Ernst Lengyel

The miR-200 family determines the epithelial phenotype of cancer cells by targeting the E-cadherin repressors ZEB1 and ZEB2

Cancer progression has similarities with the process of epithelial-to-mesenchymal transition (EMT) found during embryonic development, during which cells down-regulate E-cadherin and up-regulate Vimentin expression. By evaluating the expression of 207 microRNAs (miRNAs) in the 60 cell lines of the drug screening panel maintained by the Nation Cancer Institute, we identified the miR-200 miRNA family as an extraordinary marker for cells that express E-cadherin but lack expression of Vimentin. These findings were extended to primary ovarian cancer specimens. miR-200 was found to directly target the mRNA of the E-cadherin transcriptional repressors ZEB1 (TCF8/deltaEF1) and ZEB2 (SMAD-interacting protein 1 [SIP1]/ZFXH1B). Ectopic expression of miR-200 caused up-regulation of E-cadherin in cancer cell lines and reduced their motility. Conversely, inhibition of miR-200 reduced E-cadherin expression, increased expression of Vimentin, and induced EMT. Our data identify miR-200 as a powerful marker and determining factor of the epithelial phenotype of cancer cells.

Adipocytes promote ovarian cancer metastasis and provide energy for rapid tumor growth

Intra-abdominal tumors, such as ovarian cancer, have a clear predilection for metastasis to the omentum, an organ primarily composed of adipocytes. Currently, it is unclear why tumor cells preferentially home to and proliferate in the omentum, yet omental metastases typically represent the largest tumor in the abdominal cavities of women with ovarian cancer. We show here that primary human omental adipocytes promote homing, migration and invasion of ovarian cancer cells, and that adipokines including interleukin-8 (IL-8) mediate these activities. Adipocyte–ovarian cancer cell coculture led to the direct transfer of lipids from adipocytes to ovarian cancer cells and promoted in vitro and in vivo tumor growth. Furthermore, coculture induced lipolysis in adipocytes and β-oxidation in cancer cells, suggesting adipocytes act as an energy source for the cancer cells. A protein array identified upregulation of fatty acid–binding protein 4 (FABP4, also known as aP2) in omental metastases as compared to primary ovarian tumors, and FABP4 expression was detected in ovarian cancer cells at the adipocyte-tumor cell interface. FABP4 deficiency substantially impaired metastatic tumor growth in mice, indicating that FABP4 has a key role in ovarian cancer metastasis. These data indicate adipocytes provide fatty acids for rapid tumor growth, identifying lipid metabolism and transport as new targets for the treatment of cancers where adipocytes are a major component of the microenvironment.

Rethinking ovarian cancer: recommendations for improving outcomes.

There have been major advances in our understanding of the cellular and molecular biology of the human malignancies that are collectively referred to as ovarian cancer. At a recent Helene Harris Memorial Trust meeting, an international group of researchers considered actions that should be taken to improve the outcome for women with ovarian cancer. Nine major recommendations are outlined in this Opinion article.

Ovarian Cancer Development and Metastasis

The biology of ovarian carcinoma differs from that of hematogenously metastasizing tumors because ovarian cancer cells primarily disseminate within the peritoneal cavity and are only superficially invasive. However, since the rapidly proliferating tumors compress visceral organs and are only temporarily chemosensitive, ovarian carcinoma is a deadly disease, with a cure rate of only 30%. There are a number of genetic and epigenetic changes that lead to ovarian carcinoma cell transformation. Ovarian carcinoma could originate from any of three potential sites: the surfaces of the ovary, the fallopian tube, or the mesothelium-lined peritoneal cavity. Ovarian cacinoma tumorigenesis then either progresses along a stepwise mutation process from a slow growing borderline tumor to a well-differentiated carcinoma (type I) or involves a genetically unstable high-grade serous carcinoma that metastasizes rapidly (type II). During initial tumorigenesis, ovarian carcinoma cells undergo an epithelial-to-mesenchymal transition, which involves a change in cadherin and integrin expression and up-regulation of proteolytic pathways. Carried by the peritoneal fluid, cancer cell spheroids overcome anoikis and attach preferentially on the abdominal peritoneum or omentum, where the cancer cells revert to their epithelial phenotype. The initial steps of metastasis are regulated by a controlled interaction of adhesion receptors and proteases, and late metastasis is characterized by the oncogene-driven fast growth of tumor nodules on mesothelium covered surfaces, causing ascites, bowel obstruction, and tumor cachexia.

