In Vitro, In Vivo and Ex Vivo Irradiation Research of Low-frequency Ultrasound Combined with Chemotherapy for Ovarian Carcinoma Cells.

PURPOSE: The treatment of ovarian cancer (OC) with chemotherapy leaves resistant cancer cells which in a short time re–grow as recurrent cancer. A diverse array of resistance mechanisms for chemotherapy in tumor cells has been described but none has proven to be a viable target in a clinical setting. Cancer stem cells (CSCs) are increasingly accepted as the putative mediators of chemoresistance and relapse of cancer. This study aimed to understand the molecular mechanisms involved with chemoresistance and recurrence by investigating the roles of CSCs and their associated pathways in OC cell lines and tumor cells isolated from the ascites of OC patients. METHODS: Tumor cells were collected from chemonaive (CN) and recurrent (CR) OC patients diagnosed with advanced–stage serous OC using a novel in vitro method to obtain a distinct population of epithelial tumor cells. Flow cytometry and immunofluorescence were used to characterize the tumor population. High–resolution label–free quantitative proteomic profiling was used to define significantly differentially expressed proteins between CN and CR tumor cells. KEGG and DAVID software's were used to determine pathways associated with CR cells. The mechanisms of survival of in vitro cisplatin or paclitaxel treated ascites–derived tumor cells as well as cultured OC cell lines were determined by in vitro assays and in mouse xenografts. In another approach, the expression of embryonic stem cell factor Oct4A was determined in primary ovarian tumors and its functional role was investigated using in vitro assays and in vivo mouse models with stable knockdown (shRNA) of Oct4A in OC cell lines. RESULTS: Proteomic profiling of CN and CR tumor cells showed significant differences in proteins encoding for immune surveillance, DNA repair mechanisms, cytoskeleton rearrangement, cell–cell adhesion, cell cycle pathways, cellular transport, and proteins involved with glycine/proline/arginine synthesis in tumor cells isolated from CR relative to CN patients. Pathway analyses revealed enrichment of metabolic pathways, DNA repair mechanisms and energy metabolism in CR tumor cells. The treatment of ascites–derived OC cells with chemotherapy in vitro resulted in a CSC–like residual population with increased activation of JAK2/STAT3 pathway. Both JAK2/STAT3 activation and CSC–like characteristics were suppressed by a low dose JAK2 specific inhibitor, Momelotinib, in vitro and in vivo. This also resulted in a significantly reduced tumor burden and increased disease–free survival periods in mice in vivo. In another approach, stable knockdown of Oct4A resulted in the decreased expression of CSCs in OC cells and was consistent with decreased cell proliferation, migration and chemoresistance in vitro. In vivo Oct4A knockdown cells produced a significantly reduced tumor burden in mice resulting in a significantly increased disease–free survival periods compared to vector control cells. CONCLUSION: The above studies suggest that targeting the CSCs may prove a therapeutic option for advanced–stage OC patients. Citation Format: Nuzhat Ahmed, Emily Chan, Chantel Samardzija, Khalid Abubaker, Ardian Latifi, George Kannourakis & Jock Findlay. CANCER STEM CELLS: THE SEEDS FOR RECURRENT OVARIAN CANCER [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr DPOC-001.

