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1.
Figure 1

Figure 1. Six1 expression leads to differential regulation of genes comprising the TβRS.. From: The Six1 homeoprotein induces human mammary carcinoma cells to undergo epithelial-mesenchymal transition and metastasis in mice through increasing TGF-? signaling.

The TβRS gene list was filtered for probesets with “present” calls in more than 50% of the microarrays, then clustered using hierarchical clustering. The color scale represents the expression level of a gene above (red), below (green), or at (black) the mean expression level of that gene across all samples. Ctrl, control.

Douglas S. Micalizzi, et al. J Clin Invest. 2009 September 1;119(9):2678-2690.
2.
Figure 4

Figure 4. Six1 activates TGF-β signaling and induces EMT in vivo.. From: The Six1 homeoprotein induces human mammary carcinoma cells to undergo epithelial-mesenchymal transition and metastasis in mice through increasing TGF-? signaling.

Paraffin-embedded tissue sections from MCF7 tumors in nude mice were immunostained for Six1, E-cadherin, and Smad3. Six1 induced redistribution of E-cadherin from a membranous pattern in the control tumors to a more cytoplasmic pattern, and also increased Smad3 nuclear localization. Original magnification, ×400. Scale bar: 100 μm.

Douglas S. Micalizzi, et al. J Clin Invest. 2009 September 1;119(9):2678-2690.
3.
Figure 8

Figure 8. Six1 antibody staining correlates with activated TGF-β signaling in human breast cancer.. From: The Six1 homeoprotein induces human mammary carcinoma cells to undergo epithelial-mesenchymal transition and metastasis in mice through increasing TGF-? signaling.

Immunohistochemical staining of human breast cancer tissue arrays. Representative images show a tumor with little Six1 expression, as measured with the Atlas Six1 antibody, or nuclear Smad3, as measured using the Zymed Smad3 antibody (top row) and a tumor with both Six1 expression and nuclear Smad3 (bottom row). Original magnification, ×400. Scale bars: 100 μm.

Douglas S. Micalizzi, et al. J Clin Invest. 2009 September 1;119(9):2678-2690.
4.
Figure 5

Figure 5. Increased TGF-β signaling is necessary for elements of Six1-induced EMT.. From: The Six1 homeoprotein induces human mammary carcinoma cells to undergo epithelial-mesenchymal transition and metastasis in mice through increasing TGF-? signaling.

(A) TβRIIDN inhibited TGF-β signaling, as assessed by the TGF-β–responsive promoter 3TP after normalizing to renilla luciferase. (B and C) Inhibition of TGF-β signaling with TβRIIDN reversed E-cadherin and β-catenin relocalization as assessed by fractionation. Graphs represent quantification of Western blots for the ratio of E-cadherin or β-catenin in the soluble versus the insoluble fractions. Data are presented as mean value ± SEM in at least 3 independent experiments. (D) Inhibition of TGF-β signaling with TβRIIDN reverses β-catenin–dependent transcription, as assessed by a β-catenin–responsive reporter (TOP-flash) after normalizing to renilla luciferase. Data are presented as mean value ± SD. P values represent statistical analysis using a paired t test.

Douglas S. Micalizzi, et al. J Clin Invest. 2009 September 1;119(9):2678-2690.
5.
Figure 9

Figure 9. Six1 overexpression correlates with a shortened time to metastasis and relapse and decreased survival.. From: The Six1 homeoprotein induces human mammary carcinoma cells to undergo epithelial-mesenchymal transition and metastasis in mice through increasing TGF-? signaling.

(AC) In a study of 295 women with early-stage invasive breast carcinoma (34), high Six1 expression was associated with (A) shortened time to metastasis, (B) shortened time to relapse, and (C) shortened breast cancer–specific survival (survival). (D) In a study of 240 patients diagnosed with invasive breast cancer unselected for disease stage (35, 36), high Six1 expression was strongly associated with shortened time to relapse. In both of these datasets, the median value for Six1 expression was used to divide the samples into high (above median) and low (below median) Six1 expressors. P values were calculated by log-rank analysis.

Douglas S. Micalizzi, et al. J Clin Invest. 2009 September 1;119(9):2678-2690.
6.
Figure 2

Figure 2. Six1 induces increased TGF-β signaling.. From: The Six1 homeoprotein induces human mammary carcinoma cells to undergo epithelial-mesenchymal transition and metastasis in mice through increasing TGF-? signaling.

(A and B) MCF7-Six1 cells displayed increased levels (A) and nuclear localized (B) p-Smad3. Western blot was performed on whole cell lysate or nuclear and cytoplasmic fractions using antibodies against p-Smad3, β-actin, β-tubulin (cytoplasmic marker), and histone H1 (nuclear marker). (C and D) MCF7-Six1 cells displayed increased TGF-β–responsive 3TP-luciferase reporter activity at baseline (C) and after treatment with the indicated concentrations of TGF-β for 24 hours under serum-free conditions (D). Values are normalized to renilla luciferase. Data are presented as the mean ± SD of 3 individual clones. P values represent statistical analysis using a paired t test.

