 in breast cancer was interpreted from the survival analysis data, in which high HOXC9 expression led to considerably poorer disease-free survival and distant metastasis-free survival, especially in lymph node-positive patients. Together, the prognostic relevance of HOXC9 and the HOXC9-derived phenotypic switch between proliferative and invasive states in the breast cancer cell lines suggest that HOXC9 could be a prognostic marker in breast cancer patients with lymph node metastasis and a target for therapeutic intervention in malignant breast cancer.

Keywords: HOXC9; breast cancer; invasion.; metastasis; proliferation.

Figure 1

Effect of HOXC9 overexpression on cancer cell proliferation and invasion. (A) Western blotting to detect HOXC9 protein expression in MDA-MB-231 cells transfected with empty vector (231:emp) and HOXC9 expression vector (231:C9). Anti-DDK antibody was used. Arrow indicates the expected size of HOXC9-DDK protein. (B) RT-PCR result showing overexpression of HOXC9 in three independent MDA-MB-231 clones (231:C9 #1, 231:C9 #2, and 231:C9 #3). Two clones harboring the empty vector (231:emp #1 and 231:emp #2) were used as controls. (C) The MTT assay was performed to measure cell viability. The proliferation activity was compared between three clonal cells expressing HOXC9 (231:C9 #1, 231:C9 #2, and 231:C9 #3), and three types of control cells (231:emp #1, 231:emp #2, and parent MDA-MB-231 [231] cells). (D) The Matrigel invasion assay was performed to measure invasion ability. Representative images showing the different number of invading cells between 231:emp and 231:C9 cells are presented on the right side. (E) RT-PCR analysis of HOXC9 expression in MCF cells transfected with the empty vector (MCF7:emp) or HOXC9 expression vector (MCF:C9): proliferation (F) and invasion analyses (G) were performed. *; p < 0.05, **; p < 0.01, ***; p < 0.001 vs. parental MDA-MB-231 or MCF7:emp control cells.

Figure 2

Effect of HOXC9 knockdown on MCF7 and BT474 cells. (A) RT-PCR result showing the suppression of HOXC9 expression in si_HOXC9 treated cells. Nonspecific siRNA (si_Con) was used as the control. The MTT assay (B) and Matrigel invasion assay (C) were performed to measure cell proliferation and invasion ability, respectively. *; p < 0.05, **; p < 0.01 vs. si_Con treated cells.

Figure 3

Prognostic significance of HOXC9 expression in breast cancer patients. (A) Publically available data retrieved from GENT (http://medical-genome.kribb.re.kr/GENT) show that HOXC9 is upregulated in breast cancer tissues. breast-C: breast cancer tissues, breast-N: normal breast tissues. Each circle represents an individual tissue sample. Data are shown as boxplots. The Y axis of the plot indicates normalized expression measures. The box extends from the 25th to 75th percentiles and the line in the middle of the box is plotted at the median. Significance was analyzed using an unpaired t-test (p = 2.913E-11). (B-G) Survival analysis using the MTCI breast cancer survival analysis tool (http://glados.ucd.ie/BreastMark/). Disease free survival (DFS) (n = 1533, number of events = 767, hazard ratio = 1.164 [0.9947 - 1.363], p = 0.058) (B) and distant metastasis-free survival (DMFS) (n = 577, number of events = 187, hazard ratio = 1.758 [1.3-2.379], p = 0.000208) (C) in all patients. DFS (n = 633, number of events = 278, hazard ratio = 1.382 [1.074-1.778] p = 0.0116) (D) and DMFS (n = 257, number of events = 109, hazard ratio = 2.17 [1.483-3.176], p = 4.351e-05) (E) in lymph node-positive patients. DFS (n = 538, number of events = 163, hazard ratio = 1.03 [0.7574-1.401], p = 0.85) (F) and DMFS (n = 276, number of events = 57, hazard ratio = 1.289 [0.7646 - 2.174], p = 0.339) (G) in lymph node-negative patients.