Liat Drucker

Overexpression of tetraspanins affects multiple myeloma cell survival and invasive potential

Cellular interactions with microenviron‐mental components are critical in multiple myeloma (MM) and impede effective disease treatment. Mem‐branal‐embedded tetraspanins, associated with metastasis suppression, are underexpressed in MM. We aimed to investigate the consequences of CD81/CD82 tetraspanins over‐expression in MM cell lines. CAG and RPMI 8226 were transfected with pEGFP‐N1/C1 fusion vectors of CD81/CD82. Employing flow cytometry, immunocytochemistry, and activity assays we assessed transfected cells for: morphology, survival, death, caspases, cell cycle, proliferation, oxidative stress, adhesion, motility and invasion. Overexpressed CD81/ CD82 pEGFP‐N1 vectors reduced survival without elevation of pre‐G1 or AnnexinV+/7AAD‐ and independently of caspases. Decreased Ki67 and elevated intracellular glutathione were detected. No perturbations in cell cycle distribution were observed. The pEGFP‐C1 vectors of CD81/CD82 caused reduction of MM cell adherence with/without fibronectin, insulinlike growth factor (IGF)‐I, and matrigel. They also reduced cell motility and attenuated invasion potential’ expressed by reduced secreted MMP‐9 activity. These novel findings delineate the significance of CD81/ CD82 expression to MM cell survival and their negative effects on cell adhesion, motility, and invasion thus, supporting their role as tumor metastasis suppressors.—Tohami T., Drucker L., Shapiro H., Radnay J., and Lishner M. Overexpression of tetraspanins affects multiple myeloma cell survival and invasive potential FASEB J. 21, 691–699 (2007)

Simvastatin induces death of multiple myeloma cell lines.

Background Accumulating reports indicate that statins widely prescribed for hypercholesteromia have antineoplastic activity. We hypothesized that because statins inhibit farnesylation of Ras that is often mutated in multiple myeloma (MM), as well as the production of interleukin (IL)-6, a key cytokine in MM, they may have antiproliferative and/or proapoptotic effects in this malignancy. Methods U266, RPMI 8226, and ARH77 were treated with simvastatin (0-30 μM) for 5 days. The following aspects were evaluated: viability (IC50), cell cycle, cell death, cytoplasmic calcium ion levels, supernatant IL-6 levels, and tyrosine kinase activity. Results Exposure of all cell lines to simvastatin resulted in reduced viability with IC50s of 4.5 μM for ARH77, 8 μM for RPMI 8226, and 13 μM for U266. The decreased viability is attributed to cell-cycle arrest (U266, G1; RPMI 8226, G2M) and cell death. ARH77 underwent apoptosis, whereas U266 and RPMI 8226 displayed a more necrotic form of death. Cytoplasmic calcium levels decreased significantly in all treated cell lines. IL-6 secretion from U266 cells was abrogated on treatment with simvastatin, whereas total tyrosine phosphorylation was unaffected. Conclusions Simvastatin displays significant antimyeloma activity in vitro. Further research is warranted for elucidation of the modulated molecular pathways and clinical relevance.

Adenomatous polyposis coli I1307K mutation in Jewish patients with different ethnicity

A new mutation, I1307K, recently was reported in the adenomatous polyposis coli (APC) gene. This mutation was found to be predominant in Ashkenazi Jews, creating a hypermutable area and predisposing the development of carcinoma. The objective of the current study was to estimate the prevalence of this mutation in several of the ethnic groups that comprise the Israeli population and to elucidate the clinical features of the mutation carriers with colorectal carcinoma (CRC).

Bevacizumab attenuates major signaling cascades and eIF4E translation initiation factor in multiple myeloma cells

Multiple myeloma (MM), a malignancy of plasma cells, remains fatal despite introduction of novel therapies, partially due to humoral factors, including vascular endothelial growth factor (VEGF), in their microenvironment. The aim of this study was to explore the efficacy of anti-VEGF treatment with bevacizumab directly on MM cells. Particular attention was directed to the affect of VEGF inhibition on protein translation initiation. Experiments were conducted on MM cells (lines, bone marrow (BM) samples) cultured on plastic. Inhibition of VEGF was achieved with the clinically employed anti-VEGF antibody, bevacizumab, as a platform and its consequences on viability, proliferation, and survival was assessed. VEGF downstream signals of established importance to MM cell biology were assayed as well, with particular emphasis on translation initiation factor eIF4E. We showed that blocking VEGF is deleterious to the MM cells and causes cytostasis. This was evidenced in MM cell lines, as well as in primary BM samples (BM MM). A common bevacizumab-induced attenuation of critical signaling effectors was determined: VEGFR1, mTOR, c-Myc, Akt, STAT3, (cell lines) and eIF4E translation initiation factor (lines and BM). ERK1/2 displayed a variegated response to bevacizumab (lines). Utilizing a constitutively Akt-expressing MM model, we showed that the effect of bevacizumab on viability and eIF4E status is Akt-dependent. Of note, the effect of bevacizumab was achieved with high concentrations (2 mg/ml), but was shown to be specific. These findings demonstrate that bevacizumab has a direct influence on major pathways critically activated in MM that is independent from its established effect on angiogenesis. The cytostatic effect of VEGF inhibition on MM cells underscores its potential in combined therapy, and our findings, regarding its influence on translation initiation, suggest that drugs that unbalance cellular proteostasis may be particularly effective.

