This study identified 97 proteins significantly altered between patient matched pre-NACT (chemotherapy naïve) and post-NACT (chemotherapy treated) HGSOC tumor tissue. Pathway analysis of these alterations revealed activation of pathways associated with cell survival and metabolism in post NACT treated tumors. Aldo-keto reductase family 1 member B1 (AKR1B1) is elevated in post-NACT treated tumors and correlates with alterations in fatty acid metabolism. Notably, AKR1B1 strongly correlates with epithelial-to-mesenchymal transition (EMT) and is linked to glucose metabolism, cancer differentiation, and aggressiveness. (20). Inhibition of AKR1B1 has also been evaluated in diabetes management with inhibition of AKR1B1 being shown to prevent advanced glycation end-product accumulation and atherosclerotic lesion formation. (21) Additionally, succinate-semialdehyde dehydrogenase, mitochondrial (ALDH5A1), which is involved in glutamate metabolism, was elevated in post-NACT treated tumors and work by Tian and colleagues identified that low expression of ALDH5A1 is associated with worse overall survival in ovarian cancer. (22) In our study, elevated levels of AKR1B1 and ALDH5A1 in post-NACT treated tumors suggests alterations in proteins regulating cellular metabolism accompanies exposure of ovarian cancer cells to cytotoxic chemotherapy. In support of this finding, recent evidence has shown that HGSOC tumor cells exhibit altered cellular metabolism following multiple lines of chemotherapy treatment (23). We further observe sorbin and SH3 domain containing 2 (SORBS2) as significantly elevated in post-NACT tumors, which has also been described as elevated at the transcript level in post versus pre-NACT treated HGSOC tumors (18). Interestingly, SORBS2 has been shown to suppress metastatic spread and to promote a tumor-suppressive microenvironment in ovarian cancer models in vivo (24).
We further compared pre -NACT treated tumors from patients with residual disease following IDS (≤ 0.1-1.0 cm residual disease, R1, n = 6) versus those with no/microscopic (< 0.1cm) residual (R0, n = 14). Notably, pathways regulating cellular invasion and migration were predicted as activated in pre-NACT treated tumors from patients with (R1) versus without residual disease (R0) following IDS. Investigation of proteins altered among invasion and migration signaling pathways revealed branched chain amino-acid transaminase 1 (BCAT1) as elevated in pre-NACT tumors from R1 versus R0 patients (+ 1.11 LogFC, P = 0.021). BCAT1 has been shown to play a key role in promoting tumor cell invasion and migration in a variety of solid tumor malignancies including hepatocellular carcinoma, non-small cell lung cancer as well as ovarian cancer (25–27). Silencing BCAT1 in ovarian cancer cells results in decreased proliferation, invasion, and migration and correlates with prolonged survival time in xenograft models (27). We further investigated proteins altered between R1 vs R0 patients from pre-NACT tumors in patients with (> 0.1cm) or without residual disease in an independent cohort of HGSOC patients (n = 154) and identified 16 proteins significantly co-altered that are quantitatively correlated with the present investigation.
