Mitochondrial Proteomic Analysis of Cisplatin Resistance in Ovarian Cancer

Epithelial ovarian cancer (EOC) is the leading cause of death among women with gynecologic malignancies and accounts for approximately 6% of cancer deaths among women. Cisplatin and its analogues form the backbone of the most active chemotherapy regimens in advanced EOC; however, development of platinum resistance is common and typically marks a transition in which curing the patient is no longer possible. An emerging theme in many cancers is that mitochondrial dysfunction contributes to an aggressive carcinogenic phenotype. We hypothesized that changes in the mitochondrial proteome are required to support development of cisplatin resistance in human EOC. To investigate this hypothesis, an organellar proteomics approach was utilized to quantify alterations in protein abundance in mitochondria enriched from isogenic cisplatin-sensitive (A2780) and -resistant (A2780-CP20) human EOC cells. Protein isolates from mitochondria-enriched fractions were analyzed by high resolution liquid chromatography–tandem mass spectrometry (LC–MS/MS), and relative abundance of identified proteins was quantified by spectral counting. Pathway analyses revealed significant increases in notch signaling pathways, cell survival, and alternate apoptotic pathways in the A2780-CP20 subtype. Among the alterations identified in the mitochondrial proteomic composition in cisplatin-resistant EOC cells, activated leukocyte cell adhesion molecule (AKAP12) and A kinase anchoring protein 12 (AKAP12) were elevated, while nestin was diminished in the mitochondrial fraction of A2780-CP20 relative to A2780. This was verified by immunoblot analysis. These results confirm that important changes in the mitochondrial proteome, many of which promote evasion of apoptosis and tumor invasiveness and metastasis, are present in cisplatin-resistant EOC

Ornithine decarboxylase as a therapeutic target for endometrial cancer

<div><p>Ornithine Decarboxylase (ODC) a key enzyme in polyamine biosynthesis is often overexpressed in cancers and contributes to polyamine-induced cell proliferation. We noted ubiquitous expression of <i>ODC1</i> in our published endometrial cancer gene array data and confirmed this in the cancer genome atlas (TCGA) with highest expression in non-endometrioid, high grade, and copy number high cancers, which have the worst clinical outcomes. <i>ODC1</i> expression was associated with worse overall survival and increased recurrence in three endometrial cancer gene expression datasets. Importantly, we confirmed these findings using quantitative real-time polymerase chain reaction (qRT-PCR) in a validation cohort of 60 endometrial cancers and found that endometrial cancers with elevated <i>ODC1</i> had significantly shorter recurrence-free intervals (KM log-rank p = 0.0312, Wald test p = 5.59e-05). Difluoromethylornithine (DFMO) a specific inhibitor of ODC significantly reduced cell proliferation, cell viability, and colony formation in cell line models derived from undifferentiated, endometrioid, serous, carcinosarcoma (mixed mesodermal tumor; MMT) and clear cell endometrial cancers. DFMO also significantly reduced human endometrial cancer ACI-98 tumor burden in mice compared to controls (p = 0.0023). ODC-regulated polyamines (putrescine [Put] and/or spermidine [Spd]) known activators of cell proliferation were strongly decreased in response to DFMO, in both tumor tissue ([Put] (p = 0.0006), [Spd] (p<0.0001)) and blood plasma ([Put] (p<0.0001), [Spd] (p = 0.0049)) of treated mice. Our study indicates that some endometrial cancers appear particularly sensitive to DFMO and that the polyamine pathway in endometrial cancers in general and specifically those most likely to suffer adverse clinical outcomes could be targeted for effective treatment, chemoprevention or chemoprevention of recurrence.</p></div

Colony formation, cell viability and polyamine levels in DFMO-treated endometrial cancer cells.