Cancer of the ovary is the leading cause of death for women with gynecologic malignancies. Over 90% of ovarian cancers derive from malignant transformation of the ovarian surface epithelium (OSE), from which cells disseminate into the peritoneal cavity, invade locally or spread via lymphatics, resulting in ascites formation and abdominal distension. Whereas early-stage ovarian cancer is curable in >90% of patients, it is asymptomatic, and most patients are diagnosed with advanced disease that carries a poor prognosis. Current tools for early detection of ovarian cancer are limited: neither ovarian palpation, transvaginal ultrasonography nor serum CA125 levels have sufficient sensitivity or specificity for general screening. Among candidate biomarkers for ovarian cancer are MUC1, claudin-3, or lysophosphatidic acid (LPA), which may be elevated in malignant ascites or even serum of ovarian cancer patients. Experimentally, LPA has been shown to stimulate mitogenic signaling cascades (Fig. 1), which may be mediated, at least in part, through metalloprotease-induced cleavage of EGF-like growth factors, notably heparin-binding epidermal growth factor–like growth factor (HB-EGF), that activate the epidermal growth factor receptor (EGFR) and downstream signaling pathways, a phenomenon termed transactivation (ref. 1; Fig. 2). The pathophysiologic role of LPA or EGF-like ligands and their receptors in ovarian carcinogenesis is poorly understood. In this issue of Clinical Cancer Research, Tanaka et al. have extended their previous studies on the expression and clinical significance of molecules involved in signaling through the LPA and EGFR axes (2–4) in ovarian cancer. Together, their findings suggest a pathophysiologic role for LPA-induced a disintegrin and metalloprotease-17 (ADAM-17/TACE)–mediated HB-EGF cleavage and EGFR transactivation in ovarian cancer, identifying HB-EGF as a prognostic marker and promising therapeutic target (5). This commentary will discuss the biology of both LPA and EGFR-associated signaling in ovarian physiology and cancer from the perspective of EGFR transactivation.LPA has emerged as an important intercellular signaling molecule implicated not only in physiologic processes such as brain development or angiogenesis but also in the pathophysiology of cancer promotion (6, 7). Being generated through hydrolysis of lysophosphatidyl choline by lysophospholipase D/autotaxin or via hydrolysis of phosphatidic acid by phospholipase A2 or A1, LPA is present and bioactive in numerous extracellular fluids, including serum, ascites or malignant effusions. Binding of LPA to one of at least four different heptahelical transmembrane G protein–coupled receptors (GPCR; LPA1/endothelial differentiation gene (Edg)2, LPA2/Edg4, LPA3/Edg7, and LPA4/GPR23/P2Y9) results in activation of at least three distinct G protein subfamilies (Gq, Gi, G12/13) and initiation of multiple signaling pathways, including Ras/Raf/mitogen-activated protein kinase, phosphoinositide-3-kinase/Akt, phospholipase C/protein kinase C, or RhoA small GTPase signaling (Fig. 1). Subsequent activation of cell surface metalloproteases such as members of the ADAM family may induce cleavage of EGF-like ligand precursors and autocrine or paracrine stimulation of the human EGFR (HER/erbB) family of transmembrane tyrosine kinases (Fig. 2). This transactivation process seems to involve multiple signaling pathways, including mitogen-activated protein kinase (p38 and p44/42), protein kinase C or c-Src, and may be amplified through additional mechanisms, such as LPA-induced production of interleukin-8. Moreover, there seems to exist a signal amplification loop through activation of protein kinase C, which up-regulates LPA production. Thus, LPA induces cancer cell proliferation, survival, drug resistance, invasion, opening of intercellular tight junctions and gap junction closure, cell migration or metastasis (Fig. 1). Membrane-bound lipid ectophosphatases of the LPP family rapidly inactivate LPA by converting it to monoacylglycerol. LPA signaling may further be attenuated through receptor desensitization and ligand-induced receptor internalization, although the fate of internalized LPA receptors—degradation versus recycling—remains to be elucidated (7). Recently, LPA has also been suggested as an intracellular messenger. Although glycerolipid synthesis at the endoplasmatic reticulum or the outer mitochondrial membrane continuously involves LPA production, the kinetics of further acylation to phosphatidic acid are high, and LPA accumulation seems to be negligible at these sites. More interestingly, LPA has been shown to bind the lipid-inducible transcription factor PPARγ and induce vascular remodeling, suggesting that LPA actions may in part be independent of classical LPA receptors (Fig. 2). However, it remains unclear how the charged phospholipid LPA may traverse the plasma membrane and translocate into the nucleus in vivo, or conversely if PPARγ is activated in an indirect manner.EGFR signaling is involved in the regulation of a myriad of biological and pathophysiologic processes including proliferation, differentiation, apoptosis, angiogenesis, or metastasis and is a convergence point for diverse signaling pathways (8). Overactivity of the receptor tyrosine kinase is considered a hallmark of malignancy and has been associated with poor prognosis for cancer patients. Ligands of the EGF superfamily are synthesized as transmembrane precursors, from which mature growth factors are released through metalloprotease-mediated cleavage of the ectodomain (9). These ligands bind to and activate one or more of the four human EGFRs designated ErbB-1 (EGFR), ErbB-2 (HER-2/neu), ErbB-3, and ErbB-4, resulting in receptor homo- and heterodimerization, a necessary prelude to receptor phosphorylation and activation of three major intracellular pathways: Ras/Raf/mitogen-activated protein kinase, phosphoinositide-3-kinase/Akt, and phospholipase C/protein kinase C. Additionally, multiple other signaling molecules are stimulated; these include c-Src, the STAT family of transcription factors as well as Rho, Rac and Cdc42 small GTPases (ref. 10; Fig. 2).The EGF-like ligand HB-EGF is expressed in a wide variety of cells and has been implicated in numerous physiologic or pathologic processes, including malignant tumor growth, where its expression has been correlated with survival in various cancers of epithelial origin. Although membrane-bound ligands can activate EGFR in adjacent cells and induce proliferation in a process called juxtacrine stimulation, metalloprotease-induced shedding seems to be critical for the majority of EGFR-related processes, including GPCR-mediated EGFR transactivation (11). Recent results confirm distinct roles for soluble versus membrane-associated HB-EGF. Whereas the membrane-anchored proform promotes cell interactions and decreases migration, soluble HB-EGF induces opposite effects, promoting a transformed phenotype in terms of proliferative rates, colony-forming ability, activation of the cyclin D1 promoter or induction of matrix metalloproteases (MMP). Likewise, LPA-induced ectodomain shedding of HB-EGF has been shown to promote tumor formation of ovarian cancer cells in nude mice (5). The regulated proteolytic cleavage of proHB-EGF yields at least two fragments: the amino-terminal soluble ligand for EGFR as well as the carboxyl-terminal cell-associated remnant, HB-EGF-c. The latter has recently been shown to translocate to the nucleus, where it interacts with the promyelocytic leukemia zinc finger (PLZF) transcriptional repressor, resulting in CRM1-dependent nuclear export and functional loss of PLZF. Thus, HB-EGF shedding results in dual intracellular signaling that is in part independent of EGFR activation (Fig. 2).Proteolytic processing of EGFR ligands and their receptors is a key regulatory switch in EGFR signaling (12). Enzymes implicated in HB-EGF shedding seem to be cell/tissue type-, localization- and/or stimulus-specific; they include MMP-3/stromelysin-1, MMP-7/matrilysin, and the ADAM family members ADAM-9/meltrin-γ, ADAM-10/kuzbanian, ADAM-12/meltrin-α, and ADAM-17/TACE. ADAMs may be activated through removal of their pro-domain by a furin-type pro-protein convertase or through autocatalysis in the trans-Golgi network; additionally, proteins interacting with the cytoplasmic tails of ADAMs may influence their activity. To date, bona fide enzymes implicated in LPA-induced HB-EGF cleavage are ADAM-10 and ADAM-17 (13, 14). Importantly, tissue inhibitors of metalloproteinases can counteract the catalytic activity of various ADAMs, and the balance between the level of active metalloproteases and tissue inhibitors of metalloproteinases may determine the net metalloprotease activity in a cellular microenvironment (15).Metalloprotease-dependent release of EGF-like ligands may transactivate the EGFR in response to multiple stimuli, including cytokines, osmotic stressors, phorbol 12-myristate 13-acetate or GPCR activation (1). Besides LPA, numerous other GPCR agonists have been implicated in this process (including angiotensin-II or insulin-like growth factor I), and a role for multiple metalloproteases (including ADAM-9, -10, -12, -15, -17, or MMP-9) and EGF-like ligands (predominantly HB-EGF but also transforming growth factor-α or amphiregulin) has been suggested (16). Multiple signaling pathways seem to control ligand shedding in response to diverse stimuli, including p38 and p44/42 mitogen-activated protein kinase, protein kinase C or c-Src. A major signaling route of LPA is Gi-mediated activation of Ras and the downstream Raf/mitogen-activated protein kinase cascade in a tyrosine kinase-dependent fashion (Fig. 1). The precise role of receptor tyrosine kinases in LPA receptor-mediated Ras activation, however, is controversial with recent evidence suggesting that these receptor types may independently control distinct signaling cascades leading to Ras activation (17). Indeed, EGFR downstream pathways overlap with prototypical LPA signal transduction, and cross-talk between these cascades may occur at various levels, which may or may not involve ligand-induced receptor transactivation (Fig. 2).LPA and LPA receptors. In the normal ovary, LPA is present and active in follicular fluid, suggesting its local synthesis and physiologic function. LPA had first been related to carcinomatous growth in the mid-1990s, when excess concentrations were found in ascites of ovarian cancer patients. Although the mechanism of LPA production in ovarian cancer has not been fully identified, both lysophospholipase D/autotaxin and phospholipase A2 seem to play a role. Autotaxin and its major substrate LPC are widely produced in the human body and are abundantly present in plasma. Both are also generated in excess by a variety of human cancers and have been found in the supernatant of transformed cells, including ovary-derived cells, most likely as part of secreted microvesicles. Thus, not surprisingly, mammalian serum contains high levels of bioactive LPA. Several studies have reported 10-fold increases of serum-LPA in ovarian cancer patients compared with healthy subjects or other cancer conditions, suggesting its use as a biomarker for ovarian cancer screening, early detection, or prognosis prediction. Discrepancies between these and opposite findings that fail to establish a correlation between LPA plasma levels and carcinomatous growth may be attributable in part to differences in sample handling and/or the method of detection employed. Whether accumulation of LPA in the tumor microenvironment constitutes the major mechanism through which autotaxin might promote tumor growth and angiogenesis or whether autotaxin might exert LPA-independent effects in vivo remains to be determined. LPA alone exerts similar trophic effects as total ascites fluid from ovarian cancer patients when added to tumor cells, inducing proliferation, survival, metastasis, or cisplatinum resistance. Conversely, expression of a lipid phosphatase that hydrolyzes LPA may reduce survival, growth and tumorigenesis of ovarian cancer cells (18). Interestingly, whereas normal OSE cells only express significant amounts of the LPA receptors LPA1/Edg2 and LPA4/GPR23, LPA2/Edg4 and LPA3/Edg7 are abnormally expressed in ovarian cancer cells, suggesting differential functions for these receptors—alone or in combination—in ovarian physiology and pathology. Recently, LPA has been shown to induce ectodomain shedding of HB-EGF essential for tumor formation of ovarian cancer cells in nude mice (5), underscoring the role of this lysophospholipid in ovarian carcinogenesis.Metalloproteases. ADAMs and MMPs are widely expressed in the human body and have been found up-regulated or decreased in various cancers; both metalloprotease families may be induced by LPA. Dynamic equilibrium between metalloproteases and their inhibitors (tissue inhibitors of metalloproteinases) plays an important role in ovarian physiology (follicular growth and ovulation), and ovarian cancer cells may exploit that proteolytic potential to promote degradation of extracellular matrix components and release membrane-anchored growth factors, their receptors or adhesion molecules. Metalloproteases may thus play a role in both, initiation or termination of mitogenic signaling (19). Interestingly, metalloproteinases may be up-regulated by expression of their substrates or related enzymes (e.g., overexpression of HB-EGF or MMP-7 may induce MMP-3; ref. 20). The concomitant up-regulation of ADAM-17 and its substrate HB-EGF in ovarian cancer observed by Nakano's group in this issue of Clinical Cancer Research (2) is intriguing and warrants further validation at a cell-biological level.EGF-like ligands and their receptors. Numerous tumors overexpress the EGFR, its heterodimeric partner ErbB-2 and/or its ligands, including up to 75% of ovarian cancers, and accumulating evidence underscores the importance of EGF-like ligands for malignant transformation of ovarian epithelial cells. The prognostic impact of EGFR expression in ovarian cancer, however, remains controversial. As for HB-EGF, previous studies have reported this ligand in the stroma but not in the surface epithelium of the normal ovary (21). The fact that Tanaka et al. did not detect HB-EGF in the normal ovary might be attributable to differences in antibody sensitivity and/or specificity. Nakano's group had previously reported increased HB-EGF concentrations in the peritoneal fluid from ovarian cancer patients (4). They now relate these findings to abnormal expression of HB-EGF in malignant OSE cells (mRNA/fluorescence in situ hybridization) that express ADAM-17 (2) and in the surrounding interstitial tissue (protein/immunohistochemistry), indicating that HB-EGF produced from malignant OSE cells is constitutively secreted into the microenvironment. Interestingly, the tetraspanin CD9 has previously been detected on OSE cells only, and association of proHB-EGF with CD9 has been shown to increase HB-EGF's juxtacrine activity and cytoprotective capacity in epithelial cell models. Thus, aberrant expression of HB-EGF in OSE cells might entail interactions with different cell surface proteins than those present in stromal cells, resulting in distinct biological functions. Increasing evidence suggests the organization of metalloproteases, ligand growth factors and/or their receptors into preformed complexes within membrane microdomains and shed membrane vesicles (22). Examples include the interaction of CD9 with HB-EGF and ADAM-10 in complexes that mediate EGFR transactivation by GPCRs (14) or the association of CD44 with HB-EGF, its sheddase MMP-7 and its receptor erbB4 on the surface of tumor cell lines (23). A detailed study of the colocalization of HB-EGF and distinct ADAM proteins, especially ADAM-10, in ovarian cancer tissue is therefore warranted to judge the physiologic relevance of the presumed interaction. Furthermore, important insights into the (patho-) physiology of both GPCR and EGFR signaling axes may be gained through studies of their polarized distribution and function in normal epithelia and during loss of polarity characteristic of malignant transformation.Drugability. The link between LPA and ovarian carcinogenesis has been mostly correlative, such as elevated levels of LPA in serum or ascites fluid and in vitro studies with exogenous LPA. The study by Tanaka et al. in this issue of Clinical Cancer Research complements their previous observations, now suggesting a functional role for LPA-induced, ADAM-17-mediated HB-EGF cleavage in ovarian cancer. This first laudable dispatch raises several questions: what is the role of other membrane-anchored or soluble metalloproteases (in particular ADAM-10, ADAM-15, or MMPs) in ovarian cancer? Will cell-permeable metalloprotease inhibitors be required to block metalloprotease action in preformed intracellular complexes with their substrates? Is loss of polarity and aberrant access of LPA to its receptors of importance? Will specific inhibitors of LPA and/or erbB receptors and/or other components of the GPCR/EGFR signaling axes (e.g., metalloproteases or HB-EGF) stand the test of clinical application? Thus far, metalloprotease- or erbB receptor-targeted agents have exhibited only limited activity in ovarian cancer patients. Now, in the light of functional redundancy and incomplete overlap of LPA and EGFR signaling, the additional exploration of LPA receptors as novel drug targets seems promising. What about other signaling events? Abundant crosstalk between diverse mitogenic pathways such as EGFR, insulin-like growth factor receptor or estrogen receptor signaling has been reported, and these interactions will have to be considered in our efforts to combat the disease. The stage is set for more mechanistic studies into the complex pathophysiology of ovarian cancer, which, hopefully, will advance to effective treatment strategies that are urgently needed in the clinics.We apologize to all colleagues, whose original work could not be cited due to strict space limitations. Interested readers are referred to the references indicated in the mentioned reviews. We thank Jeffrey L. Franklin for critical review of the manuscript.

