Kwai Wa Cheng

The RAB25 small GTPase determines aggressiveness of ovarian and breast cancers

High-density array comparative genomic hybridization (CGH) showed amplification of chromosome 1q22 centered on the RAB25 small GTPase, which is implicated in apical vesicle trafficking, in approximately half of ovarian and breast cancers. RAB25 mRNA levels were selectively increased in stage III and IV serous epithelial ovarian cancers compared to other genes within the amplified region, implicating RAB25 as a driving event in the development of the amplicon. Increased DNA copy number or RNA level of RAB25 was associated with markedly decreased disease-free survival or overall survival in ovarian and breast cancers, respectively. Forced expression of RAB25 markedly increased anchorage-dependent and anchorage-independent cell proliferation, prevented apoptosis and anoikis, including that induced by chemotherapy, and increased aggressiveness of cancer cells in vivo. The inhibition of apoptosis was associated with a decrease in expression of the proapoptotic molecules, BAK and BAX, and activation of the antiapoptotic phosphatidylinositol 3 kinase (PI3K) and AKT pathway, providing potential mechanisms for the effects of RAB25 on tumor aggressiveness. Overall, these studies implicate RAB25, and thus the RAB family of small G proteins, in aggressiveness of epithelial cancers.

Dual Inhibition of Tumor Energy Pathway by 2-Deoxyglucose and Metformin Is Effective against a Broad Spectrum of Preclinical Cancer Models

Tumor cell proliferation requires both growth signals and sufficient cellular bioenergetics. The AMP-activated protein kinase (AMPK) pathway seems dominant over the oncogenic signaling pathway suppressing cell proliferation. This study investigated the preclinical efficacy of targeting the tumor bioenergetic pathway using a glycolysis inhibitor 2-deoxyglucose (2DG) and AMPK agonists, AICAR and metformin. We evaluated the in vitro antitumor activity of 2DG, metformin or AICAR alone, and 2DG in combination either with metformin or AICAR. We examined in vivo efficacy using xenograft mouse models. 2DG alone was not sufficient to promote tumor cell death, reflecting the limited efficacy showed in clinical trials. A combined use of 2DG and AICAR also failed to induce cell death. However, 2DG and metformin led to significant cell death associated with decrease in cellular ATP, prolonged activation of AMPK, and sustained autophagy. Gene expression analysis and functional assays revealed that the selective AMPK agonist AICAR augments mitochondrial energy transduction (OXPHOS) whereas metformin compromises OXPHOS. Importantly, forced energy restoration with methyl pyruvate reversed the cell death induced by 2DG and metformin, suggesting a critical role of energetic deprivation in the underlying mechanism of cell death. The combination of 2DG and metformin inhibited tumor growth in mouse xenograft models. Deprivation of tumor bioenergetics by dual inhibition of energy pathways might be an effective novel therapeutic approach for a broad spectrum of human tumors. Mol Cancer Ther; 10(12); 2350–62. ©2011 AACR.

Identification of a Phosphothionate Analogue of Lysophosphatidic Acid (LPA) as a Selective Agonist of the LPA3 Receptor

Lysophosphatidic acid (LPA) is a bioactive lysophospholipid mediator that acts through G protein-coupled receptors. Most cell lines in culture express one or more LPA receptors, making it difficult to assign a response to specific LPA receptors. Dissection of the signaling properties of LPA has been hampered by lack of LPA receptor subtype-specific agonists and antagonists. The present study characterizes an ester-linked thiophosphate derivative (1-oleoyl-2-O-methyl-rac-glycerophosphothionate, OMPT) of LPA. OMPT is a functional LPA analogue with potent mitogenic activity in fibroblasts. In contrast to LPA, OMPT does not couple to the pheromone response through the LPA1 receptor in yeast cells. OMPT induces intracellular calcium increases efficiently in LPA3 receptor-expressing Sf9 cells but poorly in LPA2 receptor-expressing cells. Guanosine 5′-O-(3-[35S]thio)triphosphate binding assays in mammalian cells showed that LPA exhibits agonistic activity on all three LPA receptor subtypes, whereas OMPT has a potent agonistic effect only on the LPA3 receptor. In transiently transfected HEK293 cells, OMPT stimulates mitogen-activated protein kinases through the LPA3 but not the LPA1 or LPA2receptors. Furthermore, OMPT-induced intracellular calcium mobilization in mammalian cells is efficiently inhibited by the LPA1/LPA3 receptor-selective antagonist VPC12249. These results establish that OMPT is an LPA3-selective agonist. OMPT binding to the LPA3 receptor in mammalian cells is sufficient to elicit multiple responses, including activation of G proteins, calcium mobilization, and activation of mitogen-activated protein kinases. Thus OMPT offers a powerful probe for the dissection of LPA signaling events in complex mammalian systems.

