Ascitic fluid shear stress in concert with hepatocyte growth factor drive stemness and chemoresistance of ovarian cancer cells via the c-Met-PI3K/Akt-miR-199a-3p signaling pathway.

Overcoming drug resistance is an inevitable challenge to the success of cancer treatment. Recently, in ovarian cancer, a highly chemoresistant tumor, we demonstrated an important role of shear stress in stem-like phenotype and chemoresistance using a three-dimensional microfluidic device, which most closely mimics tumor behavior. Here, we examined a new mechanosensitive microRNA-miR-199a-3p. Unlike most key microRNA biogenesis in static conditions, we found that Dicer, Drosha, and Exportin 5 were not involved in regulating miR-199a-3p under ascitic fluid shear stress (0.02 dynes/cm2). We further showed that hepatocyte growth factor (HGF), but not other ascitic cytokines/growth factors such as epidermal growth factor and tumor necrosis factor α or hypoxia, could transcriptionally downregulate miR-199a-3p through its primary transcript miR-199a-1 and not miR-199a-2. Shear stress in the presence of HGF resulted in a concerted effect via a specific c-Met/PI3K/Akt signaling axis through a positive feedback loop, thereby driving cancer stemness and drug resistance. We also showed that miR-199a-3p expression was inversely correlated with enhanced drug resistance properties in chemoresistant ovarian cancer lines. Patients with low miR-199a-3p expression were more resistant to platinum with a significantly poor prognosis. miR-199a-3p mimic significantly suppressed ovarian tumor metastasis and its co-targeting in combination with cisplatin or paclitaxel further decreased the peritoneal dissemination of ovarian cancer in mice. These findings unravel how biophysical and biochemical cues regulate miR-199a-3p and is important in chemoresistance. miR-199a-3p mimics may serve as a novel targeted therapy for effective chemosensitization.

A Selective ß-Catenin-Metadherin/CEACAM1-CCL3 Axis Mediates Metastatic Heterogeneity upon Tumor-Macrophage Interaction.

Tumor heterogeneity plays a key role in cancer relapse and metastasis, however, the distinct cellular behaviors and kinetics of interactions among different cancer cell subclones and the tumor microenvironment are poorly understood. By profiling an isogenic model that resembles spontaneous human ovarian cancer metastasis with an highly metastatic (HM) and non-metastatic (NM) tumor cell pair, one finds an upregulation of Wnt/ß-catenin signaling uniquely in HM. Using humanized immunocompetent mice, one shows for the first time that activated ß-catenin acts nonautonomously to modulate the immune microenvironment by enhancing infiltrating tumor-associated macrophages (TAM) at the metastatic site. Single-cell time-lapse microscopy further reveals that upon contact with macrophages, a significant subset of HM, but not NM, becomes polyploid, a phenotype pivotal for tumor aggressiveness and therapy resistance. Moreover, HM, but not NM, polarizes macrophages to a TAM phenotype. Mechanistically, ß-catenin upregulates cancer cell surface metadherin, which communicates through CEACAM1 expressed on macrophages to produce CCL3. Tumor xenografts in humanized mice and clinical patient samples both corroborate the relevance of enhanced metastasis, TAM activation, and polyploidy in vivo. The results thus suggest that targeting the ß-catenin-metadherin/CEACAM1-CCL3 positive feedback cascade holds great therapeutic potential to disrupt polyploidization of the cancer subclones that drive metastasis.

Ginsenoside-Rg1 mediates a hypoxia-independent upregulation of hypoxia-inducible factor-1α to promote angiogenesis.

Hypoxia-inducible factor (HIF-1) is the key transcription regulator for multiple angiogenic factors and is an appealing target. Ginsenoside-Rg1, a nontoxic saponin isolated from the rhizome of Panax ginseng, exhibits potent proangiogenic activity and has the potential to be developed as a new angiotherapeutic agent. However, the mechanisms by which Rg1 promotes angiogenesis are not fully understood. Here, we show that Rg1 is an effective stimulator of HIF-1α under normal cellular oxygen conditions in human umbilical vein endothelial cells. HIF-1α steady-state mRNA was not affected by Rg1. Rather, HIF-1α protein synthesis was stimulated by Rg1. This effect was associated with constitutive activation of phosphatidylinositol 3-kinase (PI3K)/Akt and its effector p70 S6 kinase (p70(S6K)), but not extracellular-signal regulated kinase 1/2. We further revealed that HIF-1α induction triggered the expression of target genes, including vascular endothelial growth factor (VEGF). The use of small molecule inhibitors LY294002 or rapamycin to inhibit PI3K/Akt and p70(S6K) activities, respectively, resulted in diminished HIF-1α activation and subsequent VEGF expression. RNA interference-mediated knockdown of HIF-1α suppressed Rg1-induced VEGF synthesis and angiogenic tube formation, confirming that the effect was HIF-1α specific. Similarly, the angiogenic phenotype could be reversed by inhibition of PI3K/Akt and p70(S6K). These results define a hypoxia-independent activation of HIF-1α, uncovering a novel mechanism for Rg1 that could play a major role in angiogenesis and vascular remodeling.