The Role of Heat Shock Protein 27 in Extravillous Trophoblast Differentiation

Trophoblast cells from placental explants differentiate in culture to extravillous trophoblast cells (EVT cells). During trophoblast differentiation heat‐shock‐protein‐27 (HSP27) mRNA and multidrug‐resistance‐protein‐5 (MRP5, transporter of cyclic nucleotides) expression are increased. HSP27 is a regulator of actin filaments structure and dynamic, has a role in cell differentiation and may affect NF‐kB activity. In this study we aimed to assess HSP27 level in trophoblast cells and its correlation with motility and differentiation related processes [MMPs activity, nitric oxide (NO), inducible nitric oxide synthase (iNOS), proliferation and MRP5 levels]. We evaluated HSP27 expression in a first trimester human trophoblast explants model designed to assess EVT cells differentiation/migration with/without 6‐mercaptopurine (6MP, an EVT inhibitor of migration). We found that HSP27 level is expressed in the nucleous and cytoplasm of non‐proliferting villous‐trophoblast cells (negative for Ki67) and in the cell periphery and cytoplasm of motile EVT cells. Moreover, 6MP decreased HSP27 nucleous expression that was associated with inhibited MMP2 activity and NO production. Also decreased iNOS expression and increased MRP5 mRNA levels were observed. In conclusion, HSP27 expression is modulated in concordance with migration dependent parameters in trophoblast cells. J. Cell. Biochem. 103: 719–729, 2008.

Co-administration of simvastatin and cytotoxic drugs is advantageous in myeloma cell lines

We have evaluated the potential application of simvastatin (Sim) combined with conventional cytotoxic drugs for the treatment of multiple myeloma. RPMI 8226 and U266 myeloma cells seeded in culture plates were treated with Sim (5 and 10 μM, respectively) combined with melphalan (Mel; 25 and 20 μM, respectively) or dexamethasone (Dex; 1 μM). We assessed cell cycle (propidium iodide staining and flow cytometric analysis), cell morphology, viability (WST1), total cell count and cell death (Trypan blue exclusion). Sim significantly enhanced the anti-myeloma activity of cytotoxic agents in vitro (p<0.05). Incubation of U266 and RPMI 8226 with Sim prior to Mel increased the cytotoxicity in an additive manner, whereas the exposure of U266 to combined Sim and Dex resulted in a synergistic amplification of the individual effects. Combined application of Dex and Sim to RPMI 8226 cells resulted in antagonistic activity. The possible roles of Ras and phosphoinositol 3-kinase are discussed.

Microenvironment factors do not afford myeloma cell lines protection from simvastatin

Abstract:  Background: The intensive interactions of myeloma cells (multiple myeloma, MM) with microenvironmental components of the bone marrow contribute significantly to their proliferation and survival. It has been shown that these signals confer drug resistance, delineating their circumvention as a primary objective in disease treatment. This study was designed to assess the effect of some major extracellular factors on the previously established anti‐neoplastic response of myeloma cells to simvastatin (Sim). Study design: RPMI8226, U266, and ARH77 seeded in culture plates precoated with fibronectin (FN)/agarose/none were treated with Sim, insulin‐like growth factor‐I (IGF‐I), interleukin‐6 (IL‐6) or combinations for 5 d. Then we assessed cell morphology, viability (WST1), cell cycle (propidium iodide, PI, staining and flow cytometric analysis), total cell count, and cell death (trypan blue exclusion), and DNA fragmentation. Results and conclusions: Reduced viability was demonstrated with Sim in all treated cell lines with and without co‐administration of IGF‐I or IL‐6 (P < 0.05). The extent of inhibition did not vary between Sim only and combinations (NS). FN did not influence cell response to Sim alone or combined with IL‐6/IGF‐I (NS). We conclude that IL‐6, IGF‐I, and FN do not afford myeloma cell lines protection from Sim modulation.

