Noor A. Lokman

The Role of Annexin A2 in Tumorigenesis and Cancer Progression

Annexin A2 is a calcium-dependent, phospholipid-binding protein found on various cell types. It is up-regulated in various tumor types and plays multiple roles in regulating cellular functions, including angiogenesis, proliferation, apoptosis, cell migration, invasion and adhesion. Annexin A2 binds with plasminogen and tissue plasminogen activator on the cell surface, which leads to the conversion of plasminogen to plasmin. Plasmin is a serine protease which plays a key role in the activation of metalloproteinases and degradation of extracellular matrix components essential for metastatic progression. We have recently found that both annexin A2 and plasmin are increased in conditioned media of co cultured ovarian cancer and peritoneal cells. Our studies suggest that annexin A2 is part of a tumor-host signal pathway between ovarian cancer and peritoneal cells which promotes ovarian cancer metastasis. Accumulating evidence suggest that interactions between annexin A2 and its binding proteins play an important role in the tumor microenvironment and act together to enhance cancer metastasis. This article reviews the current knowledge on the biological role of annexin A2 and its binding proteins in solid malignancies including ovarian cancer.

Chick Chorioallantoic Membrane (CAM) Assay as an In Vivo Model to Study the Effect of Newly Identified Molecules on Ovarian Cancer Invasion and Metastasis

The majority of ovarian cancer patients present with advanced disease and despite aggressive treatment, prognosis remains poor. Significant improvement in ovarian cancer survival will require the development of more effective molecularly targeted therapeutics. Commonly, mouse models are used for the in vivo assessment of potential new therapeutic targets in ovarian cancer. However, animal models are costly and time consuming. Other models, such as the chick embryo chorioallantoic membrane (CAM) assay, are therefore an attractive alternative. CAM assays have been widely used to study angiogenesis and tumor invasion of colorectal, prostate and brain cancers. However, there have been limited studies that have used CAM assays to assess ovarian cancer invasion and metastasis. We have therefore developed a CAM assay protocol to monitor the metastatic properties of ovarian cancer cells (OVCAR-3, SKOV-3 and OV-90) and to study the effect of potential therapeutic molecules in vivo. The results from the CAM assay are consistent with cancer cell motility and invasion observed in in vitro assays. Our results demonstrate that the CAM assay is a robust and cost effective model to study ovarian cancer cell metastasis. It is therefore a very useful in vivo model for screening of potential novel therapeutics.

Transforming growth factor-beta-induced protein secreted by peritoneal cells increases the metastatic potential of ovarian cancer cells

Ovarian cancer metastasis is characterized by the shedding of malignant cells from the surface of the ovary and their implantation onto the peritoneal surface, which lines the abdominal cavity. As the factors promoting this process are poorly understood, we investigated the ovarian cancer–peritoneal interaction by means of in vitro coculture experiments with ovarian cancer (OVCAR‐5 and SKOV‐3) and peritoneal (LP‐9) cells. One of the proteins differentially expressed in the coculture secretome was identified by MALDI‐TOF/TOF mass spectrometry as the extracellular matrix protein transforming growth factor‐beta‐induced protein (TGFBIp, also known as βig‐H3). Immunohistochemistry showed high TGFBIp levels in normal surface ovarian epithelial and peritoneal cells, whereas TGFBIp levels in primary serous ovarian carcinomas and matching metastatic implants was very low. In functional in vitro experiments, treatment with recombinant TGFBIp significantly increased the motility and invasiveness of OVCAR‐5 and SKOV‐3 cells and significantly increased ovarian cancer cell (OVCAR‐5, OVCAR‐3 and SKOV‐3) adhesion to LP‐9 cells. TGFBIp was found to be processed at both the N‐ and C‐terminus in the secretome of the ovarian cancer–peritoneal cell coculture. Plasmin inhibitors blocked TGFBIp processing and significantly reduced OVCAR‐5 cell adhesion to peritoneal cells. We conclude that TGFBIp expressed by peritoneal cells increases the metastatic potential of ovarian cancer cells. TGFBIp is therefore a potential novel therapeutic target against ovarian cancer.

