Expression of Mitochondrial Regulators PGC1α and TFAM as Putative Markers of Subtype and Chemoresistance in Epithelial Ovarian Carcinoma

<div><p>Epithelial ovarian carcinoma (EOC), the major cause of gynaecological cancer death, is a heterogeneous disease classified into five subtypes. Each subtype has distinct clinical characteristics and is associated with different genetic risk factors and molecular events, but all are treated with surgery and platinum/taxane regimes. Tumour progression and chemoresistance is generally associated with major metabolic alterations, notably altered mitochondrial function(s). Here, we report for the first time that the expression of the mitochondrial regulators PGC1α and TFAM varies between EOC subtypes; furthermore, we have identified a profile in clear-cell carcinoma consisting of undetectability of PGC1α/TFAM, and low ERα/Ki-67. By contrast, high-grade serous carcinomas were characterised by a converse state of PGC1α/TFAM, ERα positivity and a high Ki-67 index. Interestingly, loss of PGC1α/TFAM and ERα was found also in a non-clear cell EOC cell line made highly resistant to platinum in vitro. Similar to clear-cell carcinomas, these resistant cells also showed accumulation of glycogen. Altogether, our data provide mechanistic insights into the chemoresistant nature of ovarian clear-cell carcinomas. Furthermore, these findings corroborate the need to take into account the diversity of EOC and to develop subtype specific treatment strategies.</p></div

Comparison of ovarian HGSC and CCC.

<p>Representative hematoxylin and eosin staining of ovarian HGSC and CCC. We hypothesise that these subtypes represents extreme forms of EOC; HGSC generally display positive expression of PGC1α, TFAM and ERα, have a higher Ki-67 index and are more responsive to initial platinum/taxane chemotherapy. By contrast, CCC generally do not express PGC1α, TFAM and ERα, have a lower Ki-67 index and are less responsive to initial platinum/taxane chemotherapy. Magnification 400x, scale bar shows 50 µm.</p

ERα expression and intracellular levels of glycogen in SKOV-3 and SKOV-3-R.

<p>(A) Gene expression of <i>ESR1</i> evaluated by qRT-PCR (<i>n</i> = 3). The decrease in expression in SKOV-3-R was statistically significant (<i>p</i><0.001, using independent t-test). Expression levels were normalised to <i>ACTB</i>. Error bars represent S.E.M. (B) Representative western blot showing protein expression of ERα. β-tubulin was used as a loading control. (C) Glycogen levels were evaluated using a colorimetric assay (<i>n</i> = 3). The increased level in SKOV-3-R was statistically significant (<i>p</i> = 0.027, using independent t-test). Error bars represent S.E.M.</p

PGC1α and TFAM expression in EOC SKOV-3 cells and the multiresistant subline SKOV-3-R.

<p>(A) Gene expression of <i>PPARGC1A</i> and <i>TFAM</i> in SKOV-3 and SKOV-3-R cells was evaluated by qRT-PCR (<i>n</i> = 3). The decrease in expression in SKOV-3-R was statistically significant (both <i>p</i><0.001, using independent t-test). Expression levels were normalised to <i>ACTB</i>. Error bars represent S.E.M. (B) Representative western blot showing protein expression of PGC1α and TFAM. β-tubulin was used as loading control.</p

Expression status of PGC1α and TFAM in the EOC study population.

<p>Abbreviations: EOC; epithelial ovarian carcinoma, ERα; oestrogen receptor alpha, nd; not determined, ns; not significant, PR; progesterone receptor, PGC1α; Peroxisome proliferator-activated receptor gamma co-activator 1-alpha, TFAM; mitochondrial transcription factor A.</p>a<p> Across variable; tested with Fisher's exact test for subtypes, expression of ERα and PR, and Mann-Whitney U test for distribution of age and Ki-67.</p>b<p> Within variable; tested with non-parametric Chi² test.</p><p>Expression status of PGC1α and TFAM in the EOC study population.</p

Expression status of PGC1α/TFAM in EOC.

<p>Abbreviations: EOC; epithelial ovarian carcinoma, ERα; oestrogen receptor alpha, nd; not determined, ns; not significant, PR; progesterone receptor, PGC1α; Peroxisome proliferator-activated receptor gamma co-activator 1-alpha, TFAM; mitochondrial transcription factor A.</p>a<p>Across variable; tested with Fisher's exact test for subtype, expression of ERα and PR, and Kruskal-Wallis test for distribution of age and Ki-67.</p><p>Expression status of PGC1α/TFAM in EOC.</p

Expression of PGC1α and TFAM in EOC subtypes.

<p>(A) Representative immunohistochemical staining of VDAC, ERα, PGC1α and TFAM in HGSC and CCC. VDAC indicates presence of mitochondria. ERα, PGC1α and TFAM were all expressed in HGSC, whereas in CCC expression of the proteins was lost. Each HGSC and CCC quadruplicate is from one and the same patient (magnification: 400x, scale bar shows 500 µm). (B) Histogram representing number of cases with expression of PGC1α/TFAM across EOC subtypes.</p

Ki-67 index distribution in TFAM+ and TFAM- EOC tumours.

<p>EOC tumours with negative expression of TFAM (<i>n</i> = 18) showed significantly lower Ki-67 index compared to tumours with positive expression of TFAM (<i>n</i> = 35) (Mann-Whitney U, <i>p</i> = 0.014).</p

Additional file 2: of Affinity proteomic profiling of plasma for proteins associated to area-based mammographic breast density

Tables S3-S6, S8, S9 and Figures S1-S7 Additional material and methods description, results and detailed discussion. (PDF 2026 kb

Additional file 1: of Affinity proteomic profiling of plasma for proteins associated to area-based mammographic breast density

Tables S1-S2 Complete list of antibodies included in serum bead array (SBA) 1 and 2 (sheet 1 and 2). Table S7 Summary of antibody validation (sheet 3). (XLSX 71 kb