BackgroundCurrent treatment of ovarian cancer patients with chemotherapy leaves behind a residual tumor which results in recurrent ovarian cancer within a short time frame. We have previously demonstrated that a single short-term treatment of ovarian cancer cells with chemotherapy in vitro resulted in a cancer stem cell (CSC)-like enriched residual population which generated significantly greater tumor burden compared to the tumor burden generated by control untreated cells. In this report we looked at the mechanisms of the enrichment of CSC-like residual cells in response to paclitaxel treatment.MethodsThe mechanism of survival of paclitaxel-treated residual cells at a growth inhibitory concentration of 50% (GI50) was determined on isolated tumor cells from the ascites of recurrent ovarian cancer patients and HEY ovarian cancer cell line by in vitro assays and in a mouse xenograft model.ResultsTreatment of isolated tumor cells from the ascites of ovarian cancer patients and HEY ovarian cancer cell line with paclitaxel resulted in a CSC-like residual population which coincided with the activation of Janus activated kinase 2 (JAK2) and signal transducer and activation of transcription 3 (STAT3) pathway in paclitaxel surviving cells. Both paclitaxel-induced JAK2/STAT3 activation and CSC-like characteristics were inhibited by a low dose JAK2-specific small molecule inhibitor CYT387 (1 μM) in vitro. Subsequent, in vivo transplantation of paclitaxel and CYT387-treated HEY cells in mice resulted in a significantly reduced tumor burden compared to that seen with paclitaxel only-treated transplanted cells. In vitro analysis of tumor xenografts at protein and mRNA levels demonstrated a loss of CSC-like markers and CA125 expression in paclitaxel and CYT387-treated cell-derived xenografts, compared to paclitaxel only-treated cell-derived xenografts. These results were consistent with significantly reduced activation of JAK2 and STAT3 in paclitaxel and CYT387-treated cell-derived xenografts compared to paclitaxel only-treated cell derived xenografts.ConclusionsThis proof of principle study demonstrates that inhibition of the JAK2/STAT3 pathway by the addition of CYT387 suppresses the ‘stemness’ profile in chemotherapy-treated residual cells in vitro, which is replicated in vivo, leading to a reduced tumor burden. These findings have important implications for ovarian cancer patients who are treated with taxane and/or platinum-based therapies.