Whole–genome characterization of chemoresistant ovarian cancer

Patients with high-grade serous ovarian cancer (HGSC) have experienced little improvement in overall survival, and standard treatment has not advanced beyond platinum-based combination chemotherapy, during the past 30 years. To understand the drivers of clinical phenotypes better, here we use whole-genome sequencing of tumour and germline DNA samples from 92 patients with primary refractory, resistant, sensitive and matched acquired resistant disease. We show that gene breakage commonly inactivates the tumour suppressors RB1, NF1, RAD51B and PTEN in HGSC, and contributes to acquired chemotherapy resistance. CCNE1 amplification was common in primary resistant and refractory disease. We observed several molecular events associated with acquired resistance, including multiple independent reversions of germline BRCA1 or BRCA2 mutations in individual patients, loss of BRCA1 promoter methylation, an alteration in molecular subtype, and recurrent promoter fusion associated with overexpression of the drug efflux pump MDR1.

Let-7 expression defines two differentiation stages of cancer

The early phases of carcinogenesis resemble embryonic development, often involving the reexpression of embryonic mesenchymal genes. The NCI60 panel of human tumor cell lines can genetically be subdivided into two superclusters (SCs) that correspond to CD95 Type I and II cells. SC1 cells are characterized by a mesenchymal and SC2 cells by an epithelial gene signature, suggesting that SC1 cells represent less differentiated, advanced stages of cancer. miRNAs are small 20- to 22-nucleotide-long noncoding RNAs that inhibit gene expression at the posttranscriptional level. By performing miRNA expression analysis on 10 Type I and 10 Type II cells, we have determined that SC1 cells express low and SC2 cells high levels of the miRNA let-7, respectively, suggesting that let-7 is a marker for less advanced cancers. Expression of the let-7 target high-mobility group A2 (HMGA2), an early embryonic gene, but not of classical epithelial or mesenchymal markers such as E-cadherin or vimentin, inversely correlated with let-7 expression in SC1 and SC2 cells. Using ovarian cancer as a model, we demonstrate that expression of let-7 and HMGA2 is a better predictor of prognosis than classical markers such as E-cadherin, vimentin, and Snail. These data identify loss of let-7 expression as a marker for less differentiated cancer.

Rac1 in human breast cancer : overexpression, mutation analysis, and characterization of a new isoform, Rac1b

Rac1 is a member of the Ras superfamily of small guanosine triphosphatases (GTPases) that act as molecular switches to control cytoskeletal rearrangements and cell growth. Analogous to Ras, constitutively activating point mutations of Rac1 cause tumorigenic transformation of cell lines. However, there is no information about whether Rac1 is also mutated in vivo. After RT–PCR of Rac1, several clones of seven benign and 10 malignant breast cancer tissues as well as eight breast cancer cell lines were sequenced. Only single-nucleotide polymorphisms of Rac1 could be detected, and none of these corresponded to constitutively activating point mutations that have been used in cell lines for transformation. While sequencing Rac1 in breast tissues, a new Rac1 isoform with an insertion of 19 codons within the reading frame of Rac1 close to switch region II was identified and named Rac1b. The Rac1b protein acts like a fast cycling GTPase in GTP binding and hydrolysis assays. In Northern and Western blot experiments both Rac1 RNA and Rac1 protein had a significantly higher expression in breast cancer tissues compared to normal breast tissue samples. Immunohistochemical staining of Rac1 showed weak Rac1 expression in benign breast disease but high expression level in ductal carcinoma-in-situ, primary breast cancer, and lymph node metastases. In addition, breast tumor cells from patients with recurrent disease had Rac1 expression at the plasma membrane, suggesting activation of Rac1, in patients with aggressive breast cancer.

