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Rampant centrosome amplification underlies more aggressive disease course of triple negative breast cancers.

Pannu V, Mittal K, Cantuaria G, Reid MD, Li X, Donthamsetty S, McBride M, Klimov S, Osan R, Gupta MV, Rida PC, Aneja R - Oncotarget (2015)

Bottom Line: Our data establish differences in incidence and severity of CA between TNBC and non-TNBC cell lines and clinical specimens.We found strong correlation between CA and aggressiveness markers associated with metastasis in 20 pairs of grade-matched TNBC and non-TNBC specimens (p < 0.02).Time-lapse imaging of MDA-MB-231 cells harboring amplified centrosomes demonstrated enhanced migratory ability.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Georgia State University, Atlanta, GA 30303, USA.

ABSTRACT
Centrosome amplification (CA), a cell-biological trait, characterizes pre-neoplastic and pre-invasive lesions and is associated with tumor aggressiveness. Recent studies suggest that CA leads to malignant transformation and promotes invasion in mammary epithelial cells. Triple negative breast cancer (TNBC), a histologically-aggressive subtype shows high recurrence, metastases, and mortality rates. Since TNBC and non-TNBC follow variable kinetics of metastatic progression, they constitute a novel test bed to explore if severity and nature of CA can distinguish them apart. We quantitatively assessed structural and numerical centrosomal aberrations for each patient sample in a large-cohort of grade-matched TNBC (n = 30) and non-TNBC (n = 98) cases employing multi-color confocal imaging. Our data establish differences in incidence and severity of CA between TNBC and non-TNBC cell lines and clinical specimens. We found strong correlation between CA and aggressiveness markers associated with metastasis in 20 pairs of grade-matched TNBC and non-TNBC specimens (p < 0.02). Time-lapse imaging of MDA-MB-231 cells harboring amplified centrosomes demonstrated enhanced migratory ability. Our study bridges a vital knowledge gap by pinpointing that CA underlies breast cancer aggressiveness. This previously unrecognized organellar inequality at the centrosome level may allow early-risk prediction and explain higher tumor aggressiveness and mortality rates in TNBC patients.

No MeSH data available.


Related in: MedlinePlus

Comparison of extent of centrosome amplification in grade-matched TNBC and non-TNBC patients(A) Representative confocal micrographs depicting the status of centrosome amplification in histological Grade I, Grade II and Grade III TNBC and non-TNBC tissues. Centrosomes were labelled with γ-tubulin antibody (green) and DNA was stained with DAPI (blue) Yellow arrows indicate numerical amplification and white arrows indicate structural amplification. Scale bar 5 μm. (B) Bar graph representation of number of cells out of 500 showing supernumerary centrosomes (> 2 or ≤ 2) in TNBC and non-TNBC tissue samples. (C) Box whisker graph representation of average volume of centrosomes in normal, TNBC and non-TNBC tissue samples. At least 500 cells were counted in each case (ANOVA, p < 0.05).
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Figure 2: Comparison of extent of centrosome amplification in grade-matched TNBC and non-TNBC patients(A) Representative confocal micrographs depicting the status of centrosome amplification in histological Grade I, Grade II and Grade III TNBC and non-TNBC tissues. Centrosomes were labelled with γ-tubulin antibody (green) and DNA was stained with DAPI (blue) Yellow arrows indicate numerical amplification and white arrows indicate structural amplification. Scale bar 5 μm. (B) Bar graph representation of number of cells out of 500 showing supernumerary centrosomes (> 2 or ≤ 2) in TNBC and non-TNBC tissue samples. (C) Box whisker graph representation of average volume of centrosomes in normal, TNBC and non-TNBC tissue samples. At least 500 cells were counted in each case (ANOVA, p < 0.05).