Growth-regulated oncogene alpha (GROalpha), a member of the chemokine superfamily, is commonly expressed in transformed cells and contributes to angiogenesis and tumorigenesis. Here, we report that increased GROalpha levels are detected in the plasma and ascites of ovarian cancer patients. Ovarian cancer cell lines in culture express and secrete GROalpha. However, when they are starved in serum-free medium, ovarian cancer cells ceased producing GROalpha, suggesting that GROalpha is not constitutively expressed but rather is produced in response to exogenous growth factors in ovarian cancer cells. The prototype peptide growth factors present in serum such as platelet-derived growth factor, insulin-like growth factor I, and insulin do not stimulate GROalpha production by ovarian cancer cells. In contrast, lysophosphatidic acid (LPA), a glycerol backbone phospholipid mediator present in serum and ascites of ovarian cancer patients, is a potent inducer of GROalpha expression in ovarian cancer cell lines. Treatment of ovarian cancer cells with LPA leads to transcriptional activation of the GROalpha gene promoter and robust accumulation of GROalpha protein in culture supernatants. The action of LPA on GROalpha expression is mediated by LPA receptors, particularly the LPA(2) receptor in that ectopic expression of these receptors restores the LPA-dependent GROalpha production in nonresponsive cells. Down-regulation of LPA(2) expression by small interfering RNA (siRNA) in ovarian cancer cells desensitizes GROalpha production in response to LPA. The effect of serum on GROalpha production is also significantly decreased by siRNA inhibition of LPA(2) expression. These studies identify LPA as a primary regulator of GROalpha expression in ovarian cancer.