To investigate if proteins altered in patients with residual disease also correlated with disease prognosis, we evaluated the relationship of proteins altered between R1 versus R0 patients with proteins associated with PFS in a publicly available cohort of global proteomic data from 154 HGSOC patients (16). We further assessed if proteins significantly correlating with PFS did so independently of patient age, disease stage and residual disease status as these clinical variables are known to correlate with disease outcome (19) including residual disease, as this is a significant prognostic factor determining cancer related outcome and it also has bias that can be associated with experience of the surgeon and complexity of the surgery tolerated by the patient. Our goal for this analysis was to prioritize proteins that may have an increased likelihood of influencing tumor cell biology resulting in altered invasive and metastatic potential that could in part contribute to higher disease burden. We identified proteins significantly altered in R1 versus R0 tissues that correlate with PFS; Glutathione Synthetase (GSS), Fermitin Family Member 2 (FERMT2), Perilipin 2 (PLIN2) and Methylthioribose-1-phosphate isomerase (MRI1) were altered in pre-NACT tumors. GSS and FERMT2 were also among proteins significantly elevated in patients with (> 0.1cm) or without (no macroscopic) residual disease in an independent cohort of 154 tumors from chemo-naïve HGSOC patients. GSS, which we observed to be associated with an increased risk of disease progression, is an enzyme involved in the glutathione biosynthesis pathway and has been described as a potent antioxidant protecting cells from oxidative damage by free radicals and detoxification (28). FERMT2 was upregulated in R1 versus R0 chemotherapy naïve tissues as well as in an independent cohort of HGSOC patients with (> 0.1cm) versus without (no macroscopic) residual disease (n = 154), and that correlated with an increased risk of disease progression independent of patient age at diagnosis, residual disease status as well as disease stage. FERMT2, also known as Kindlin-2, has been associated with poor survival in lung adenocarcinoma (29) and hepatocellular carcinoma (30) and FERMT2 as well as other fermitin family members have been shown to promote EMT and metastasis through the ß catenin pathway (31–33). In contrast, we observed increased abundance of MRI1 in pre-NACT tissue samples from R1 vs R0 patients and this protein was also correlated with a lower risk of disease progression. MRI1 plays an essential role in methionine formation, which in turn functions in protein translation, methylation, and protection against reactive oxygen species through the generation of glutathione. (34, 35) As MRI1 is related to promoting glutathione signaling, and as noted above for GSS, the specific role of MRI1 in decreasing the risk of disease progression in ovarian cancer will require further investigation to understand whether this protein promotes tumor cell survival through mitigating oxidative stress. Notably, both GSS and MRI1 did not remain significantly correlated with PFS following adjustment for common covariates of disease progression, such as patient age, disease stage and residual disease status. Perilipin 2 (PLIN2) was correlated with an increased risk of disease progression and was elevated in in patients with (R1) or without (R0) residual disease in pre-NACT tissues. The PLIN family are associated with controlling lipolysis (36). Research by Liu et al demonstrates that PLIN2 binds not only to lipids but other Wnt signaling components, with depletion in the presence of lipids affecting the normal association of PLIN2 (36). The Wnt/ß-catenin pathway is one of the major signaling pathways thought to be involved in EMT, with alterations in this pathway affecting Wnt pathway proteins on the cell membrane, cytoplasm, and nucleus (37). Additional work by Arend et al (37) evaluated niclosamide, an FDA-approved derivative for treating tapeworm infections, as a target of the Wnt/ ß-catenin pathway in ovarian cancer cells. Use of niclosamide, along with two synthetically manufactured niclosamide analogs, significantly inhibited proliferation in two chemoresistant ovarian cancer cell lines, demonstrating the potential of drug repurposing for chemoresistant epithelial ovarian cancer (38).These proteins may serve as biomarkers to stratify patients for whom optimal cytoreduction is not achievable, to provide prognostic information, or serve as future drug targets.
Among the strengths of this study includes the use of human ovarian tumors with chemonaïve and NACT/chemotherapy-treated tumors collected from the same patient. This novel sample set allows for documenting in situ changes that occur with administration of NACT in a clinical setting. Our study is limited by a small sample size (n = 20) and limited clinical data regarding patient outcomes. Another limitation is that many of our patients were predominantly of Hispanic race and thus is not necessarily representative of all populations.
The differences demonstrated between chemotherapy naïve (e.g. pre-NACT) and chemotherapy exposed (e.g. post-NACT) tissues represents a unique opportunity to investigate candidate molecular marker(s) to stratify patients with high disease distribution or who may not respond well to conventional chemotherapy. In addition, identification of adaptive tumor changes may also inform preclinical investigation of interventions aimed at overcoming resistance to NACT. Our identification of a protein signature associated with R0 debulking status in the pretreatment setting was another exciting observation with potential clinical utility. A test predictive of R0 at diagnostic laparoscopy could better inform patient counselling at diagnosis as well as forecast whether > 3 cycles of chemotherapy may be needed to optimize the chance of achieving R0 at IDS. Our exploratory efforts have not only identified proteins associated with aggressive tumor biology, but also set the stage for follow-up investigation of a patient cohort that has had more regimented decision making in selection of patients for primary cytoreduction versus NACT. Validation of our exploratory findings and identification of additional targets could enable development of a predictive signatures for ovarian cancer patient surgical outcomes.