Placenta-breast cancer cell interactions promote cancer cell epithelial mesenchymal transition via TGFβ/JNK pathway

Women diagnosed with pregnancy associated breast cancer often have advanced cancer with metastases and reduced expression of ERα compared to non-pregnant women. Nevertheless, metastases to the placenta are uncommon. Previously, we demonstrated that breast cancer cells (MCF-7/T47D) migrated from ex vivo human placental explant implantation sites. We aimed to analyze the effect of factors produced during placental implantation or as a result of the interaction between the implanted placentae to cancer cells on cancer cells migration and aggressiveness. We collected supernatants from implanted placentae and placental-breast cancer cells cocultures and analyzed their effects on cancer cells phenotype and pathways. Supernatants collected from breast cancer cells served as controls. We found that supernatants collected from implanted placentae induced modest cancer cells migration that was not accompanied by epithelial to mesenchymal transition (EMT), supported breast cancer cells survival and elevated MCF-7 cell number. The coculture supernatant induced excessive motility and EMT of the MCF-7 cells. This EMT was mediated by Smad3 and JNK/ERK activation. Both placenta and coculture supernatants reduced ERα expression in the cancer cells. Finally, we showed that MCF-7 cocultured with the human placental explants underwent continuous activation of JNK and Smad3 pathways and the EMT process, which led to their migration away from the placental implantation sites. These findings may explain the reduced ERα and elevated metastases found in breast cancer during pregnancy and highlights pathways involved in it.

Multiple myeloma and bone marrow mesenchymal stem cells’ crosstalk: Effect on translation initiation

Multiple myeloma (MM) malignant plasma cells reside in the bone marrow (BM) and convert it into a specialized pre‐neoplastic niche that promotes the proliferation and survival of the cancer cells. BM resident mesenchymal stem cells (BM‐MSCs) are altered in MM and in vitro studies indicate their transformation by MM proximity is within hours. The response time frame suggested that protein translation may be implicated. Thus, we assembled a co‐culture model of MM cell lines with MSCs from normal donors (ND) and MM patients to test our hypothesis. The cell lines (U266, ARP‐1) and BM‐MSCs (ND, MM) were harvested separately after 72 h of co‐culture and assayed for proliferation, death, levels of major translation initiation factors (eIF4E, eIF4GI), their targets, and regulators. Significant changes were observed: BM‐MSCs (ND and MM) co‐cultured with MM cell lines displayed elevated proliferation and death as well as increased expression/activity of eIF4E/eIF4GI; MM cell lines co‐cultured with MM‐MSCs also displayed higher proliferation and death rates coupled with augmented translation initiation factors; in contrast, MM cell lines co‐cultured with ND‐MSCs did not display elevated proliferation only death and had no changes in eIF4GI levels/activity. eIF4E expression was increased in one of the cell lines. Our study demonstrates that there is direct dialogue between the MM and BM‐MSCs populations that includes translation initiation manipulation and critically affects cell fate. Future research should be aimed at identifying therapeutic targets that may be used to minimize the collateral damage to the cancer microenvironment and limit its recruitment into the malignant process.

Hormone-dependent placental manipulation of breast cancer cell migration

BACKGROUND Breast cancer during pregnancy is often more advanced than in non-pregnant women. Nevertheless, no case of metastasis inside the placenta has been reported. Previously, we showed that placental-explants eliminated breast cancer cells from their surroundings, due to cell-death and elevated migration. Our objective was to find the underlying mechanisms of these phenomena. METHODS AND RESULTS Our model contained Michigan Cancer Foundation 7 (MCF7) or T47D cells co-cultured with and without human placental explants. Microarray analysis, validated by quantitative PCR, of MCF7 following their placental co-culture suggested activation of estrogen (E(2)) signaling. As extensive cross-talk exists between E(2) and progesterone, their involvement in mediating placental effects on breast cancer cells was tested. Indeed, addition of E(2) and progesterone receptor (ER and PR) inhibitors to the co-culture system reduced cancer cell motility, yet did not alter cell-cycle or death. E(2) and progesterone concentrations in placental media were found to be similar to those of early pregnancy blood levels. Interestingly, placental-breast cancer co-culture media contained lower progesterone (P < 0.05) and higher E(2) (200%, P < 0.05) levels than placentae cultured separately. Placental supernatant and E(2) and progesterone at placental levels were sufficient to increase MCF7 and T47D migration and invasion (P < 0.05), yet did not alter MCF7 cell-cycle or death. Furthermore, placental supernatant elevated p38 and Jun N-terminal kinase (JNK) phosphorylation in both cell lines (P < 0.05). Inhibitors of JNK, ER and PR reversed MCF7 and T47D motility induced by the placenta, suggesting their involvement. CONCLUSIONS We suggest that E(2) and progesterone contribute to cell migration away from placental areas. We hypothesize that they may increase metastatic spread to other organs in pregnancy.