Amplification of MDS1/EVI1 and EVI1, Located in the 3q26.2 Amplicon, Is Associated with Favorable Patient Prognosis in Ovarian Cancer

Increased copy number involving chromosome 3q26 is a frequent and early event in cancers of the ovary, lung, head and neck, cervix, and BRCA1 positive and basal breast cancers. The p110alpha catalytic subunit of phosphoinositide-3-kinase (PI3KCA) and protein kinase Ciota (PKCiota) have previously been shown as functionally deregulated by 3q copy number increase. High-resolution array comparative genomic hybridization of 235 high-grade serous epithelial ovarian cancers using contiguous bacterial artificial chromosomes across 3q26 delineated an approximately 2 Mb-wide region at 3q26.2 encompassing PDCD10 to MYNN (chr3:168722613-170908630). Ecotropic viral integration site-1 (EVI1) and myelodysplastic syndrome 1 (MDS1) are located at the center of this region, and their DNA copy number increases are associated with at least 5-fold increased RNA transcript levels in 83% and 98% of advanced ovarian cancers, respectively. Moreover, MDS1/EVI1 and EVI1 protein levels are increased in ovarian cancers and cancer cell lines. EVI1 and MDS1/EVI1 gene products increased cell proliferation, migration, and decreased transforming growth factor-beta-mediated plasminogen activator inhibitor-1 promoter activity in ovarian epithelial cells. Intriguingly, the increases in EVI1 DNA copy number and MDS1/EVI1 transcripts are associated with improved patient outcomes, whereas EVI1 transcript levels are associated with a poor patient survival. Thus, the favorable patient prognosis associated with increased DNA copy number seems to be as a result of high-level expression of the fusion transcript MDS1/EVI1. Collectively, these studies suggest that MDS1/EVI1 and EVI1, previously implicated in acute myelogenous leukemia, contribute to the pathophysiology of epithelial ovarian cancer.

Regulation of Gonadotropin-Releasing Hormone and Its Receptor Gene Expression by 17β-Estradiol in Cultured Human Granulosa-Luteal Cells

There is evidence that GnRH and its binding sites are expressed in numerous extrapituitary tissues, including the primate ovary. However, the factors that regulate ovarian GnRH and its receptor (GnRH-R) remain poorly characterized. Since gonadal steroids are key regulators of ovarian functions, the present study investigated the role of 17β-estradiol (E2) in regulating GnRH and GnRH-R messenger RNA (mRNA) from human granulosa-luteal cells (hGLCs). RT-PCR was used to isolate the ovarian GnRH-R transcript equivalent to the full-length coding region in the pituitary from hGLCs. Sequence analysis revealed that the ovarian GnRH-R mRNA is identical to its pituitary counterpart. Basal expression studies indicated that GnRH and GnRH-R mRNA levels significantly increased with time in vitro, reaching levels of 160% and 170% on day 8 and 10 of culture, respectively (P < 0.05). Treatment with various concentrations of estradiol (1–100 nm) for 24 h resulted in a dose-dependent decrease (P < 0.05) in GnRH and GnRH-R mR...

