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Paired-like Homeodomain Transcription factor 2 expression by breast cancer bone marrow disseminated tumor cells is associated with early recurrent disease development.

Pillai SG, Dasgupta N, Siddappa CM, Watson MA, Fleming T, Trinkaus K, Aft R - Breast Cancer Res. Treat. (2015)

Bottom Line: Suppression of PITX2 expression significantly reduced invasiveness in MDAMB231 cells.Three genes-NKD1, LEF1, and DKK4-were significantly downregulated in response to PITX2 suppression.Furthermore, PITX2 likely plays a role in the metastatic process through its effect on the expression of genes associated with the Wnt/beta-Catenin signaling pathway.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, MO, 63110, USA.

ABSTRACT
The presence of disseminated tumor cells (DTCs) in the bone marrow (BM) of breast cancer patients is prognostic for early relapse. In the present study, we analyzed the gene expression profiles from BM cells of breast cancer patients to identify molecular signatures associated with DTCs and their relevance to metastatic outcome. We analyzed BM from 30 patients with stage II/III breast cancer by gene expression profiling and correlated expression with metastatic disease development. A candidate gene, PITX2, was analyzed for expression and phenotype in breast cancer cell lines. PITX2 was knocked down in the MDAMB231 cell lines for gene expression analysis and cell invasiveness. Expression of various signaling pathway molecules was confirmed by RT-PCR. We found that the expression of Paired-like Homeobox Transcription factor-2 (PITX2) is absent in the BM of normal healthy volunteers and, when detected in the BM of breast cancer patients, is significantly correlated with early metastatic disease development (p = 0.0062). Suppression of PITX2 expression significantly reduced invasiveness in MDAMB231 cells. Three genes-NKD1, LEF1, and DKK4-were significantly downregulated in response to PITX2 suppression. Expression of PITX2 in BM of early-stage breast cancer patients is associated with risk for early disease recurrence. Furthermore, PITX2 likely plays a role in the metastatic process through its effect on the expression of genes associated with the Wnt/beta-Catenin signaling pathway.

No MeSH data available.


Related in: MedlinePlus

Matrigel invasion assays showing reduced invasion in MDAMB231 cells with PITX2 knockdown. aTop panel shows the cells in the matrigel invasion chamber prior to the clearing of the top chamber. Cells present both at the top chamber and invaded cells in the bottom chamber are visible. Bottom panel shows cells after clearing the top chamber; thus, only those cells invaded to bottom chamber are visible. Different derivatives of cells used in the experiment are indicated. Statistical significance by paired t test in comparison with non-transduced MDAMB231 cells are shown. (MDAMB231- non-transduced parental cells, pLKO vector—transduced with empty vector, Non-targeting—transduced with a non-targeting sequence, B2M—transduced with shRNA of an unrelated gene beta-2 microglobulin, ko- PITX2—transduced with shRNA of PITX2.) b Percentage of cells present in bottom chamber at 24 and 48 h. c Expression levels of PITX2 in the cells used for experiment as determined by qRT–PCR
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Fig3: Matrigel invasion assays showing reduced invasion in MDAMB231 cells with PITX2 knockdown. aTop panel shows the cells in the matrigel invasion chamber prior to the clearing of the top chamber. Cells present both at the top chamber and invaded cells in the bottom chamber are visible. Bottom panel shows cells after clearing the top chamber; thus, only those cells invaded to bottom chamber are visible. Different derivatives of cells used in the experiment are indicated. Statistical significance by paired t test in comparison with non-transduced MDAMB231 cells are shown. (MDAMB231- non-transduced parental cells, pLKO vector—transduced with empty vector, Non-targeting—transduced with a non-targeting sequence, B2M—transduced with shRNA of an unrelated gene beta-2 microglobulin, ko- PITX2—transduced with shRNA of PITX2.) b Percentage of cells present in bottom chamber at 24 and 48 h. c Expression levels of PITX2 in the cells used for experiment as determined by qRT–PCR

Mentions: To determine whether PITX2 plays a role in cell invasiveness, we performed gene knockdown experiments using the MDAMB231 cell line. MDAMB231 is highly invasive and has relatively high expression of all three of PITX2 isoforms. Using a lentivirus shRNA system, PITX2 expression was reduced to near undetectable level as determined by qRT–PCR. Clonal cell lines with the highest level of knockdown were selected for invasion assays. MDAMB231 cells stably transduced with empty vector, a non-targeting sequence, or shRNA targeting an unrelated gene, beta-2 microglobulin, were included as controls. The relative expression of PITX2 in each cell type used is shown in Fig. 3c. The knockdown cells (KO) had undetectable expression of PITX2, while the control cell lines expressed similar levels of PITX2. The percentage of cells which migrated into the lower chamber of the invasion cassette is shown in Fig. 3b. Cells with reduced PITX2 expression showed a significant reduction in invasion compared to all control cells, both at 24 and 48 h after plating (Fig. 3a, b). There was a 63.8 % reduction in invasion in PITX2 deficient cells at 24 h and 72.6 % reduction at 48 h compared to the parental cells (p < 0.0001). These data suggest that loss of PITX2 expression attenuates the invasive phenotype of MDAMB231 cells.Fig. 3


Paired-like Homeodomain Transcription factor 2 expression by breast cancer bone marrow disseminated tumor cells is associated with early recurrent disease development.

