Table 1.
Characteristics of biopsy and residual triple negative breast tumor samples.
Fig 1.
Reciprocal regulation of AnxA6 and GRF2 by non-selective calcium channel blockers.
A) BT-549 (AnxA6 high) and empty vector or AnxA6 expressing HCC1806 (AnxA6-low) cells were treated with Ni2+ or Ca2+ for 72 h and AnxA6, AnxA2 and GRF2 protein assessed by immunoblotting. B) Densitometric analysis of AnxA6 and GRF2 expression. Bars represent fold change relative to control cells from two independent experiments. C and D) Effects of Ni2+ concentration and time on the reciprocal expression of AnxA6 and GRF2. AnxA6 expressing HCC1806 cells were treated with the indicated concentrations of Ni2+ for 72 h (C) or at 0.5 mM Ni2+ for the indicated times (D); Densitometric analysis of the expression of AnxA6, GRF2 and AnxA2 from a representative experiment (C and D, left panels). E) HCC1806 cells were treated with the indicated concentrations of bepridil or amlodipine for 72 h and the expression of AnxA6 and GRF2 assessed by western blotting. F and G) Densitometric analysis of the protein bands in bepridil treated (F) and amlodipine treated cells from a representative experiment (G). Flag-AnxA6: flag-tagged recombinant AnxA6, End-AnxA6: endogenously expressed AnxA6.
Fig 2.
Expression of AnxA6 and proliferation markers in normal breast and breast disease tissues.
A) Thin sections of formalin-fixed paraffin-embedded tissues in a broad-spectrum breast disease TMA were stained with antibodies against the indicated proteins. Shown are representative stained tissues. B) The stained tissues were digitally scanned and digitally analyzed using the Tissue IA software. Shown are boxplots depicting the mean staining intensity and distribution of AnxA6, Ki67, SOS1 and GRF2 staining intensity in the normal and the variety of breast disease tissues. * indicates p<0.05 for relative staining intensity of each protein in the indicated subgroup of breast disease tissues compared to normal breast tissues.
Fig 3.
Expression of AnxA6 and proliferation markers in malignant and metastasis breast cancer tissues.
Tissues were processed as described in Fig 2 and the malignant and metastasis tissues stratified into TNBC and non-TNBC subsets. Shown are representative stained tumor tissues (A) and analysis of the staining intensities of the indicated proteins by using the Tissues IA software (B). * indicates p<0.05 for the mean staining intensity of each protein in the TNBC subset compared to the non-TNBC subset.
Fig 4.
Expression status of AnxA6 and proliferation markers in response to cytotoxic chemotherapy.
A) Formalin-fixed paraffin-embedded TNBC tissues from primary and residual tumors following chemotherapy were stained with antibodies against the indicated proteins. Slides were processed as in Fig 2, and representative stained tumor tissues. B) The staining intensity of the indicated proteins was quantified by using the Tissues IA software. The p-values represent the mean staining intensity of the respective proteins in the primary tumors (Biopsies) compared to that in the residual tumors (Residual). C) ROC assessment of the staining intensity of AnxA6 and other proliferation markers in biopsies from patients with residual disease versus the staining intensity in the matching residual tumors. AUC: area under the curve.
Table 2.
Pearson correlations between the expression levels of proliferation markers in biopsies and residual tumors.
Fig 5.
Association of the expression status of AnxA6 and Ki67 with distant relapse free survival of breast cancer patients following chemotherapy.
Kaplan-Meier plots showing the relationship between high (red) and low (blue) AnxA6 or Ki67 expression and distant relapse free (DRFS) survival of TNBC patients (A) and non-TNBC patients (B).
Fig 6.
The reciprocal expression of AnxA6 and GRF2 delineates rapidly growing basal-like from invasive mesenchymal-like TNBC cell lines.
A) Whole cell extracts were prepared from TNBC cell lines (n = 19) and normal breast epithelial cells (n = 2). The blots were analyzed by western blotting and densitometry as recently reported in Whalen et al., 2019 (Ref. 17). The ratio of GRF2:AnxA6 was used to stratify the cell lines according to their potential for rapid growth or invasiveness. B) A plot of the reciprocal expression of AnxA6 and GRF2 delineating TNBC cells with a higher potential for rapid growth (low AnxA6/high GRF2) from those with a higher potential for invasiveness (high AnxA6/low GRF2). C) A heat map of cells from Group 1 (n = 4) and Group 3 (n = 5) showing the clustering of the TNBC cell lines from each group and the discernible differences in their gene expression profiles.
Fig 7.
Reciprocal expression of AnxA6 and GRF2 in TNBC PDX models.
A) Whole tissue extracts were prepared from patient derived xenografts (n = 19), and equal amounts of protein were analyzed by western blotting using antibodies to the indicated proteins. B) Densitometric analysis of the expression of the indicated proteins. Shown are protein band intensities normalized to β-actin from a representative experiment. C) The ratio of GRF2:AnxA6 was used to classify the tumors into potentially rapidly growing or invasive tumors as in Fig 6. D) A plot of the reciprocal expression of AnxA6 and GRF2 based on the GRF2:AnxA6 ratio for each PDX sample and showing two major groups: Group 1 with a strong potential for rapid growth and Group 3 with a strong potential for invasiveness, separated by a more diverse Group 2 with varied potentials for growth and invasiveness.
Fig 8.
Distinct phenotypes of AnxA6 expressing and AnxA6 down regulated TNBC cells.
Schematic showing 3D cultures in growth factor-reduced matrigel and the transformation of the AnxA6-high/GRF2-low invasive BT-549 TNBC cells into AnxA6-low/GRF2-high rapidly growing BT-549 TNBC cells following down regulation of AnxA6. It should be noted that parental BT-549 cells are poorly tumorigenic while AnxA6 depletion or loss in BT-549 leads to high tumorigenicity (see Ref. 17).