<p><b>A</b>, Colony counts in a panel of endometrial cancer and immortal normal endometrial epithelial cells including ACI-98 (undifferentiated), MSU-15 (clear cell), ACI-61 (endometrioid) ACI-70 (MMT), HEC-1-A (endometrioid), EM E6/E7 TERT1 (normal immortalized endometrial epithelial), ECC-1 (endometrioid) and ACI-45 (carcinosarcoma, MMT). Cells are ordered most highly sensitive on left to least sensitive on the right. <b>B</b>, Representative colony plates for most (ACI-98) and least (ACI-45) sensitive cells. <b>C</b> and <b>D</b>, ACI-98 cells highly sensitive to wide range of doses of DFMO while ACI-45 cells are non-sensitive even at very high doses (5 mM) as measured by MTS assay. The percent values (%) shown are DFMO to Control. Intracellular polyamine levels (putrescine, spermidine, spermine) were analyzed in ACI-98 (<b>E</b>) and ACI-45 (<b>F</b>) cells either untreated (control) or DFMO-treated (0.25 mM) using RP-HPLC. Intracellular polyamines were quantified and expressed as nmol/mg protein. Standard errors are indicated (+/- S.E.). Put, putrescine; Spd, spermidine; Spm, spermine.</p

<i>ODC1</i> expression in endometrial cancers.

<p>A-C in LCM (laser capture micro-dissected) samples. <b>A</b>, Endometrioid (E, n = 139) and Serous (S, n = 37) types and normal epithelial tissues (N, n = 12); <b>B</b>, FIGO Stages I and II (n = 133), III (n = 24), and IV (n = 18); <b>C</b>, Grade 1 (G1, n = 42), Grade 2 (G2, n = 65), and Grade 3 (G3, n = 69). <u>Signal: Affymetrix signal normalized to target value of 500.</u> <b>D</b>, In TCGA samples of four molecular sub-types: Copy number low (MSS, n = 90), MSI high (n = 65), POLE ultra-mutant (n = 17) and Copy number high—serous like (n = 60). ***p < 0.001; **p < 0.01; *p< 0.05. <u>Counts: Normalized RNA-seq counts.</u></p

<i>ODC1</i> mRNA expression overall survival (OS) and recurrence free interval (RFI).

<p>Kaplan-Meier diagrams and Wald statistics showing increased <i>ODC1</i> is significantly associated with shorter OS (<b>A</b>) in TCGA (RNA-seq) (n = 232), LCM cases (Affymetrix) (n = 188) and in Spectrum (qRT-PCR) (n = 60) samples (<b>B</b>) <i>ODC1</i> mRNA is significantly associated with recurrence for TCGA and qRT-PCR cohorts but is not significant in the Affymetrix LCM dataset. “<u>High” and “Low” indicate above and below the median expression level.</u></p

Effect of oral DFMO on polyamine levels in tumor-bearing mice.

<p>Intracellular polyamine levels (putrescine, spermidine, spermine) were analyzed in tumor tissue (<b>A</b>) and blood plasma (<b>B</b>) of DFMO-treated and untreated mice using reverse-phase HPLC. Intracellular polyamines were quantified and expressed as nmol/mg protein (<b>A</b>) or nmol/ml plasma (<b>B</b>). Standard errors are indicated (+/- S.E.). Put, putrescine; Spd, spermidine; Spm, spermine.</p

Elevated AKAP12 in Paclitaxel-Resistant Serous Ovarian Cancer Cells Is Prognostic and Predictive of Poor Survival in Patients

A majority of high-grade (HG) serous ovarian cancer (SOC) patients develop resistant disease despite high initial response rates to platinum/paclitaxel-based chemotherapy. We identified shed/secreted proteins in preclinical models of paclitaxel-resistant human HGSOC models and correlated these candidate proteins with patient outcomes using public data from HGSOC patients. Proteomic analyses of a HGSOC cell line secretome was compared to those from a syngeneic paclitaxel-resistant variant and from a line established from an intrinsically chemorefractory HGSOC patient. Associations between the identified candidate proteins and patient outcome were assessed in a discovery cohort of 545 patients and two validation cohorts totaling 795 independent SOC patients. Among the 81 differentially abundant proteins identified (<i>q</i> < 0.05) from paclitaxel-sensitive vs -resistant HGSOC cell secretomes, AKAP12 was verified to be elevated in all models of paclitaxel-resistant HGSOC. Furthermore, elevated AKAP12 transcript expression was associated with worse progression-free and overall survival. Associations with outcome were observed in three independent cohorts and remained significant after adjusted multivariate modeling. We further provide evidence to support that differential gene methylation status is associated with elevated expression of AKAP12 in taxol-resistant ovarian cancer cells and ovarian cancer patient subsets. Elevated expression and shedding/secretion of AKAP12 is characteristic of paclitaxel-resistant HGSOC cells, and elevated AKAP12 transcript expression is a poor prognostic and predictive marker for progression-free and overall survival in SOC patients