Pillai SG, Dasgupta N, Siddappa CM, Watson MA, Fleming T, Trinkaus K, Aft R - Breast Cancer Res. Treat. (2015)

Matrigel invasion assays showing reduced invasion in MDAMB231 cells with PITX2 knockdown. aTop panel shows the cells in the matrigel invasion chamber prior to the clearing of the top chamber. Cells present both at the top chamber and invaded cells in the bottom chamber are visible. Bottom panel shows cells after clearing the top chamber; thus, only those cells invaded to bottom chamber are visible. Different derivatives of cells used in the experiment are indicated. Statistical significance by paired t test in comparison with non-transduced MDAMB231 cells are shown. (MDAMB231- non-transduced parental cells, pLKO vector—transduced with empty vector, Non-targeting—transduced with a non-targeting sequence, B2M—transduced with shRNA of an unrelated gene beta-2 microglobulin, ko- PITX2—transduced with shRNA of PITX2.) b Percentage of cells present in bottom chamber at 24 and 48 h. c Expression levels of PITX2 in the cells used for experiment as determined by qRT–PCR
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Related In: Results  -  Collection

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Fig3: Matrigel invasion assays showing reduced invasion in MDAMB231 cells with PITX2 knockdown. aTop panel shows the cells in the matrigel invasion chamber prior to the clearing of the top chamber. Cells present both at the top chamber and invaded cells in the bottom chamber are visible. Bottom panel shows cells after clearing the top chamber; thus, only those cells invaded to bottom chamber are visible. Different derivatives of cells used in the experiment are indicated. Statistical significance by paired t test in comparison with non-transduced MDAMB231 cells are shown. (MDAMB231- non-transduced parental cells, pLKO vector—transduced with empty vector, Non-targeting—transduced with a non-targeting sequence, B2M—transduced with shRNA of an unrelated gene beta-2 microglobulin, ko- PITX2—transduced with shRNA of PITX2.) b Percentage of cells present in bottom chamber at 24 and 48 h. c Expression levels of PITX2 in the cells used for experiment as determined by qRT–PCR
Mentions: To determine whether PITX2 plays a role in cell invasiveness, we performed gene knockdown experiments using the MDAMB231 cell line. MDAMB231 is highly invasive and has relatively high expression of all three of PITX2 isoforms. Using a lentivirus shRNA system, PITX2 expression was reduced to near undetectable level as determined by qRT–PCR. Clonal cell lines with the highest level of knockdown were selected for invasion assays. MDAMB231 cells stably transduced with empty vector, a non-targeting sequence, or shRNA targeting an unrelated gene, beta-2 microglobulin, were included as controls. The relative expression of PITX2 in each cell type used is shown in Fig. 3c. The knockdown cells (KO) had undetectable expression of PITX2, while the control cell lines expressed similar levels of PITX2. The percentage of cells which migrated into the lower chamber of the invasion cassette is shown in Fig. 3b. Cells with reduced PITX2 expression showed a significant reduction in invasion compared to all control cells, both at 24 and 48 h after plating (Fig. 3a, b). There was a 63.8 % reduction in invasion in PITX2 deficient cells at 24 h and 72.6 % reduction at 48 h compared to the parental cells (p < 0.0001). These data suggest that loss of PITX2 expression attenuates the invasive phenotype of MDAMB231 cells.Fig. 3

Bottom Line: Suppression of PITX2 expression significantly reduced invasiveness in MDAMB231 cells.Three genes-NKD1, LEF1, and DKK4-were significantly downregulated in response to PITX2 suppression.Furthermore, PITX2 likely plays a role in the metastatic process through its effect on the expression of genes associated with the Wnt/beta-Catenin signaling pathway.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, MO, 63110, USA.

ABSTRACT
The presence of disseminated tumor cells (DTCs) in the bone marrow (BM) of breast cancer patients is prognostic for early relapse. In the present study, we analyzed the gene expression profiles from BM cells of breast cancer patients to identify molecular signatures associated with DTCs and their relevance to metastatic outcome. We analyzed BM from 30 patients with stage II/III breast cancer by gene expression profiling and correlated expression with metastatic disease development. A candidate gene, PITX2, was analyzed for expression and phenotype in breast cancer cell lines. PITX2 was knocked down in the MDAMB231 cell lines for gene expression analysis and cell invasiveness. Expression of various signaling pathway molecules was confirmed by RT-PCR. We found that the expression of Paired-like Homeobox Transcription factor-2 (PITX2) is absent in the BM of normal healthy volunteers and, when detected in the BM of breast cancer patients, is significantly correlated with early metastatic disease development (p = 0.0062). Suppression of PITX2 expression significantly reduced invasiveness in MDAMB231 cells. Three genes-NKD1, LEF1, and DKK4-were significantly downregulated in response to PITX2 suppression. Expression of PITX2 in BM of early-stage breast cancer patients is associated with risk for early disease recurrence. Furthermore, PITX2 likely plays a role in the metastatic process through its effect on the expression of genes associated with the Wnt/beta-Catenin signaling pathway.

No MeSH data available.


Related in: MedlinePlus