Epithelial ovarian cancer is the fourth most common cause of cancer death among women in the developed world. Currently only 30% of ovarian cancer patients remain free from disease long term. Paclitaxel is an integral component of primary therapy for ovarian cancer, but less than half of ovarian cancers respond to the drug. Enhancing the response to primary therapy with paclitaxel could improve outcomes for women with the disease. In recent years, we have identified several kinases that regulate the sensitivity of cancer cells to paclitaxel by modulating cancer metabolism, enhancing microtubule stability, inhibiting centrosome splitting or blocking AKT/survivin signaling. We performed siRNA kinome-screens to identify molecular targets whose decreased expression overcomes paclitaxel resistance and increases paclitaxel sensitivity in ovarian cancer cells. Upon assessing cell viability, we showed that 20% of the potential kinase targets whose knockdown modulates paclitaxel sensitivity participate in glucose and energy metabolism. Among these, a leading candidate was 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 2 (PFKFB2), an isoform of the glycolytic enzyme phosphofructokinase (PFK2). Many cancer cells depend on glucose for survival. Glycolysis involves ten metabolic reactions catalyzed by enzymes whose expression is frequently increased during malignant transformation. Many reports document the role of oncogenic signaling in regulating the activity of metabolic enzymes to support the enhanced macromolecule synthesis required for rapid proliferation of cancer cells. Cancer cells express altered levels of the different PFK-2/FBPase-2 isoenzymes and even modulate their relative kinase and bisphosphatase activities according to metabolic needs in a spatial and/or temporal manner. PFKFB2 is a bifunctional glycolytic enzyme that regulates the level of fructose-2,6-bisphosphate (Fru-2,6-BP) and is overexpressed in a fraction of ovarian and breast cancers. We found that knockdown of PFKFB2 inhibited clonogenic growth and enhanced paclitaxel sensitivity in ovarian and breast cancer cell lines with wild-type TP53 (wtTP553). Additionally, PFKFB2 siRNA or PFKFB2 shRNA significantly inhibited tumor growth and enhanced sensitivity to paclitaxel in xenografts derived from two ovarian cancer cell lines. Knockdown of PFKFB2 increased the rate of glycolysis, but decreased the flow of intermediates through the pentose-phosphate pathway in cancer cell lines with wtTP53, decreasing NADPH. Reactive oxygen species (ROS) accumulated after PFKFB2 knockdown, which stimulated phosphorylation of Janus kinase (JNK), induced G1 cell cycle arrest, and initiated apoptosis that depended upon upregulation of p21Cip1 and Puma. Thus, PFKFB2 is a glycolytic enzyme that drives tumor cell growth and enhances paclitaxel resistance by inhibiting TP53-dependent G1 cell cycle arrest and apoptosis. These findings highlight the interaction of cancer-altered metabolism with cell proliferation and chemosensitivity, which may provide a novel target in patients with ovarian and breast cancers where TP53 function remains intact. We also found that knockdown of kinases that regulate microtubule stability could sensitize ovarian cancer cells to paclitaxel treatment. In previous studies, baseline microtubule stability correlated with response to paclitaxel in ovarian cancer cell lines and both parameters could be enhanced by knockdown of individual kinases. Using the initial 14 kinase targets, we performed assays of microtubule stability, apoptosis, and cell cycle arrest with all possible pairs of kinase siRNA which enhance paclitaxel sensitivity across multiple cell lines and found that dual knockdown of IKBKB/STK39 had the greatest effect on enhancing paclitaxel stability. Our study documents the impact of sequentially silencing IKBKB and STK39 on paclitaxel sensitivity, providing a rationale for siRNA-based therapy. Different siRNAs are in the developmental pipeline for a variety of diseases including cancer, and more than a dozen are in phase I or II clinical trials. One of the most promising candidates to emerge from our kinase screen was salt inducible kinase 2 (SIK2), a serine/threonine kinase that is required for bipolar mitotic spindle formation and normal mitotic progression. With Dr. Ahmed Ahmed, we previously found knockdown of SIK2 induces polyploidy by blocking centrosome splitting and inhibits PI3K, sensitizing cells to paclitaxel. We demonstrated that knockdown of SIK2 inhibits xenograft growth and potentiates paclitaxel activity in vivo. Subsequently, we have partnered with Arrien Pharmaceuticals to test a small-molecule inhibitor of SIK2. We have found that novel SIK2 inhibitors, ARN-3236 and ARN3261, block the activity of SIK2 kinase and inhibit ovarian cancer cell growth, enhancing paclitaxel sensitivity in cultured cells and in xenografts. ARN-3261 will enter first-in-human trials at MD Anderson early next year. Taken together, our results support the development of small-molecule kinase inhibitors that modulate glycolysis, enhance microtubule stability, induce polyploidy, and inhibit AKT/survivin signaling, providing novel routes to enhance primary paclitaxel-based therapy for ovarian cancer and to improve patient outcomes. Citation Format: Zhen Lu. Kinase-mediated modulation of paclitaxel sensitivity in ovarian cancer. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr IA08.

