Enhances Tumor Cell Survival Research Articles

83: Molecular Imaging of HIF-1α and pVHL Interaction in Living Subjects Using Firefly Luciferase Complementation - Improvement Over the Renilla Luciferase System

Tumor hypoxia induces genomic and proteomic changes which enhance tumor cell survival and dissemination. Clinically, hypoxic tumors are more resistant to therapy and portends for a worse outcome. Under hypoxia, HIF-1α (Hypoxia-inducible factor) regulates the activation of genes promoting malignant progression. Under normoxia, HIF-1α is hydroxylated by the prolyl hydroxylases (PHDs) and is targeted for ubiquitin mediated degradation by interacting with the von Hippel Lindau (VHL) protein complex. The study of HIF-1α and pVHL interaction has been challenging due to the transient nature of the interaction between HIF-1α and pVHL and the rapid rate of HIF-1α degradation. Here, we present a novel method of studying the interaction between HIF-1α and pVHL using the split firefly luciferase complementation based bioluminescence system and compare this to the split Renilla luciferase system. HIF-1α and pVHL split Renilla and firefly luciferase chimeras were generated by fusing the amino- and carboxy-terminal regions of the luciferase to HIF-1α and pVHL, respectively. In vitro split luciferase activity was assayed by co-transfecting the split luciferase expression plasmids into mammalian cells and measuring the complementation based luciferase activity using a luminometer. Imaging studies were performed in nude mice implanted with HIF-1α and pVHL split firefly luciferase expressing mammalian cells using an optical cooled charge-coupled devise camera. The split firefly luciferase fragments, in the absence of HIF-1α and pVHL, produce minimal complementation. Upon HIF-1α proline hydroxylation, pVHL interacts with hydroxylated-HIF-1α, leading to complementation of the split luciferase fragments and generation of an active enzyme. HIF-1α proline hydroxylation by the PHDs is a requisite step, and interfering with proline hydroxylation by using 1) HIF-1α mutants (P402A and P564G), 2) small molecule inhibitors of proline hydroxylation, or 3) genetic silencing of PHD2 using the shRNA approach leads to inhibition of HIF-1α and pVHL mediated split luciferase activity. The interaction between HIF-1α and pVHL was also imaged in living subjects. Confirming our cell culture results, the HIF-1α and pVHL split luciferase interaction in live mice also requires HIF-1α hydroxylation. In comparison to the HIF-1α and pVHL split Renilla luciferase system, the split firefly luciferase system has several advantages, including a lower background, more stable bioluminescence signal, easier administration of the luciferase substrate, and less dependence on the size of the HIF-1a construct used. The split firefly luciferase complementation based bioluminescence system represents a novel approach for studying the interaction between HIF-1α and pVHL in vivo and has several advantages over the split Renilla system. We have established a new technique for visualizing the interaction between an E3 ubiquitin ligase and its substrate and investigating the importance of post-translational modifications in protein-protein interaction. This system will allow high-throughput screening to identify compounds that modulate the interaction between HIF-1α and pVHL, and imaging studies will allow for intra-tumor target validation assessment in live animals.

Notch1 Signaling Promotes Primary Melanoma Progression by Activating Mitogen-Activated Protein Kinase/Phosphatidylinositol 3-Kinase-Akt Pathways and Up-regulating N-Cadherin Expression

Cellular signaling mediated by Notch receptors results in coordinated regulation of cell growth, survival, and differentiation. Aberrant Notch activation has been linked to a variety of human neoplasms. Here, we show that Notch1 signaling drives the vertical growth phase (VGP) of primary melanoma toward a more aggressive phenotype. Constitutive activation of Notch1 by ectopic expression of the Notch1 intracellular domain enables VGP primary melanoma cell lines to proliferate in a serum-independent and growth factor-independent manner in vitro and to grow more aggressively with metastatic activity in vivo. Notch1 activation also enhances tumor cell survival when cultured as three-dimensional spheroids. Such effects of Notch signaling are mediated by activation of the mitogen-activated protein kinase (MAPK) and Akt pathways. Both pathways are activated in melanoma cells following Notch1 pathway activation. Inhibition of either the MAPK or the phosphatidylinositol 3-kinase (PI3K)-Akt pathway reverses the Notch1 signaling-induced tumor cell growth. Moreover, the growth-promoting effect of Notch1 depends on mastermind-like 1. We further showed that Notch1 activation increases tumor cell adhesion and up-regulates N-cadherin expression. Our data show regulation of MAPK/PI3K-Akt pathway activities and expression of N-cadherin by the Notch pathway and provide a mechanistic basis for Notch signaling in the promotion of primary melanoma progression.