MicroRNA-194 promotes prostate cancer metastasis by inhibiting SOCS2

Serum levels of miR-194 have been reported to predict prostate cancer recurrence after surgery, but its functional contributions to this disease have not been studied. Herein, it is demonstrated that miR-194 is a driver of prostate cancer metastasis. Prostate tissue levels of miR-194 were associated with disease aggressiveness and poor outcome. Ectopic delivery of miR-194 stimulated migration, invasion, and epithelial-mesenchymal transition in human prostate cancer cell lines, and stable overexpression of miR-194 enhanced metastasis of intravenous and intraprostatic tumor xenografts. Conversely, inhibition of miR-194 activity suppressed the invasive capacity of prostate cancer cell lines in vitro and in vivo Mechanistic investigations identified the ubiquitin ligase suppressor of cytokine signaling 2 (SOCS2) as a direct, biologically relevant target of miR-194 in prostate cancer. Low levels of SOCS2 correlated strongly with disease recurrence and metastasis in clinical specimens. SOCS2 downregulation recapitulated miR-194-driven metastatic phenotypes, whereas overexpression of a nontargetable SOCS2 reduced miR-194-stimulated invasion. Targeting of SOCS2 by miR-194 resulted in derepression of the oncogenic kinases FLT3 and JAK2, leading to enhanced ERK and STAT3 signaling. Pharmacologic inhibition of ERK and JAK/STAT pathways reversed miR-194-driven phenotypes. The GATA2 transcription factor was identified as an upstream regulator of miR-194, consistent with a strong concordance between GATA2 and miR-194 levels in clinical specimens. Overall, these results offer new insights into the molecular mechanisms of metastatic progression in prostate cancer. Cancer Res; 77(4); 1021-34. ©2016 AACR.

CIB2 Negatively Regulates Oncogenic Signaling in Ovarian Cancer via Sphingosine Kinase 1

Sphingosine kinase 1 (SK1) is a key regulator of the cellular balance between proapoptotic and prosurvival sphingolipids. Oncogenic signaling by SK1 relies on its localization to the plasma membrane, which is mediated by the calcium and integrin binding protein CIB1 via its Ca2+-myristoyl switch function. Here we show that another member of the CIB family, CIB2, plays a surprisingly opposite role to CIB1 in the regulation of SK1 signaling. CIB2 bound SK1 on the same site as CIB1, yet it lacks the Ca2+-myristoyl switch function. As a result, CIB2 blocked translocation of SK1 to the plasma membrane and inhibited its subsequent signaling, which included sensitization to TNFα-induced apoptosis and inhibition of Ras-induced neoplastic transformation. CIB2 was significantly downregulated in ovarian cancer and low CIB2 expression was associated with poor prognosis in ovarian cancer patients. Notably, reintroduction of CIB2 in ovarian cancer cells blocked plasma membrane localization of endogenous SK1, reduced in vitro neoplastic growth and tumor growth in mice, and suppressed cell motility and invasiveness both in vitro and in vivo Consistent with the in vitro synergistic effects between the SK1-specific inhibitor SK1-I and standard chemotherapeutics, expression of CIB2 also sensitized ovarian cancer cells to carboplatin. Together, these findings identify CIB2 as a novel endogenous suppressor of SK1 signaling and potential prognostic marker and demonstrate the therapeutic potential of SK1 in this gynecologic malignancy. Cancer Res; 77(18); 4823-34. ©2017 AACR.

Annexin A2 and S100A10 are independent predictors of serous ovarian cancer outcome

Annexin A2, a calcium phospholipid binding protein, has been shown to play an important role in ovarian cancer metastasis. This study examined whether annexin A2 and S100A10 can be used as prognostic markers in serous ovarian cancer. ANXA2 and S100A10 gene expressions were assessed in publicly available ovarian cancer data sets and annexin A2 and S100A10 protein expressions were assessed by immunohistochemistry in a uniform cohort of stage III serous ovarian cancers (n = 109). Kaplan-Meier and Cox regression analyses were performed to assess the relationship between annexin A2 or S100A10 messenger RNA (mRNA) and protein expressions with clinical outcome. High ANXA2 mRNA levels in stage III serous ovarian cancers were associated with reduced progression-free survival (PFS; P = 0.023) and overall survival (OS; P = 0.0038), whereas high S100A10 mRNA levels predicted reduced OS (P = 0.0019). Using The Cancer Genome Atlas data sets, ANXA2 but not S100A10 expression was associated with higher clinical stage (P = 0.005), whereas both ANXA2 and S100A10 expressions were associated with the mesenchymal molecular subtype (P < 0.0001). Kaplan-Meier and Cox regression analyses showed that high stromal annexin A2 immunostaining was significantly associated with reduced PFS (P = 0.013) and OS (P = 0.044). Moreover, high cytoplasmic S100A10 staining was significantly associated with reduced OS (P = 0.027). Multivariate Cox regression analysis showed stromal annexin A2 (P = 0.009) and cytoplasmic S100A10 (P = 0.016) levels to be independent predictors of OS. Patients with high stromal annexin A2 and high cytoplasmic S100A10 expressions had a 3.4-fold increased risk of progression (P = 0.02) and 7.9-fold risk of ovarian cancer death (P = 0.04). Our findings indicate that together annexin A2 and S100A10 expressions are powerful predictors of serous ovarian cancer outcome.