Adenoviral vectors with E1A regulated by tumor-specific promoters are selectively cytolytic for breast cancer and melanoma.

Hsa_circ_0001535 is involved in biological processes in various tumors. However, the biological effects and related mechanism of hsa_circ_0001535 in ovarian cancer (OC) is unclear. This work is aimed to probe the biological function and underlying mechanism of hsa_circ_0001535 in OC, especially sponged with mi-RNA, require further elucidation. Hsa_circ_0001535 expression in OC tissues and cell lines were examined by qRT-PCR. Hsa_circ_0001535 overexpression model was constructed by lentivirus-mediatedtransfection in two OC cell lines, and the biological functions of hsa_circ_0001535 were evaluated by CCK-8, transwell assay and Western blot. Dual luciferase reporter gene assay was respectively used to explore the relationship between hsa_circ_0001535 and miR-593-3p, as well as miR-593-3p and PTEN. The expression of miR-593-3p and PTEN were detected by qRT-PCR in two OC cell lines and OC tissues. Hsa_circ_0001535 was down-regulated in OC tissues and cell lines. Hsa_circ_0001535 overexpression inhibited proliferation, migration and EMT marker expression in OC cells. Of interest, hsa_circ_0001535 targeted miR-593-3p and reduced its RNA level in OC cells. PTEN was a target gene of miR-593-3p, which was up-regulated by inhibiting miR-593-3p in OC cells. Furthermore, miR-593-3p mimic treatment reversed the up-regulation of PTEN by hsa_circ_0001535 overexpression in OC cells. The above results showed that hsa_circ_0001535 acted as a molecular sponge for miR-593-3p to repress miR-593-3p expression, and promoted the expression of PTEN, thus inhibited proliferation and migration of OC cells. Our research provides a potential therapeutic target for ovarian cancer patients.

Ovarian cancer is considered to be the most lethal type of gynecological cancer. During the advanced stages of ovarian cancer, an accumulation of ascites is observed. Fucosylation has been classified as an abnormal post-translational modification that is present in many diseases, including ovarian cancer. Ovarian cancer cells that are cultured with ascites stimulation change their morphology; concomitantly, the fucosylation process is altered. However, it is not known which fucosylated proteins are modified. The goal of this work was to identify the differentially fucosylated proteins that are expressed by ovarian cancer cell lines that are cultured with ovarian cancer patients' ascites. Aleuria aurantia lectin was used to detect fucosylation, and some changes were observed, especially in the cell membrane. Affinity chromatography and mass spectrometry (MALDI-TOF) were used to identify 6 fucosylated proteins. Four proteins (Intermediate filament family orphan 1 [IFFO1], PHD finger protein 20-like protein 1 [PHF20L1], immunoglobulin gamma 1 heavy chain variable region partial [IGHV1-2], and Zinc finger protein 224 [ZNF224]) were obtained from cell cultures stimulated with ascites, and the other two proteins (Peregrin [BRPF1] and Dystrobrevin alpha [DTNA]) were obtained under normal culture conditions. The fucosylated state of some of these proteins was further analyzed. The experimental results show that the ascites of ovarian cancer patients modulated the fucosylation process. The PHD finger protein 20-like protein 1, Zinc finger protein 224 and Peregrin proteins colocalize with fucosylation at different levels.