The Targeting of Phosphoinositide-3 Kinase Attenuates Pulmonary Metastatic Tumor Growth Following Laparotomy

We aimed to characterize a potential role for phosphatidylinositol 3-kinase (PI3k) in leading to accelerated postoperative metastatic tumor growth. PI3k enhances tumor cell survival in part by phosphorylating Akt and reducing apoptosis. Postoperatively, apoptosis is reduced within local recurrences and distant metastases. This reduction is associated with the phenomenon of accelerated postoperative tumor growth. Balb/c mice underwent a tail vein injection of 1x10 metastatic murine mammary adenocarcinoma 4T1 cells. Animals were divided into the following treatment groups (n=10/group): group A, controls; group B, DMSO intraperitoneally (IP) daily from days 14 to 21; group C, IP LY294002 daily from days 14 to 21; group D, laparotomy only; group E, laparotomy followed by IP DMSO for 7 days; and group F, laparotomy followed by LY294002 IP for 7 days. All laparotomies were performed on day 14. Animals were killed at day 28. Metastatic tumor burden was assessed using the lung/body weight ratio and a histologic metastatic index. Mitotic counts and apoptotic indices were established using a combination of hematoxylin and eosin histology and TUNEL immunohistochemistry. A parallel survival study was performed, and PI3k activity was assessed using western blots for phospho-Akt. Laparotomy was associated with increased systemic tumor burden (P=0.001). Postoperatively, LY294002 significantly attenuated metastatic tumor growth (P<0.001). Effective PI3k inhibition was confirmed by demonstrating a reduced Akt phosphorylation. Moreover, PI3k inhibition led to reduced proliferation, increased apoptosis (P<0.001), and enhanced postoperative survival (P<0.001). Targeting PI3k with postoperative LY294002 significantly attenuates the acceleration in postoperative metastatic tumor growth seen following laparotomy.

Mechanism of metastasis by membrane type 1-matrix metalloproteinase in hepatocellular carcinoma

To investigate the precise role of membrane type 1-matrix metalloproteinase (MT1-MMP) in hepatocellular carcinoma (HCC) metastasis. Human HCC cells Hep3B with overexpression of MT1-MMP were established by stable transfection, and compared with control cells carrying the empty vector. Cells were examined in vivo for their differences in the metastatic ability of athymic nude mice, and analyzed in vitro for their differences in invasion ability by invasion chamber coated with Matrigel, adhesion towards collagen I and migration through culture chamber. Cell proliferation and apoptosis in adherent and suspension status were evaluated by MTT and flow cytometry analysis. We found that overexpression of MT1-MMP could increase intrahepatic metastasis in nude mice with orthotopic implantation of HCC cells (incidence of 100% [MT1-MMP transfectants] vs 40% [vector control transfectants], P<0.05). MT1-MMP could also enhance cell invasion through Matrigel (107.7 vs 39.3 cells/field, P<0.001), adhesion towards matrix (0.30 vs 0.12 absorbance unit at 540 nm, P<0.001), cell migration (89.3 vs 39.0 cells/field, P<0.001), and cell proliferation (24.3 vs 40.5 h/doubling, P<0.001). We also observed that MT1-MMP supported cell survival (71.4% vs 23.9%, P<0.001) with reduced apoptosis (43.7% vs 51.0%, P<0.05) in an attachment-free environment. MT1-MMP overexpression could enhance metastasis. In addition to its active role in matrix degradation during tumor invasion, MT1-MMP enhances tumor cell survival upon challenge of detachment, which is important during metastasis when cells enter the circulation.