Selectins: An Important Family of Glycan-Binding Cell Adhesion Molecules in Ovarian Cancer.

Ovarian cancer is the most lethal gynecological malignancy worldwide. Unlike most other tumor types that metastasize via the vasculature, ovarian cancer metastasizes predominantly via the transcoelomic route within the peritoneal cavity. As cancer metastasis accounts for the majority of deaths, there is an urge to better understand its determinants. In the peritoneal cavity, tumor-mesothelial adhesion is an important step for cancer dissemination. Selectins are glycan-binding molecules that facilitate early steps of this adhesion cascade by mediating heterotypic cell-cell interaction under hydrodynamic flow. Here, we review the function and regulation of selectins in peritoneal carcinomatosis of ovarian cancer, and highlight how dysregulation of selectin ligand biogenesis affects disease outcome. Further, we will introduce the latest tools in studying selectin-glycan interaction. Finally, an overview of potential therapeutic intervention points that may lead to the development of efficacious therapies for ovarian cancer is provided.

Sialyl Lewisx-P-selectin cascade mediates tumor-mesothelial adhesion in ascitic fluid shear flow.

Organ-specific colonization suggests that specific cell-cell recognition is essential. Yet, very little is known about this particular interaction. Moreover, tumor cell lodgement requires binding under shear stress, but not static, conditions. Here, we successfully isolate the metastatic populations of cancer stem/tumor-initiating cells (M-CSCs). We show that the M-CSCs tether more and roll slower than the non-metastatic (NM)-CSCs, thus resulting in the preferential binding to the peritoneal mesothelium under ascitic fluid shear stress. Mechanistically, this interaction is mediated by P-selectin expressed by the peritoneal mesothelium. Insulin-like growth factor receptor-1 carrying an uncommon non-sulfated sialyl-Lewisx (sLex) epitope serves as a distinct P-selectin binding determinant. Several glycosyltransferases, particularly α1,3-fucosyltransferase with rate-limiting activity for sLex synthesis, are highly expressed in M-CSCs. Tumor xenografts and clinical samples corroborate the relevance of these findings. These data advance our understanding on the molecular regulation of peritoneal metastasis and support the therapeutic potential of targeting the sLex-P-selectin cascade.

New insights into the role of soluble E-cadherin in tumor angiogenesis.

A key to successful metastasis is the formation of new vasculature, known as angiogenesis. Therefore, it is of great interest to unravel the underlying molecular mechanisms of tumor angiogenesis. Cadherins are a major class of cell surface receptors. The loss of cadherins, especially E-cadherin, is a well-established marker for tumor metastasis. Loss of E-cadherin is also a defining characteristic of several carcinomas, such as lobular carcinoma of the breast, and de-differentiated endometrioid carcinoma of the endometrium and ovary, which are known to be associated with more aggressive tumor behavior. Although E-cadherin is synthesized as a transmembrane molecule, its extracellular domain can be enzymatically cleaved off and released as a soluble E-cadherin (sE-cad), and this accounts for the loss of E-cadherin function or expression that has been implicated in tumor progression and metastasis. Importantly, sE-cad is present at high levels in the serum and malignant ascites of ovarian carcinoma patients. Nevertheless, little is known about how this essential protein dictates metastasis. Hitherto, many studies have given attention only to the dominant negative role of the loss of E-cadherin in weakening cell-cell adhesion, however, it is not known if sE-cad has biological activity in itself. In addition, the release mechanism of sE-cad has remained elusive. Here we show for the first time that sE-cad is a pivotal regulator of angiogenesis. We further show that exosomes are a novel major platform for the cleavage and release of sE-cad in vitro, in vivo and in patients' derived samples (Nat Commun, 9: 2270).

Soluble E-cadherin promotes tumor angiogenesis and localizes to exosome surface.