Interleukin-12-Mediated Tumoricidal Activity of Patient Lymphocytes in an Autologous in Vitro Ovarian Cancer Assay System

Simple SummaryOvarian cancer has the highest lethality among gynecological tumors. Therefore, it is essential to find reliable biomarkers to improve early detection. This is the first report describing ADAM17 detection in serum and ascites fluid of ovarian cancer patients. A high ADAM17 concentration in serum at primary diagnosis is associated with early FIGO stages and predicts complete resection of the tumor mass. In addition, ADAM17 and CA-125 complement each other, especially in the diagnosis of early stages. In summary, ADAM17 appears to be a promising screening marker for detecting early-stage ovarian cancer.Ovarian cancer has the highest mortality rate among gynecological tumors. This is based on late diagnosis and the lack of early symptoms. To improve early detection, it is essential to find reliable biomarkers. The metalloprotease ADAM17 could be a potential marker, as it is highly expressed in many solid tumors, including ovarian and breast cancer. The aim of this work is to evaluate the relevance of ADAM17 as a potential diagnostic blood-based biomarker in ovarian cancer. Ovarian cancer cell lines IGROV-1 and A2780, as well as primary patient-derived tumor cells obtained from tumor tissue and ascitic fluid, were cultured to analyze ADAM17 abundance in the culture supernatant. In a translational approach, a cohort of 117 well-characterized ovarian cancer patients was assembled and ADAM17 levels in serum and corresponding ascitic fluid were determined at primary diagnosis. ADAM17 was quantified by enzyme-linked immunosorbent assay (ELISA). In the present study, ADAM17 was detected in the culture supernatant of ovarian cancer cell lines and primary cells. In addition, ADAM17 was found in serum and ascites of ovarian cancer patients. ADAM17 level was significantly increased in ovarian cancer patients compared to an age-matched control group (p < 0.0001). Importantly early FIGO I/II stages, which would not have been detected by CA-125, were associated with higher ADAM17 concentrations (p = 0.007). This is the first study proposing ADAM17 as a serum tumor marker in the setting of a gynecological tumor disease. Usage of ADAM17 in combination with CA-125 and other markers could help detect early stages of ovarian cancer.

Long non-coding RNA (LncRNA) MALAT1 is an important regulatory molecule in many diseases, especiallyin ovarian cancer. We aimed at exploring the function of MALAT1 in ovarian cancer and at clarifying its mechanisms. The expression level of MALAT1 in ovarian cancer tissues, para-carcinoma tissues and ovarian cancer cell lines were analyzed by Real-time polymerase chain reaction (RT-PCR). The cell proliferation rate was detected by CCK8 assay in SKOV3 and HO8910 cells. Transwell was used to detect the invasion and migration activities in SKOV3 and HO8910 cells. The cell cycle distribution and apoptosis rate were measured by flow cytometry analysis. The expression level of Dvl2, GSK-3β, β-catenin and cyclin D1 were detected by RT-PCR and Western blot. The relative expression level of MALAT1 was identified to be aberrantly up-regulated in ovarian cancer tissues and cell lines. The high expression level of MALAT1 was associated with poor prognosis in ovarian cancer patients. The down-regulation of MALAT1 inhibited cell proliferation, invasion and migration, arrested cell cycle progression in S phase and induced cell apoptosis in ovarian cancer cell lines. Meanwhile, the down-regulation of MALAT1 decreased the expression level of DVL2, β-catenin and cyclin D1 and increased the expression level of GSK-3β in SKOV3 and HO8910 cells. Moreover, the inhibitory effect of MALAT1 down-regulation in cell invasion and migration was reversed by SKL2001 activating Wnt/β-catenin signal pathway and enhanced by XAV939 inhibiting Wnt/β-catenin signal pathway. MALAT1 was overexpressed in ovarian cancer and associated to the poor prognosis. The down-regulation of MALAT1 inhibited cell proliferation, invasion and migration, arrested cell cycle progression in S phase and induced cell apoptosis by restraining the activation of Wnt/β-catenin signaling pathway in ovarian cancer cells.

Treatment for malignant ascites in advanced ovarian cancer (OC) patients remains controversial. The objective of this study was to investigate the efficacy of combined continuous circulatory hyperthermic intraperitoneal chemotherapy (HIPEC) preceded or followed by cytoreductive surgery (CRS) for malignant ascites in OC patients. Female OC patients (n=32) with malignant ascites were divided based on stable (n=17) or unstable (n=15) vital signs. Stable patients were treated with CRS immediately followed by HIPEC (CRS+HIPEC). Unstable patients were treated using B-mode ultrasound-guided HIPEC followed by delayed CRS upon vital sign stability (HIPEC+dCRS). All patients were followed up until death or until December 2012. Median follow-up was 29months. All patients showed ascite regression [objective remission rates (ORR)=100%]. Among stable patients, CRS+HIPEC was successful in 14/17 (83.4%). Among unstable patients, HIPEC+dCRS was successful in 13/15 (86.7%). Median survival times were 19 and 17months in the stable and unstable groups, respectively. No significant differences in CRS rates, ascites ORR, Karnofsky performance status scores, or survival rates were observed between groups (P>0.05). Cytoreductive surgery with immediate HIPEC and HIPEC with dCRS, determined by vital sign stability, may lead to similar outcomes in OC patients with malignant ascites.

