Maureen P. Lynch

Induction of interleukin-8 preserves the angiogenic response in HIF-1alpha-deficient colon cancer cells.

Hypoxia inducible factor-1 (HIF-1) is considered a crucial mediator of the cellular response to hypoxia through its regulation of genes that control angiogenesis. It represents an attractive therapeutic target in colon cancer, one of the few tumor types that shows a clinical response to antiangiogenic therapy. But it is unclear whether inhibition of HIF-1 alone is sufficient to block tumor angiogenesis. In HIF-1α knockdown DLD-1 colon cancer cells (DLD-1HIF-kd), the hypoxic induction of vascular endothelial growth factor (VEGF) was only partially blocked. Xenografts remained highly vascularized with microvessel densities identical to DLD-1 tumors that had wild-type HIF-1α (DLD-1HIF-wt). In addition to the preserved expression of VEGF, the proangiogenic cytokine interleukin (IL)-8 was induced by hypoxia in DLD-1HIF-kd but not DLD-1HIF-wt cells. This induction was mediated by the production of hydrogen peroxide and subsequent activation of NF-κB. Furthermore, the KRAS oncogene, which is commonly mutated in colon cancer, enhanced the hypoxic induction of IL-8. A neutralizing antibody to IL-8 substantially inhibited angiogenesis and tumor growth in DLD-1HIF-kd but not DLD-1HIF-wt xenografts, verifying the functional significance of this IL-8 response. Thus, compensatory pathways can be activated to preserve the tumor angiogenic response, and strategies that inhibit HIF-1α may be most effective when IL-8 is simultaneously targeted.

Leptin Signaling Promotes the Growth of Mammary Tumors and Increases the Expression of Vascular Endothelial Growth Factor (VEGF) and Its Receptor Type Two (VEGF-R2)

To gain insight into the mechanism(s) by which leptin contributes to mammary tumor (MT) development we investigated the effects of leptin, kinase inhibitors, and/or leptin receptor antagonists (LPrA2) on 4T1 mouse mammary cancer cells in vitro and LPrA2 on 4T1-MT development in vivo. Leptin increases the expression of vascular endothelial growth factor (VEGF), its receptor (VEGF-R2), and cyclin D1 through phosphoinositide 3-kinase, Janus kinase 2/signal transducer and activator of transcription 3, and/or extracellular signal-activated kinase 1/2 signaling pathways. In contrast to leptin-induced levels of cyclin D1 the changes in VEGF or VEGF-R2 were more dependent on specific signaling pathways. Incubation of 4T1 cells with anti-VEGF-R2 antibody increased leptin-mediated VEGF expression suggesting an autocrine/paracrine loop. Pretreatment of syngeneic mice with LPrA2 prior to inoculation with 4T1 cells delayed the development and slowed the growth of MT (up to 90%) compared with controls. Serum VEGF levels and VEGF/VEGF-R2 expression in MT were significantly lower in mice treated with LPrA2. Interestingly, LPrA2-induced effects were more pronounced in vivo than in vitro suggesting paracrine actions in stromal, endothelial, and/or inflammatory cells that may impact the growth of MT. Although all the mechanism(s) by which leptin contributes to tumor development are unknown, it appears leptin stimulates an increase in cell numbers, and the expression of VEGF/VEGF-R2. Together, these results provide further evidence suggesting leptin is a MT growth-promoting factor. The inhibition of leptin signaling could serve as a potential adjuvant therapy for treatment of breast cancer and/or provide a new target for the designing strategies to prevent MT development.