The limitations of current anti-angiogenic therapies necessitate other targets with complimentary mechanisms. Here, we show for the first time that soluble E-cadherin (sE-cad) (an 80-kDa soluble form), which is highly expressed in the malignant ascites of ovarian cancer patients, is a potent inducer of angiogenesis. In addition to ectodomain shedding, we provide further evidence that sE-cad is abundantly released in the form of exosomes. Mechanistically, sE-cad-positive exosomes heterodimerize with VE-cadherin on endothelial cells and transduce a novel sequential activation of ß-catenin and NFκB signaling. In vivo and clinical data prove the relevance of sE-cad-positive exosomes for malignant ascites formation and widespread peritoneal dissemination. These data advance our understanding of the molecular regulation of angiogenesis in ovarian cancer and support the therapeutic potential of targeting sE-cad. The exosomal release of sE-cad, which represents a common route for externalization in ovarian cancer, could potentially be biomarkers for diagnosis and prognosis.

Exosomes: Emerging biomarkers and targets for ovarian cancer.

The limitations of current chemotherapies have motivated research in developing new treatments. Growing evidence shows that interaction between tumors and their microenvironment, but not tumor cells per se, is the key factor in tumor progression and therefore of obvious scientific interest and therapeutic value. Exosomes are small (30-100 nm) extracellular vesicles which have emerged as key mediators of intercellular communication between tumor cells and major cell types in the tumor microenvironment such as fibroblasts, endothelial cells, and immune cells as well as noncellular extracellular matrices through paracrine mechanisms. This review is to highlight the emerging role of exosomes in particular types of cancer, such as ovarian cancer, owing to its unique route of metastasis, which is capable of rapidly translating exosome research for clinical applications in diagnosis, prognosis, and potential treatment.

BRCA1 deficiency induces protective autophagy to mitigate stress and provides a mechanism for BRCA1 haploinsufficiency in tumorigenesis.

Stress adaptation has profound impacts on malignant progression and response to treatment. BRCA1 is an important modulator of cellular stress, but our understanding of its mechanisms of action remains incomplete. Here we identify autophagy as an essential mechanism protecting BRCA1 deficient cancer cells from metabolic stress and allow their survival, which may underlie its significant cancer-promoting properties. We showed that targeted inhibition of endogenous BRCA1 using small interfering RNA caused significant autophagy in response to serum starvation and endoplasmic reticulum stress, whereas overexpression of BRCA1 did not, confirming that the effect was BRCA1 specific. We demonstrated that Beclin 1 was activated in BRCA1 deficient cells, suggesting involvement of a canonical pathway. Importantly, BRCA1 deficient cells were highly dependent on autophagy for survival, and rapidly underwent cell death upon disruption of autophagy. Notably, this dependence on protective autophagy extended to their tissue of origin, as ovarian surface epithelial cells from women testing positive for BRCA1 mutations, in contrast to those with no mutations, robustly induced autophagy to mitigate the stress and promote their survival. These findings highlight a novel role for BRCA1 in protective autophagy, which may make its essential contribution to tumorigenesis and prognosis.

c-Met overexpression contributes to the acquired apoptotic resistance of nonadherent ovarian cancer cells through a cross talk mediated by phosphatidylinositol 3-kinase and extracellular signal-regulated kinase 1/2.

Ovarian cancer is the most lethal gynecologic cancer mainly because of widespread peritoneal dissemination and malignant ascites. Key to this is the capacity of tumor cells to escape suspension-induced apoptosis (anoikis), which also underlies their resistance to chemotherapy. Here, we used a nonadherent cell culture model to investigate the molecular mechanisms of apoptotic resistance of ovarian cancer cells that may mimic the chemoresistance found in solid tumors. We found that ovarian cancer cells acquired a remarkable resistance to anoikis and apoptosis induced by exposure to clinically relevant doses of two front-line chemotherapeutic drugs cisplatin and paclitaxel when grown in three-dimensional than monolayer cultures. Inhibition of the hepatocyte growth factor (HGF) receptor c-Met, which is frequently overexpressed in ovarian cancer, by a specific inhibitor or small interfering RNA blocked the acquired anoikis resistance and restored chemosensitivity in three-dimensional not in two-dimensional cultures. These effects were found to be dependent on both phosphatidylinositol 3-kinase (PI3K)/Akt and extracellular signal-regulated kinase (ERK) 1/2 signaling pathways. Inhibitors of PI3K/Akt abrogated ERK1/2 activation and its associated anoikis resistance in response to HGF, suggesting a signaling relay between these two pathways. Furthermore, we identified a central role of Ras as a mechanism of this cross talk. Interestingly, Ras did not lie upstream of PI3K/Akt, whereas PI3K/Akt signaling to ERK1/2 involved Ras. These findings shed new light on the apoptotic resistance mechanism of nonadherent ovarian cancer ascites cells and may have important clinical implications.