Mentions: Our in silico data analysis yielded significantly elevated CAI in TNBC women compared to non-TNBC. Thus, we visualized amplified centrosomes in grade-matched tissues from TNBC (n = 59) and non-TNBC (n = 116) patients (Fig. 2A) employing multicolor confocal immunofluorescence microscopy. Centrosomes were labelled by γ-tubulin (green) antibody, wherein centrosomal aberrations were determined by abnormal number of γ-tubulin spots (more than two) as well as by increased volume over normal centrosomal volume in breast epithelial cells from normal adjacent tissues. Fig. 2A shows representative confocal immunomicrographs depicting centrosomes (γ-tubulin, green) and nuclei (DAPI, blue) from normal breast tissue and breast cancer tissue from grade-matched (Grade II) TNBC and non-TNBC patients. Cells with more than two centrosomes were estimated by examining and counting centrosomes in atleast 500 cells/slide. Fig. 2B shows that the number of cells harboring extra centrosomes were twice as much higher in TNBC samples (62, n = 30) versus non-TNBC (30, n = 98) (p < 0.05) in a grade-matched background. We next measured centrosomal volumes in all specimens using the three-dimensional measurement module from the Zeiss imaging software. While mean centrosomal volume in normal breast epithelial cells was 0.22 μm3 (ranging from 0.08 to 0.76 μm3), the mean volume of γ-tubulin-stained spots analyzed in at least 500 tumor cells from each patient sample was 4.85 μm3, which is ~15 times higher than the centrosomal volume in normal cells. Fig. 2C shows that centrosome volume was significantly higher in TNBC samples (n = 30) (average = 6.8 μm3) compared to grade-matched (Grade II) non-TNBC (n = 98) samples (average = 4.2 μm3) (p < 0.05). Additionally, we calculated the total centrosome amplification as a percentage by adding percent cells harboring more than two centrosomes and percent cells harboring centrosomes with volume larger than 0.76 μm3. Our analysis revealed that on an average, ~68% of cells in TNBC samples exhibited centrosome amplification as compared to ~45% in non-TNBC samples (p < 0.05).


Rampant centrosome amplification underlies more aggressive disease course of triple negative breast cancers.

Pannu V, Mittal K, Cantuaria G, Reid MD, Li X, Donthamsetty S, McBride M, Klimov S, Osan R, Gupta MV, Rida PC, Aneja R - Oncotarget (2015)

Comparison of extent of centrosome amplification in grade-matched TNBC and non-TNBC patients(A) Representative confocal micrographs depicting the status of centrosome amplification in histological Grade I, Grade II and Grade III TNBC and non-TNBC tissues. Centrosomes were labelled with γ-tubulin antibody (green) and DNA was stained with DAPI (blue) Yellow arrows indicate numerical amplification and white arrows indicate structural amplification. Scale bar 5 μm. (B) Bar graph representation of number of cells out of 500 showing supernumerary centrosomes (> 2 or ≤ 2) in TNBC and non-TNBC tissue samples. (C) Box whisker graph representation of average volume of centrosomes in normal, TNBC and non-TNBC tissue samples. At least 500 cells were counted in each case (ANOVA, p < 0.05).
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Related In: Results  -  Collection