Lymph node metastasis of primary endometrial cancers: Associated proteins revealed by MALDI imaging

Metastasis is a crucial step of malignant progression and is the primary cause of death from endometrial cancer. However, clinicians presently face the challenge that conventional surgical‐pathological variables, such as tumour size, depth of myometrial invasion, histological grade, lymphovascular space invasion or radiological imaging are unable to predict with accuracy if the primary tumour has metastasized. In the current retrospective study, we have used primary tumour samples of endometrial cancer patients diagnosed with (n = 16) and without (n = 27) lymph node metastasis to identify potential discriminators. Using peptide matrix assisted laser desorption/ionisation mass spectrometry imaging (MALDI‐MSI), we have identified m/z values which can classify 88% of all tumours correctly. The top discriminative m/z values were identified using a combination of in situ sequencing and LC‐MS/MS from digested tumour samples. Two of the proteins identified, plectin and α‐Actin‐2, were used for validation studies using LC‐MS/MS data independent analysis (DIA) and immunohistochemistry. In summary, MALDI‐MSI has the potential to identify discriminators of metastasis using primary tumour samples.

MALDI Mass Spectrometry Imaging Reveals Decreased CK5 Levels in Vulvar Squamous Cell Carcinomas Compared to the Precursor Lesion Differentiated Vulvar Intraepithelial Neoplasia

Vulvar cancer is the fourth most common gynecological cancer worldwide. However, limited studies have been completed on the molecular characterization of vulvar squamous cell carcinoma resulting in a poor understanding of the disease initiation and progression. Analysis and early detection of the precursor lesion of HPV-independent vulvar squamous cell carcinoma (VSCC), differentiated vulvar intraepithelial neoplasia (dVIN), is of great importance given dVIN lesions have a high level of malignant potential. Here we present an examination of adjacent normal vulvar epithelium, dVIN, and VSCC from six patients by peptide Matrix-assisted laser desorption/ionization Mass Spectrometry Imaging (MALDI-MSI). The results reveal the differential expression of multiple peptides from the protein cytokeratin 5 (CK5) across the three vulvar tissue types. The difference observed in the relative abundance of CK5 by MALDI-MSI between the healthy epithelium, dVIN, and VSCC was further analyzed by immunohistochemistry (IHC) in tissue from eight VSCC patients. A decrease in CK5 immunostaining was observed in the VSCC compared to the healthy epithelium and dVIN. These results provide an insight into the molecular fingerprint of the vulvar intraepithelial neoplasia that appears to be more closely related to the healthy epithelium than the VSCC.

Keratin 5 overexpression is associated with serous ovarian cancer recurrence and chemotherapy resistance.

This study investigated the clinical significance of keratin 5 and 6 expression in serous ovarian cancer progression and chemotherapy resistance. KRT5 and KRT6 (KRT6A, KRT6B & KRT6C) gene expression was assessed in publically available serous ovarian cancer data sets, ovarian cancer cell lines and primary serous ovarian cancer cells. Monoclonal antibodies which detect both K5/6 or only K5 were used to assess protein expression in ovarian cancer cell lines and a cohort of high grade serous ovarian carcinomas at surgery (n = 117) and after neoadjuvant chemotherapy (n = 21). Survival analyses showed that high KRT5 mRNA in stage III/IV serous ovarian cancers was significantly associated with reduced progression-free (HR 1.38, P < 0.0001) and overall survival (HR 1.28, P = 0.013) whilst high KRT6 mRNA was only associated with reduced progression-free survival (HR 1.2, P = 0.031). Both high K5/6 (≥ 10%, HR 1.78 95% CI; 1.03−2.65, P = 0.017) and high K5 (≥ 10%, HR 1.90, 95% CI; 1.12−3.19, P = 0.017) were associated with an increased risk of disease recurrence. KRT5 but not KRT6C mRNA expression was increased in chemotherapy resistant primary serous ovarian cancer cells compared to chemotherapy sensitive cells. The proportion of serous ovarian carcinomas with high K5/6 or high K5 immunostaining was significantly increased following neoadjuvant chemotherapy. K5 can be used to predict serous ovarian cancer prognosis and identify cancer cells that are resistant to chemotherapy. Developing strategies to target K5 may therefore improve serous ovarian cancer survival.

WOMEN IN CANCER THEMATIC REVIEW: Ovarian cancer–peritoneal cell interactions promote extracellular matrix processing

Ovarian cancer has a distinct tendency for metastasising via shedding of cancerous cells into the peritoneal cavity and implanting onto the peritoneum that lines the pelvic organs. Once ovarian cancer cells adhere to the peritoneal cells, they migrate through the peritoneal layer and invade the local organs. Alterations in the extracellular environment are critical for tumour initiation, progression and intra-peritoneal dissemination. To increase our understanding of the molecular mechanisms involved in ovarian cancer metastasis and to identify novel therapeutic targets, we recently studied the interaction of ovarian cancer and peritoneal cells using a proteomic approach. We identified several extracellular matrix (ECM) proteins including, fibronectin, TGFBI, periostin, annexin A2 and PAI-1 that were processed as a result of the ovarian cancer-peritoneal cell interaction. This review focuses on the functional role of these proteins in ovarian cancer metastasis. Our findings together with published literature support the notion that ECM processing via the plasminogen-plasmin pathway promotes the colonisation and attachment of ovarian cancer cells to the peritoneum and actively contributes to the early steps of ovarian cancer metastasis.