Survivin enhances radiation resistance in primary human glioblastoma cells via caspase-independent mechanisms

The observed radioresistance of human glioblastoma multiforme (GBM) poses a major challenge, which, if overcome, may lead to significant advances in the management of this patient population. There is accumulating evidence from correlative studies that Survivin expression is associated with increased malignant potential of human gliomas. The purpose of this study was to investigate whether Survivin plays a direct role in mediating radiation resistance in primary human glioma cell lines, and, if so, investigating the underlying mechanisms. Our panel of GBM cell lines included two that were relatively radiation resistant (GM20 and GM21) and two that were more radiation sensitive (GM22 and GM23), which demonstrated differential levels of Survivin expression between the two groups. Through the use of adenoviral vectors containing either dominant-negative (pAd-S(T34A)) or wild-type Suvrivin (pAd-S(WT)), we were able to inactivate or overexpress Survivin, respectively. Our findings suggest that Survivin plays a critical role in mediating radiation resistance in primary GBM cells, in part through suppression of apoptotic cell death via a caspase-independent manner. We have identified novel mechanisms by which Survivin may enhance tumor cell survival upon radiation exposure such as regulation of double-strand DNA break repair and tumor cell metabolism, which were most evident in the radiation-resistant cell lines. These differences in Survivin function both in radiation-resistant vs radiation-sensitive cell lines and in the presence vs absence of radiation exposure warrant further investigation and highlight potentially important mechanisms of radiation resistance in these tumors.

The role of nuclear factor-κb in the biology and treatment of multiple myeloma

Increased nuclear factor (NF)-κB activity is associated with enhanced tumor cell survival in multiple myeloma (MM). The function of NF-κB is inhibited through binding to its inhibitor, Iκb. Release of activated NF-κB follows proteasome-mediated degradation of IκBα resulting from phosphorylation of the inhibitor and finally conjugation with ubiquitin. We report that myeloma tumor cells show enhanced NF-κB activity. In addition, these patients possess polymorphisms of IκBα at sites important in the degradation of the inhibitor protein. Exposure of myeloma cells to chemotherapy leads to an increase in IκBα phosphorylation and reduces the levels of this inhibitor of NF-kB function. Chemoresistant myeloma cell-lines have increased NF-κB activity compared to sensitive lines. An inhibitor of NF-κB activity, the proteasome inhibitor PS-341 (Millenium Inc, Boston, MA), showed consistent antitumor activity against chemoresistant and sensitive myeloma cells. The sensitivity of chemoresistant myeloma cells to chemotherapeutic agents was markedly increased (100,000- to 1,000,000-fold) when combined with a noncytotoxic dose of PS-341. In contrast, this combination had little growth inhibitory effect on normal hematopoietic cells. Similar effects were observed using a dominant negative super-repressor for IκBα. These results suggest that inhibition of NF-κB with PS-341 may overcome chemoresistance and allow doses of chemotherapeutic agents to be markedly reduced with antitumor effects without significant toxicity.

Follicular Dendritic Cells Protect Malignant B Cells from Apoptosis Induced by Anti-Fas and Antineoplastic Agents

The observation that follicular dendritic cells (FDC) reduce apoptosis in B cells prompted the hypothesis that FDC might enhance tumor cell survival by protecting malignant B cells from apoptotic death. To test this notion, apoptosis was induced in B cell lymphomas by anti-Fas or various antineoplastic agents in the presence and absence of FDC. Apoptosis was detected and quantified by TUNEL analysis. Induction of apoptosis with anti-Fas, etoposide, cyclophosphamide, and busulfan was markedly antagonized by FDC at FDC to B cell ratios of >/=1:16. For example, treatment with 10 ng/ml anti-Fas caused 60-90% of A20 cells to undergo apoptosis in 6 h, whereas addition of FDC reduced apoptosis to background levels (3-15%). Similarly, treatment with busulfan induced apoptosis in 55-80% of A20 cells, whereas addition of FDC reduced B cell death to </=15%; moreover, depletion of FDC abrogated the protective actions. In contrast, the apoptosis-inducing effect of Adriamycin was not reversed by FDC. The ability to block apoptosis induced by anti-Fas or busulfan was not limited to A20 but was observed in four other malignant pre-B cell or B cell lines. The mechanism by which FDC spare malignant B cells from apoptosis did not involve alterations in levels of Bcl-2, Bcl-XL, or Bax. Collectively, these data raise the possibility that FDC may enhance tumor cell survival by protecting malignant B cells against apoptosis induced by anti-Fas and some but not all chemotherapeutic agents.