IL-15 super-agonist (ALT-803) enhances natural killer (NK) cell function against ovarian cancer

Adolescent ovarian cancer (OC) has high malignancy. Long non-coding RNAs (lncRNAs) have been implicated in the pathogenesis of various malignancies, but their role in adolescent OC remains poorly understood. This study aims to assess the modulatory role of Exosome-transmitted lncRNA Actin filament-associated protein 1 Antisense RNA 1 (AFAP1-AS1) on the activity of OC cells. We recruited a cohort of 40 adolescent patients diagnosed with OC and a control group of 40 healthy individuals. Serum samples were collected from both groups prior surgical intervention. Exosomes from peripheral blood and ascites were collected via differential centrifugation. The expression levels of AFAP1-AS1 in OC tissues and cell lines (IOSE-80, CAOV3, and SKOV3) were quantified using quantitative reverse-transcription polymerase chain reaction (qRT-PCR). The exosomal particle size and surface markers were characterized through nanoparticle tracking analysis and transmission electron microscopy. Furthermore, siRNA-mediated knockdown of AFAP1-AS1 was performed in IOSE-80, CAOV3, and SKOV3 cell lines. Functional assays, including wound-healing experiments and Transwell migration assays, were conducted to evaluate cellular migration and metastasis. Our findings demonstrate that the expression of AFAP1-AS1 is significantly upregulated in OC patients' serum exosomes and ascitic fluid, correlating with unfavorable pathological features such as advanced federation international of gynecology and obstetrics (FIGO) stage and larger tumor diameter. In-vitro experiments revealed that OC cell lines and primary human OC cells showed enhanced proliferation and metastasis when exposed to ascites-derived exosomes enriched in AFAP1-AS1. Importantly, we observed that AFAP1-AS1 can be transmitted to neighboring cells via exosomal pathways. Additionally, exosomes isolated from ascites treated with siRNA targeting AFAP1-AS1 can inhibit cellular migration and invasion. Our data provide evidence for the oncogenic role of AFAP1-AS1, which is transmitted via exosomes. This finding has significant implications for understanding the molecular mechanisms of AFAP1-AS1 in the pathogenesis of adolescent ovarian cancer.

Breast cancer is the most common cause and epithelial ovarian cancer the fourth most common cause of cancer death among women in the developed world. While breast cancer can be cured in 70% of cases, only 30% of ovarian cancer patients remain free from disease long term. Paclitaxel is an integral component of primary therapy for both forms of cancer, but less than half of breast and ovarian cancers respond to the drug. Enhancing the response to primary therapy with paclitaxel could improve outcomes for women with both diseases. In recent years several kinases have been identified that regulate the sensitivity of cancer cells to paclitaxel by inhibiting centrosome splitting or enhancing microtubule stability. Much less attention has been given to kinases that affect paclitaxel sensitivity by modulating cancer cell metabolism. We previously performed siRNA kinome-screens to identify molecular targets whose decreased expression overcomes paclitaxel resistance and increases paclitaxel sensitivity in ovarian cancer cells. We showed that 20% of the potential kinase targets whose knockdown modulates paclitaxel sensitivity participate in glucose and energy metabolism. Among these, a leading candidate was 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 2 (PFKFB2), an isoform of the glycolytic enzyme phosphofructokinase (PFK2). PFKFB2 is overexpressed in a fraction of ovarian and breast cancers. Knockdown of PFKFB2 inhibited clonogenic growth of ovarian and breast cancer cell lines and enhanced paclitaxel sensitivity in the cell lines with wt TP53. Liposome encapsulated PFKFB2 siRNA significantly inhibited tumor growth and enhanced sensitivity to paclitaxel in xenografts derived from ovarian cancer cell lines. Knockdown of PFKFB2 increased glycolysis, decreasing the flow of glycolytic intermediates to the pentose-phosphate pathway with reduced G6PD activity in wt TP53 cancer cell lines. With decreased NADPH, ROS accumulated after PFKFB2 knockdown, stimulating phosphorylation of JNK, inducing G1 cell cycle arrest, and initiating apoptosis dependent upon upregulation of p21Cip1 and Puma which are downstream targets of TP53. Our studies have shown for the first time that PFKFB2, a glycolytic enzyme, drives tumor cell growth and regulates paclitaxel sensitivity by inducing apoptosis and G1 cell cycle arrest. These findings highlight a remarkable degree of coordination between cancer metabolism with cell proliferation and chemo-sensitivity, which may provide a novel target in patients with ovarian cancers and breast cancers where TP53 function remains intact. Citation Format: Hailing Yang, Shu Zhang, Weiqun Mao, Ahmed A. Ahmed, Nicholas B. Jennings, Cristian Rodriguez-Aguayo, Gabriel Lopez-Berestein, Anil K. Sood, Xiao-Feng Le, Zhen Lu, Robert C. Bast. Silencing pfkfb2 enhances paclitaxel sensitivity by modulating metabolism of p53 wt ovarian and breast cancer cells and xenografts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3564. doi:10.1158/1538-7445.AM2017-3564