HIF-1α and HIF-2α have divergent roles in colon cancer

Hypoxia‐inducible factor (HIF)‐1 and HIF‐2 are heterodimeric transcription factors that mediate the cellular response to hypoxia. Their key regulatory subunits, HIF‐1α and HIF‐2α, are induced similarly by hypoxia, but their functional roles in cancer may be distinct and isoform‐specific. SW480 colon cancer cells with stable expression of siRNA to HIF‐1α or HIF‐2α or both were established. HIF‐1α‐deficient cells displayed lower rates of proliferation and migration, but HIF‐2α‐deficient cells exhibited enhanced anchorage independent growth in a soft agar assay. Xenograft studies revealed that HIF‐1α deficiency inhibited overall tumor growth, whereas deficiency of HIF‐2α stimulated tumor growth. In human colon cancer tissues, expression of HIF‐1α and to a lesser extent, HIF‐2α, was linked to upregulation of VEGF and tumor angiogenesis. However, loss of expression of HIF‐2α but not HIF‐1α was strongly correlated with advanced tumor stage. DNA microarray analysis identified distinct sets of HIF‐1α and HIF‐2α target genes that may explain these phenotypic differences. Collectively, these findings suggest that HIF isoforms may have differing cellular functions in colon cancer. In particular, HIF‐1α promoted the growth of SW480 colon cancer cells but HIF‐2α appeared to restrain growth. Consequently, therapeutic approaches that target HIF may need to consider these isoform‐specific properties.

Caspase-3 is a pivotal mediator of apoptosis during regression of the ovarian corpus luteum

Because caspase-3 is considered a primary executioner of apoptosis and has been implicated as a mediator of luteal regression, we hypothesized that corpora lutea (CL) derived from caspase-3 null mice would exhibit a delayed onset of apoptosis during luteal regression, when compared with CL derived from wild-type (WT) mice. To test this hypothesis, ovulation was synchronized in immature (postpartum d 24 –27) WT and caspase-3-deficient female littermates by exogenous gonadotropins. Individual CL were isolated by manual dissection, 30 h after ovulation, and placed in organ culture dishes in the absence of serum and growth factors. At the time of isolation (0 h) and after 24, 48, and 72 h in culture, the CL were removed and assessed for the presence of processed (active) caspase-3 enzyme and for apoptosis by multiple criteria. There was no evidence of active caspase-3 enzyme or apoptosis in either WT or caspase-3-deficient CL before culture. However, CL derived from the WT mice exhibited a time-dependent increase in the level of active caspase-3 and apoptosis during culture. By comparison, CL derived from caspase-3-deficient mice, cultured in parallel, failed to exhibit any detectable active caspase-3 and showed attenuated rates of apoptosis. To extend these findings derived from ex vivo culture experiments, ovaries were collected from WT and caspase-3 null female littermates at 2, 4, or 6 d post ovulation, and the occurrence of apoptosis within the CL was analyzed. Whereas ovaries of WT mice had only residual luteal tissue at d 6 post ovulation, ovaries collected from caspase-3-deficient mice retained many CL, at d 6 post ovulation, that were similar in size to those observed in the early luteal phase of WT mice. Importantly, there was no dramatic increase in apoptosis in CL of caspase-3-deficient mice at any time point examined post ovulation, indicating that the involution process had indeed been delayed. In contrast, the levels of progesterone declined regardless of genotype. These data provide the first direct evidence that caspase-3 is functionally required for apoptosis to proceed normally during luteal regression. However, caspase-3 is not a direct mediator of the decrease in steroidogenesis associated with luteolysis. (Endocrinology 143: 1495–1501, 2002)

Signaling mechanisms in tumor necrosis factor alpha-induced death of microvascular endothelial cells of the corpus luteum

The microvasculature of the corpus luteum (CL), which comprises greater than 50% of the total number of cells in the CL, is thought to be the first structure to undergo degeneration via apoptosis during luteolysis. These studies compared the apoptotic potential of various cytokines (tumor necrosis factor α, TNFα; interferon gamma, IFNγ; soluble Fas ligand, sFasL), a FAS activating antibody (FasAb), and the luteolytic hormone prostaglandin F2α (PGF2α) on CL-derived endothelial (CLENDO) cells. Neither sFasL, FasAb nor PGF2α had any effect on CLENDO cell viability. Utilizing morphological and biochemical parameters it was evident that TNFα and IFNγ initiated apoptosis in long-term cultures. However, TNFα was the most potent stimulus for CLENDO cell apoptosis at early time points. Unlike many other studies described in non-reproductive cell types, TNFα induced apoptosis of CLENDO cells occurs in the absence of inhibitors of protein synthesis. TNFα-induced death is typically associated with acute activation of distinct intracellular signaling pathways (e.g. MAPK and sphingomyelin pathways). Treatment with TNFα for 5–30 min activated MAPKs (ERK, p38, and JNK), and increased ceramide accumulation. Ceramide, a product of sphingomyelin hydrolysis, can serve as an upstream activator of members of the MAPK family independently in numerous cell types, and is a well-established pro-apoptotic second messenger. Like TNFα, treatment of CLENDO cells with exogenous ceramide significantly induced endothelial apoptosis. Ceramide also activated the JNK pathway, but had no effect on ERK and p38 MAPKs. Pretreatment of CLENDO cells with glutathione (GSH), an intracellular reducing agent and known inhibitor of reactive oxygen species (ROS) or TNFα-induced apoptosis, significantly attenuated TNFα-induced apoptosis. It is hypothesized that TNFα kills CLENDO cells through elevation of reactive oxygen species, and intracellular signals that promote apoptosis.