CD95 promotes tumour growth

CD95 (also called Fas and APO-1) is a prototypical death receptor that regulates tissue homeostasis mainly in the immune system through the induction of apoptosis. During cancer progression CD95 is frequently downregulated or cells are rendered apoptosis resistant, raising the possibility that loss of CD95 is part of a mechanism for tumour evasion. However, complete loss of CD95 is rarely seen in human cancers and many cancer cells express large quantities of CD95 and are highly sensitive to CD95-mediated apoptosis in vitro. Furthermore, cancer patients frequently have elevated levels of the physiological ligand for CD95, CD95L. These data raise the possibility that CD95 could actually promote the growth of tumours through its non-apoptotic activities. Here we show that cancer cells in general, regardless of their CD95 apoptosis sensitivity, depend on constitutive activity of CD95, stimulated by cancer-produced CD95L, for optimal growth. Consistently, loss of CD95 in mouse models of ovarian cancer and liver cancer reduces cancer incidence as well as the size of the tumours. The tumorigenic activity of CD95 is mediated by a pathway involving JNK and Jun. These results demonstrate that CD95 has a growth-promoting role during tumorigenesis and indicate that efforts to inhibit its activity rather than to enhance it should be considered during cancer therapy.

Loss of E-Cadherin Promotes Ovarian Cancer Metastasis via α5-Integrin, which Is a Therapeutic Target

E-cadherin loss is frequently associated with ovarian cancer metastasis. Given that adhesion to the abdominal peritoneum is the first step in ovarian cancer dissemination, we reasoned that down-regulation of E-cadherin would affect expression of cell matrix adhesion receptors. We show here that inhibition of E-cadherin in ovarian cancer cells causes up-regulation of alpha(5)-integrin protein expression and transcription. When E-cadherin was blocked, RMUG-S ovarian cancer cells were able to attach and invade more efficiently. This greater efficiency could, in turn, be inhibited both in vitro and in vivo with an alpha(5)beta(1)-integrin-blocking antibody. When E-cadherin is silenced, alpha(5)-integrin is up-regulated through activation of an epidermal growth factor receptor/FAK/Erk1-mitogen-activated protein kinase-dependent signaling pathway and not through the canonical E-cadherin/beta-catenin signaling pathway. In SKOV-3ip1 ovarian cancer xenografts, which express high levels of alpha(5)-integrin, i.p. treatment with an alpha(5)beta(1)-integrin antibody significantly reduced tumor burden, ascites, and number of metastasis and increased survival by an average of 12 days when compared with IgG treatment (P < 0.0005). alpha(5)-Integrin expression was detected by immunohistochemistry in 107 advanced stage ovarian cancers using a tissue microarray annotated with disease-specific patient follow-up. Ten of 107 tissues (9%) had alpha(5)-integrin overexpression, and 39% had some level of alpha(5)-integrin expression. The median survival for patients with high alpha(5)-integrin levels was 26 months versus 35 months for those with low integrin expression (P < 0.05). Taken together, we have identified alpha(5)-integrin up-regulation as a molecular mechanism by which E-cadherin loss promotes tumor progression, providing an explanation for how E-cadherin loss increases metastasis. Targeting this integrin could be a promising therapy for a subset of ovarian cancer patients.

The initial steps of ovarian cancer cell metastasis are mediated by MMP-2 cleavage of vitronectin and fibronectin

Most patients (80%) with ovarian cancer (OvCa) present with metastatic disease. Attachment of OvCa cells to peritoneum and omentum represents the first rate-limiting step for metastatic spread. Therefore, identifying factors regulating cell attachment in the abdominal cavity is critical to the development of therapeutic agents. We show here that MMP-2 expression was upregulated in OvCa cells upon attachment to their microenvironment. Downregulation of MMP-2 mRNA or pharmacological inhibition of MMP-2 proteolytic function, in both human OvCa primary cells and cell lines, reduced attachment of OvCa cells to a 3D organotypic model of metastatic OvCa, full human omentum or peritoneum, and in vivo to mouse peritoneum and omentum. Absence of MMP-2 in the host did not alter OvCa adhesion, as determined utilizing mice harboring homozygous null mutations in either the Mmp2 or Mmp9 genes. Conversely, adhesion induced upregulation of MMP-2 mRNA in OvCa cells. MMP-2 inhibition in OvCa cells through pharmacological or antibody treatment prior to i.p. dissemination in nude mice significantly decreased tumor growth and metastasis and extended survival. MMP-2 enhanced peritoneal adhesion of OvCa cells through cleavage of ECM proteins fibronectin (FN) and vitronectin (Vn) into small fragments and increased binding of OvCa cells to these FN and Vn fragments and their receptors, alpha5beta1 and alphaVbeta3 integrin. These findings indicate that MMP-2 expressed by metastatic OvCa cells functionally regulates their attachment to peritoneal surfaces.