Gonadotropin-releasing hormone activates mitogen-activated protein kinase in human ovarian and placental cells

Considering that the action of gonadotropin-releasing hormone (GnRH) may be mediated via different signaling pathways in extrapituitary tissues, in the present study we investigated the role of the human GnRH receptor (GnRHR) in activating mitogen-activated protein kinases (MAPKs), which regulate cell growth, division, and differentiation. The phosphorylation state of p44 and p42 MAPKs was examined using antibodies that distinguish phospho-p44/42 MAPK (P-MAPK, Thr(202)/Tyr(204)) from total p44/42 MAPK (T-MAPK, activated plus inactivated) in human ovarian and placental cells. Cell cultures were treated with various concentrations of a GnRH agonist, (D-Ala(6))-GnRH, for 5 min. (D-Ala(6))-GnRH stimulated a rapid activation of P-MAPK in human granulosa-luteal cells (hGLCs) and immortalized extravillous trophoblast (IEVT) cells. Interestingly, (D-Ala(6))-GnRH treatment of ovarian cancer (OVCAR-3) and placental carcinoma (JEG-3) cells induced a biphasic regulatory pattern in P-MAPK activity. In contrast, no change of T-MAPK levels was observed following addition of the GnRH agonist in the ovarian and placental cells examined. The physiological implication of MAPK activation by GnRH in the ovarian and placental cells was also investigated. Human GLCs were treated with (D-Ala(6))-GnRH for 24 h, and progesterone secretion was measured by an established RIA. (D-Ala(6))-GnRH induced a significant decrease in progesterone secretion with maximum inhibition (a 45% decrease over basal level) at 10(-7) M. This inhibitory effect was completely reversed by pretreatment with MAPK/ERK kinase 1 (MEK1) inhibitor (PD98059), suggesting the involvement of the MAPK pathway in hGLCs. Placental JEG-3 cells were treated with (D-Ala(6))-GnRH for 24 h, and betahCG mRNA level was measured using Northern blot analysis. (D-Ala(6))-GnRH stimulated the expression of betahCG mRNA to 160% of control value in JEG-3 cells. In contrast to the ovarian cells, pretreatment of JEG-3 cells with PD98059 failed to block the stimulatory effect of GnRH on betahCG mRNA level, suggesting that other signaling pathway(s) may play a more dominant role in GnRH-induced betahCG mRNA expression. To our knowledge, this is the first demonstration that (1) GnRH induces activation of the MAPK signaling pathway in normal and carcinoma cells of the human ovary and placenta, and (2) MAPK mediates the direct action of GnRH on progesterone production in hGLCs.

The Emerging Role of the RAB25 Small GTPase in Cancer

RAB25, a member of the rat sarcoma (RAS) family of small GTPase, has been implicated in the pathophysiology of ovarian, breast and other cancers. Its role in endosomal transport and recycling of cell‐surface receptors and signaling proteins presents a novel paradigm for the disruption of cellular pathways and promotion of tumor development and aggressiveness. Variations in structure and post‐translational modifications control the localization of RAS superfamily proteins to specific subcellular compartments and recruitment of downstream effectors, allowing these small GTPases to function as sophisticated modulators of a complex and diverse range of cellular processes. Here, we review the link between RAB25 and tumor development and current knowledge regarding its possible roles in cancer.

Expression of the messenger RNA for gonadotropin-releasing hormone and its receptor in human cancer cell lines

The presence of gonadotropin-releasing hormone (GnRH) binding sites in biopsy samples of human epithelial ovarian cancer and ovarian tumor cell lines as well as the demonstration of the inhibitory effects of GnRH analogues on the growth of these cells raised the possibility that GnRH is produced locally by ovarian cancer cells. In order to investigate an autocrine/paracrine regulatory mechanism in human carcinomas, we have studied the expression of GnRH and GnRHR mRNA in human ovarian epithelial cell lines (OVCAR-3 and SKOV-3), human choriocarcinoma cell line (JEG-3) and human hepatocarcinoma cell line (HepG 2). Using primers corresponding to published human GnRH and GnRHR cDNA sequences, predicted PCR products were obtained from these cell lines by reverse transcription-polymerase chain reaction (RT-PCR) and confirmed by Southern hybridization. Sequencing analysis of GnRH PCR products showed that their sequences have 100% identity to the published human GnRH cDNA sequence. These results indicated that GnRH and GnRHR genes are expressed in all the cell lines tested in the present study, and strengthen the concept that GnRH may act as an autocrine regulator on the growth of cancer cells.