The tumor microenvironment as a determinant of drug response and resistance.

Classically, studies of drug resistance in cancer have focused on the molecular biology of single cancer cells. These types of studies have provided important information regarding certain drug resistance mechanisms, including mechanisms that reduce intracellular drug accumulation, alter or repair drug-induced damage, and reduce drug-induced apoptosis. While these cellular mechanisms undoubtedly contribute to the overall phenomenon of drug resistance, it is now evident that the tumor cell microenvironment also influences how a tumor cell behaves and responds to cytotoxic drugs or radiation. Two different forms of tumor cell-environmental interaction may explain how some tumor cells survive initial drug exposure and eventually express classical mechanisms of drug resistance. The first form involves soluble mediators, such as interleukins, that are secreted by non-tumor, stromal cells. Interleukin-6 (IL-6) is a classical example of how a soluble mediator secreted by the tumor microenvironment is capable of enhancing tumor cell survival and perhaps blocking apoptosis. The second form of tumor cell-environment interaction requires direct cell contact and has been given the term cell-adhesion-mediated drug resistance (CAM-DR). In this case, binding extracellular matrix ligands in the tumor microenvironment may activate cell adhesion molecules, such as the integrins, and these interactions result in the activation of signal transduction pathways that block drug-induced apoptosis. Interrupting the tumor cell-environment interactions or the associated signal transduction pathways may represent a new approach for the treatment of cancer. Copyright 1999 Harcourt Publishers Ltd.

Bcl-2 expression in the spectrum of preinvasive breast lesions.

The bcl-2 gene encodes for a protein that blocks apoptosis. Although the bcl-2 protein has been identified in some invasive breast cancers, its expression in preinvasive breast lesions has not been well characterized. We studied bcl-2 expression using immunohistochemistry in cases of normal breast tissue (n = 10), ductal hyperplasia (n = 18), atypical ductal hyperplasia (ADH) (n = 10), atypical lobular hyperplasia/lobular carcinoma in situ (lobular neoplasia, LN) (n = 10), and ductal carcinoma in situ (DCIS) (n = 42). We also related bcl-2 expression to p53 expression in these lesions because the p53 gene is altered in many invasive breast cancers and is also involved in the regulation of apoptosis. bcl-2 was consistently expressed in the epithelial cells in all normal breast tissue, ductal hyperplasia, ADH, and LN lesions. In contrast, bcl-2 expression was present in 76% of the DCIS cases and was related to the histologic grade of DCIS. Staining for bcl-2 was observed in 100% of the well differentiated DCIS cases, 90% of intermediately differentiated cases, and 33% of poorly differentiated cases (P < 0.001). No immunoreactivity for the p53 protein was seen in any of the cases of normal breast tissue, ductal hyperplasia, ADH, or LN lesions. However, 24% of the DCIS cases were p53 positive. The bcl-2+/p53- phenotype, as seen in all cases of normal breast tissue, ductal hyperplasia, ADH, and LN was also observed in 67% of the DCIS cases. In contrast, the remaining 33% of DCIS cases showed combinations of bcl-2 and p53 expression that differed from that of normal breast epithelium and the other pathologic lesions studied. Most lesions with altered bcl-2/p53 phenotypes, including all bcl-2-/p53+ cases, represented examples of poorly differentiated DCIS. The bcl-2 protein is consistently expressed in normal breast epithelium, ductal hyperplasia, ADH, and LN. bcl-2 expression is variable in DCIS. Among DCIS cases, bcl-2 is most common in well differentiated and intermediately differentiated lesions. Most DCIS lesions are bcl-2+ and p53-, similar to normal epithelium, benign proliferative lesions, and LN. However, a minority of DCIS lesions show combinations of bcl-2 and p53 expression that differ from normal breast epithelium and from the other pathologic lesions studied. It is possible that such lesions may represent a subset of DCIS in which the regulation of apoptosis is no longer under normal control mechanisms, resulting in enhanced tumor cell survival and tumor growth.