Leptin regulation of proangiogenic molecules in benign and cancerous endometrial cells.

Several proangiogenic/proinflammatory factors involved in endometrial cancer are regulated by leptin, but the signaling mechanisms responsible for these leptin‐induced actions are largely unknown. Here, we report that in benign (primary and HES) and cancerous‐endometrial epithelial cells (EEC) (An3Ca, SK‐UT2 and Ishikawa), leptin in a dose‐dependent manner regulates vascular endothelial growth factor, (VEGF); interleukin‐1 beta, (IL‐1β); leukemia inhibitory factor, (LIF) and their respective receptors, VEGFR2, IL‐1R tI and LIFR. Remarkably, leptin induces a greater increase in VEGF/VEGFR2 and LIF levels in cancer than in benign cells. However, IL‐1β was only increased by leptin in benign primary‐EEC. Cancer‐EEC expressed higher levels of leptin receptor (full‐length OB‐Rb and short isoforms) in contrast to benign primary‐EEC. Leptin‐mediated activation of JAK2 (janus kinase 2) was upstream to the activation of PI‐3K (phosphatidylinositol‐3 kinase) and/or MAPK (mitogen‐activated protein kinase) signaling pathways. Leptin induction of cytokines/receptors generally involved JAK2 and MAPK activation, but PI‐3K phosphorylation was required for leptin increase of LIF, IL‐1/IL‐1R tI. Leptin‐mediated activation of mTOR (mammalian target of Rapamycin), mainly linked to MAPK, played a central role in leptin regulation of all cytokines and receptors. These results suggest that leptins effects are cell‐specific and could confer a proliferative or cell survival advantage or possibly promote endometrial thickness. Leptins effects on proangiogenic molecules were more evident in malignant versus benign cells and may imply that there is an underlying shift in leptin‐induced cell signaling pathways in endometrial cancer cells.

CD95 Rapidly Clusters in Cells of Diverse Origins

We have shown that CD95-mediated cell death requires a clustering of the receptor in distinct sphingolipid-rich domains of the cell membrane (Grassmé et al., 2000, Cremesti et al., 2000). These domains form in response to acid sphingomyelinase (ASM)-induced ceramide generation. However, recent studies challenged the finding of early CD95 clustering (Algeciras-Schimnich et al., 2002). Here, six independent groups tested clustering of CD95 in diverse cell type including primary cells ex vivo and established cell lines. The studies show clustering of CD95 within seconds to minutes in all cell types tested by the different groups. In addition, clustering of CD95 was detected after stimulation of cells using three agonistic anti-CD95 antibodies (CH11, APO-1-3 and JO2), CD95 ligand and stimuli that induce an upregulation and activation of the endogenous CD95/CD95 ligand system. The data confirm our previous studies and suggest rapid, i.e. within seconds to minutes, CD95 clustering as a general phenomenon occurring in many cell types.