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Show All Figures
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Figure 2: Comparison of extent of centrosome amplification in grade-matched TNBC and non-TNBC patients(A) Representative confocal micrographs depicting the status of centrosome amplification in histological Grade I, Grade II and Grade III TNBC and non-TNBC tissues. Centrosomes were labelled with γ-tubulin antibody (green) and DNA was stained with DAPI (blue) Yellow arrows indicate numerical amplification and white arrows indicate structural amplification. Scale bar 5 μm. (B) Bar graph representation of number of cells out of 500 showing supernumerary centrosomes (> 2 or ≤ 2) in TNBC and non-TNBC tissue samples. (C) Box whisker graph representation of average volume of centrosomes in normal, TNBC and non-TNBC tissue samples. At least 500 cells were counted in each case (ANOVA, p < 0.05).
Mentions: Our in silico data analysis yielded significantly elevated CAI in TNBC women compared to non-TNBC. Thus, we visualized amplified centrosomes in grade-matched tissues from TNBC (n = 59) and non-TNBC (n = 116) patients (Fig. 2A) employing multicolor confocal immunofluorescence microscopy. Centrosomes were labelled by γ-tubulin (green) antibody, wherein centrosomal aberrations were determined by abnormal number of γ-tubulin spots (more than two) as well as by increased volume over normal centrosomal volume in breast epithelial cells from normal adjacent tissues. Fig. 2A shows representative confocal immunomicrographs depicting centrosomes (γ-tubulin, green) and nuclei (DAPI, blue) from normal breast tissue and breast cancer tissue from grade-matched (Grade II) TNBC and non-TNBC patients. Cells with more than two centrosomes were estimated by examining and counting centrosomes in atleast 500 cells/slide. Fig. 2B shows that the number of cells harboring extra centrosomes were twice as much higher in TNBC samples (62, n = 30) versus non-TNBC (30, n = 98) (p < 0.05) in a grade-matched background. We next measured centrosomal volumes in all specimens using the three-dimensional measurement module from the Zeiss imaging software. While mean centrosomal volume in normal breast epithelial cells was 0.22 μm3 (ranging from 0.08 to 0.76 μm3), the mean volume of γ-tubulin-stained spots analyzed in at least 500 tumor cells from each patient sample was 4.85 μm3, which is ~15 times higher than the centrosomal volume in normal cells. Fig. 2C shows that centrosome volume was significantly higher in TNBC samples (n = 30) (average = 6.8 μm3) compared to grade-matched (Grade II) non-TNBC (n = 98) samples (average = 4.2 μm3) (p < 0.05). Additionally, we calculated the total centrosome amplification as a percentage by adding percent cells harboring more than two centrosomes and percent cells harboring centrosomes with volume larger than 0.76 μm3. Our analysis revealed that on an average, ~68% of cells in TNBC samples exhibited centrosome amplification as compared to ~45% in non-TNBC samples (p < 0.05).

Bottom Line: Our data establish differences in incidence and severity of CA between TNBC and non-TNBC cell lines and clinical specimens.We found strong correlation between CA and aggressiveness markers associated with metastasis in 20 pairs of grade-matched TNBC and non-TNBC specimens (p < 0.02).Time-lapse imaging of MDA-MB-231 cells harboring amplified centrosomes demonstrated enhanced migratory ability.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Georgia State University, Atlanta, GA 30303, USA.

ABSTRACT
Centrosome amplification (CA), a cell-biological trait, characterizes pre-neoplastic and pre-invasive lesions and is associated with tumor aggressiveness. Recent studies suggest that CA leads to malignant transformation and promotes invasion in mammary epithelial cells. Triple negative breast cancer (TNBC), a histologically-aggressive subtype shows high recurrence, metastases, and mortality rates. Since TNBC and non-TNBC follow variable kinetics of metastatic progression, they constitute a novel test bed to explore if severity and nature of CA can distinguish them apart. We quantitatively assessed structural and numerical centrosomal aberrations for each patient sample in a large-cohort of grade-matched TNBC (n = 30) and non-TNBC (n = 98) cases employing multi-color confocal imaging. Our data establish differences in incidence and severity of CA between TNBC and non-TNBC cell lines and clinical specimens. We found strong correlation between CA and aggressiveness markers associated with metastasis in 20 pairs of grade-matched TNBC and non-TNBC specimens (p < 0.02). Time-lapse imaging of MDA-MB-231 cells harboring amplified centrosomes demonstrated enhanced migratory ability. Our study bridges a vital knowledge gap by pinpointing that CA underlies breast cancer aggressiveness. This previously unrecognized organellar inequality at the centrosome level may allow early-risk prediction and explain higher tumor aggressiveness and mortality rates in TNBC patients.

No MeSH data available.


Related in: MedlinePlus