Rab25 increases cellular ATP and glycogen stores protecting cancer cells from bioenergetic stress

Cancer cells are metabolically stressed during tumour progression due to limited tumour vascularity and resultant nutrient, growth factor and oxygen deficiency that can induce cell death and inhibit tumour growth. We demonstrate that Rab25, a small GTPase involved in endosomal recycling, that is genomically amplified in multiple tumour lineages, is a key regulator of cellular bioenergetics and autophagy. RAB25 enhanced survival during nutrient stress by preventing apoptosis and autophagy via binding and activating AKT leading to increased glucose uptake and improved cellular bioenergetics. Unexpectedly, Rab25 induced the accumulation of glycogen in epithelial cancer cells, a process not previously identified. Strikingly, an increase in basal ATP levels combined with AKT‐dependent increases in glucose uptake and glycogen storage allowed maintenance of ATP levels during bioenergetic stress. The clinical relevance of these findings was validated by the ability of a Rab25‐dependent expression profile enriched for bioenergetics targets to identify patients with a poor prognosis. Thus, Rab25 is an unexpected regulator of cellular bioenergetics implicated as a useful biomarker and potential therapeutic target.

Differential expression of activin/inhibin subunit and activin receptor mRNAs in normal and neoplastic ovarian surface epithelium (OSE)

Ovarian surface epithelium (OSE) is the tissue of origin for the majority of ovarian cancers. The mechanism underlying the neoplastic transformation of OSE to ovarian cancer is poorly understood. Activin, a member of the transforming growth factor-beta superfamily, has been shown to increase cell proliferation in ovarian cancer cells. The present study was carried out to investigate the expression and regulation of activin/inhibin subunits and activin receptors in normal and neoplastic OSE. Using reverse transcriptase-polymerase chain reaction and Southern blot analysis, the mRNA levels of alpha, betaA and betaB subunits and activin receptor type IIA and IIB were analyzed in normal OSE and the ovarian cancer cell line, OVCAR-3 cells. The alpha and betaA subunits were highly expressed in normal OSE when compared to OVCAR-3 cells. By contrast, betaB subunit was highly expressed in OVCAR-3 cells, when compared to normal OSE cells. Interestingly, activin receptor IIB mRNA levels were significantly higher in OVCAR-3 when compared to normal OSE cells, whereas activin receptor IIA mRNA levels were the same in both cell types. To characterize the growth modulatory role of activin during neoplastic progression, normal OSE and OVCAR-3 cells were treated with recombinant human activin A (rh-activin A). At concentrations of 1,10 and 100 ng/ml, rh-activin A stimulated the growth of OVCAR-3 cells, but not of normal OSE. Treatment with follistatin, binding protein of activin, attenuates the stimulatory effect of activin. To determine whether the growth stimulatory action of activin in the neoplastic OSE is mediated via an autocrine regulatory mechanism, OVCAR-3 cells were treated with rh-activin A in a dose- and time-dependent manner and the expression levels of activin/inhibin subunits and activin receptors were investigated. Treatments with activin increased the alpha and betaA subunit mRNA levels in a dose- and time-dependent manner. However, no difference was observed in levels of betaB subunit, or in activin receptor type IIA and IIB mRNAs following activin treatments in OVCAR-3 cells. Taken together, these results suggest that different levels of activin/inhibin and activin receptor isoforms are expressed in normal and neoplastic OSE cells. In addition, the altered expression of the activin/inhibin subunits, as well as the cell proliferative effect of activin observed in OVCAR-3 but not in normal OSE cells, indicate that activin may act as an autocrine regulator of neoplastic OSE progression.