Loss of Cables, a Cyclin-Dependent Kinase Regulatory Protein, Is Associated with the Development of Endometrial Hyperplasia and Endometrial Cancer

Endometrial cancer is the most common gynecological cancer in Western industrialized countries. Cables, a cyclin-dependent kinase binding protein, plays a role in proliferation and/or differentiation. Cables mutant mice are viable, but develop endometrial hyperplasia and carcinoma in situ at a young age. Exposure to chronic low levels of estrogen results in development of endometrial cancer, similar to that observed in the postmenopausal female. In vitro and in vivo studies demonstrate that levels of Cables mRNA in benign human endometrial epithelium are up-regulated by progesterone and down-regulated by estrogen. Furthermore, nuclear immunostaining for Cables is lost in a high percentage of cases of human endometrial hyperplasia and adenocarcinoma, which are likely the product of unopposed estrogen. The loss of Cables immunostaining in the human endometrial cancer samples correlates with a marked decrease in Cables mRNA. Ectopic expression of Cables in human endometrial cells dramatically slows cell proliferation. Collectively, these data provide evidence that Cables is hormonally regulated and is involved in regulating endometrial cell proliferation. In addition, loss or suppression of Cables may be an early step in the development of endometrial cancer.

Acid sphingomyelinase involvement in tumor necrosis factor α-regulated vascular and steroid disruption during luteolysis in vivo

TNF is well known for its role in inflammation, including direct effects on the vasculature. TNF also is implicated in the regulation of reproduction by its actions to affect ovarian steroidogenic cells and to induce apoptosis of corpus luteum (CL)-derived endothelial cells in vitro. We hypothesized that the disruption of TNF signaling would postpone the regression of the highly vascularized CL in vivo, and this effect could be replicated in mutant mouse models lacking TNF receptor (TNFRI−/−) and/or a critical enzyme of TNF signaling, acid sphingomyelinase (ASMase−/−). In the current study, the treatment of pseudopregnant mice with the luteolytic mediator prostaglandin F2-α (PGF) significantly increased TNF in the ovaries when compared with saline-treated controls. Treatment with PGF also reduced serum progesterone (P4) concentrations and caused involution of the CL. However, pretreatment of pseudopregnant mice with Etanercept (ETA), a TNF-neutralizing antibody, inhibited the PGF-induced decrease in P4 and delayed luteal regression. A similar outcome was evident in pseudopregnant TNFRI−/− animals. Treatment of luteal microvascular endothelial cells (MVECs) with TNF provoked a significant increase in ASMase activity when compared with the corresponding controls. Furthermore, TNF-induced MVEC death was inhibited in the ASMase−/− mice. The ASMase−/− mice displayed no obvious evidence of luteal regression 24 h after treatment with PGF and were resistant to the PGF-induced decrease in P4. Together these data provide evidence that TNF plays an active role in luteolysis. Further studies are required to determine the deleterious effects of anti-inflammatory agents on basic ovarian processes.

Defining the extent of cables loss in endometrial cancer subtypes and its effectiveness as an inhibitor of cell proliferation in malignant endometrial cells in vitro and in vivo

Loss of Cables expression is associated with a high incidence of endometrial hyperplasia and endometrial adenocarcinoma in humans. The Cables mutant mouse develops endometrial hyperplasia and following exposure to chronic estrogen develops early endometrial adenocarcinoma. The objectives of the current study were to determine if: 1) loss of Cables expression occurred in high grade endometrioid adenocarcinoma, uterine serous and clear cell carcinoma as observed in endometrial hyperplasia and low grade endometrial adenocarcinoma; 2) overexpression of Cables inhibited cell proliferation in endometrial cancer (EC) cells in vitro and in vivo; and 3) progesterone could regulate the expression of Cables mRNA. Hyperplastic endometrium and low and high grade endometrioid adenocarcinoma showed loss of Cables expression when compared to benign control secretory endometrium. Loss of Cables expression in serous and clear cell tumors was similar to that observed in endometrioid adenocarcinomas with greater than 80% showing loss of protein expression. Treatment of EC lines with progesterone increased cables expression in low-grade EC whereas it had no effect on cables expression in cells derived from high-grade EC. The progesterone-induced increase in cables was abrogated in the presence of a progesterone receptor (PR) antagonist, suggesting the PR mediates the increase. Cables overexpression inhibited cell proliferation of well differentiated EC cells and had no effect on the poorly differentiated EC cells. The capacity to form tumors was dramatically reduced in the Cables overexpressing cell lines compared to those cells containing the control vector. Collectively these results suggest that Cables is an important regulator of cell proliferation and loss of Cables expression contributes to the development of all types of EC.