Douglas S. Micalizzi, et al. J Clin Invest. 2009 September 1;119(9):2678-2690.
7.
Figure 6

Figure 6. Six1 overexpression promotes metastasis in an orthotopic xenograft model.. From: The Six1 homeoprotein induces human mammary carcinoma cells to undergo epithelial-mesenchymal transition and metastasis in mice through increasing TGF-? signaling.

(AH) NOD/Scid mice injected orthotopically with MCF7-Six1 cells exhibited distant metastases to lymph nodes, as detected by whole body imaging of ZsGreen fluorescence (AE) and confirmed in a subset of mice by histology (H&E stain; FH). (A) Representative ZsGreen fluorescent image of primary tumor. (B and C) Fluorescent image of tumor cells within lumbar (B, arrow) and axillary (C, arrow) lymph nodes. (D and E) Fluorescent image of lumbar (D) and axillary (E) lymph nodes upon dissection (original magnification, ×5). (F) H&E staining of primary tumor showing a poorly differentiated carcinoma (original magnification, ×400). (G) Tumor deposits within distant lymph node (asterisk indicates tumor cells in subcapsular sinus; original magnification, ×100). (H) Large axillary metastasis (asterisk), consistent with lymph node replaced by tumor (original magnification, ×100). Scale bars: 2 mm (AC), 1 mm (D and E), 100 μm (F), 200 μm (G and H).

Douglas S. Micalizzi, et al. J Clin Invest. 2009 September 1;119(9):2678-2690.
8.
Figure 7

Figure 7. Six1 increases metastatic burden in an experimental metastasis model dependent on TGF-β signaling.. From: The Six1 homeoprotein induces human mammary carcinoma cells to undergo epithelial-mesenchymal transition and metastasis in mice through increasing TGF-? signaling.

(A) Bioluminescent imaging of nude mice 48 days after intracardiac injection of either MCF7-Ctrl or MCF7-Six1 cells into the left ventricle. The luminescence signal is represented by the overlaid false-color image, with intensity of the signal indicated by the scale. The anesthetized mice were imaged 10 minutes after intraperitoneal injection of D-luciferin using the IVIS200 (Caliper LS). (B) Quantification of the total body luminescent signal (in photons/second) for each group at the indicated days after injection, presented as the mean ± SEM. *P < 0.05; **P < 0.01. (C) Kaplan-Meier curve representing the overall survival of the injected mice. Statistical analysis was performed using the log-rank test. (D) Quantification of luminescent signal (in photons/second) on day 48 after injection, with the cell lines indicated. Data points represent the luminescent signal of individual animals. Horizontal bars represent the median values. Statistical analysis of luminescence data from days 4–62 was performed using linear mixed models with group-by-time interaction and an unstructured variance-covariance matrix. Interaction contrasts across the 4 animal groups were obtained for each of the 5 days after injection. (E) Kaplan-Meier curve representing the overall survival of the injected mice. Statistical analysis was performed using the log-rank test.

Douglas S. Micalizzi, et al. J Clin Invest. 2009 September 1;119(9):2678-2690.
9.
Figure 3

Figure 3. Six1 induces features of EMT in MCF7 mammary carcinoma cells.. From: The Six1 homeoprotein induces human mammary carcinoma cells to undergo epithelial-mesenchymal transition and metastasis in mice through increasing TGF-? signaling.

(A) MCF7-Six1 cells displayed differential regulation of EMT-associated genes. Gene expression from MCF7-Six1 and MCF7-Ctrl clones was analyzed by microarray analysis performed in duplicate for each clone. A gene list generated by a priori search of EMT-associated genes was filtered for probesets with a “present” call in at least 50% of the microarrays, then clustered using hierarchical clustering. The color scale represents the expression level of a gene above (red), below (green), or at (black) the mean expression level of that gene across all samples. (B) MCF7-Six1 clones displayed increased fibronectin, decreased cytokeratin 18, and no change in total E-cadherin, as determined by Western blot of whole cell lysates. (C) MCF7-Six1 cells showed a shift of E-cadherin and β-catenin from the insoluble (cytoskeleton-associated) fraction to the soluble (cytosolic) fraction. Fractions were analyzed by Western blot using antibodies against E-cadherin, β-catenin, and β-actin. (D and E) Quantification of Western blot of subcellular localization. (F) MCF7-Six1 cells had increased activity of the β-catenin–responsive luciferase reporter TOP-flash. Values were normalized to renilla luciferase activity. (G) Six1 expression decreased cell adhesion to matrix proteins in MCF7 cells. The relative number of adhering cells was quantified by crystal violet staining. For DG, data are presented as mean ± SD of 3 individual clones. P values represent statistical analysis using a paired t test.

Douglas S. Micalizzi, et al. J Clin Invest. 2009 September 1;119(9):2678-2690.

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