THE ROLE AND MECHANISM OF THE HOMEOBOX GENE DLX4 IN TRANSFORMING GROWTH FACTOR-B RESISTANCE IN CANCER

Transforming growth factor-b (TGF-b) is a cytokine that plays essential roles in regulating embryonic development and tissue homeostasis. In normal cells, TGF-b exerts an anti-proliferative effect. TGF-b inhibits cell growth by controlling a cytostatic program that includes activation of the cyclin-dependent kinase inhibitors p15Ink4B and p21WAF1/Cip1 and repression of c-myc. In contrast to normal cells, many tumors are resistant to the anti-proliferative effect of TGF-b. In several types of tumors, particularly those of gastrointestinal origin, resistance to the anti-proliferative effect of TGF-b has been attributed to TGF-b receptor or Smad mutations. However, these mutations are absent from many other types of tumors that are resistant to TGF-b-mediated growth inhibition. The transcription factor encoded by the homeobox patterning gene DLX4 is overexpressed in a wide range of malignancies. In this study, I demonstrated that DLX4 blocks the anti-proliferative effect of TGF-b by disabling key transcriptional control mechanisms of the TGF-b cytostatic program. Specifically, DLX4 blocked the ability of TGF-b to induce expression of p15Ink4B and p21WAF1/Cip1 by directly binding to Smad4 and to Sp1. Binding of DLX4 to Smad4 prevented Smad4 from forming transcriptional complexes with Smad2 and Smad3, whereas binding of DLX4 to Sp1 inhibited DNA-binding activity of Sp1. In addition, DLX4 induced expression of c-myc, a repressor of p15Ink4B and p21WAF1/Cip1 transcription, independently of TGF-b signaling. The ability of DLX4 to counteract key transcriptional control mechanisms of the TGF-b cytostatic program could explain in part the resistance of tumors to the anti-proliferative effect of TGF-b. This study provides a molecular explanation as to why tumors are resistant to the anti-proliferative effect of TGF-b in the absence of mutations in the TGF-b signaling pathway. Furthermore, this study also provides insights into how aberrant activation of a developmental patterning gene promotes tumor pathogenesis.

TYPE I INSULIN-LIKE GROWTH FACTOR RECEPTOR TYROSINE KINASE AS A MOLECULAR TARGET IN MANTLE CELL LYMPHOMA

Mantle cell lymphoma (MCL) is an aggressive B-cell lymphoid malignancy representing 5-10% of all non-Hodgkin’s lymphomas. It is distinguished by the t(11;14)(q13;q32) chromosomal translocation that juxtaposes the proto-oncogene CCND1, which encodes cyclin D1 at 11q13 to the IgH gene at 14q32. MCL patients represent about 6% of all new cases of Non-Hodgkin’s lymphomas per year or about 3,500 new cases per year. MCL occurs more frequently in older adults – the average age at diagnosis is the mid-60s with a male-to-female ratio of 2-3:1. It is typically characterized by the proliferation of neoplastic B-lymphocytes in the mantle zone of the lymph node follicle that have a prominent inclination to disseminate to other lymphoid tissues, bone marrow, peripheral blood and other organs. MCL patients have a poor prognosis because they develop resistance/relapse to current non-specific therapeutic regimens. It is of note that the exact molecular mechanisms underlying the pathogenesis of MCL are not completely known. It is reasonable to anticipate that better characterization of these mechanisms could lead to the development of specific and likely more effective therapeutics to treat this aggressive disease. The type I insulin-like growth factor receptor (IGF-IR) is thought to be a key player in several different solid malignancies such as those of the prostate, breast, lung, ovary, skin and soft tissue. In addition, recent studies in our lab showed evidence to support a pathogenic role of IGF-IR in some types of T-cell lymphomas and chronic myeloid leukemia. Constitutively active IGF-IR induces its oncogenic effects through the inhibition of apoptosis and induction of transformation, metastasis, and angiogenesis. Previous studies have shown that signaling through IGF-IR leads to the vi activation of multiple signaling transduction pathways mediated by the receptor-associated tyrosine kinase domain. These pathways include PI3K/Akt, MAP kinase, and Jak/Stat. In the present study, we tested the possible role of IGF-IR in MCL. Our results demonstrate that IGF-IR is over-expressed in mantle cell lymphoma cell lines compared with normal peripheral blood B- lymphocytes. Furthermore, inhibition of IGF-IR by the cyclolignan picropodophyllin (PPP) decreased cell viability and cell proliferation in addition to induction of apoptosis and G2/M cell cycle arrest. Screening of downstream oncogenes and apoptotic proteins that are involved in both IGF-IR and MCL signaling after treatment with PPP or IGF-IR siRNA showed significant alterations that are consistent with the cellular changes observed after PPP treatment. Therefore, our findings suggest that IGF-IR signaling contributes to the survival of MCL and thus may prove to be a legitimate therapeutic target in the future.

Understanding acquired resistance to Lapatinib in breast cancer cells

Signaling through epidermal growth factor receptor (EGFR/ErbB) family members plays a very important role in regulating proliferation, development, and malignant transformation of mammary epithelial cells. ErbB family members are often over-expressed in human breast carcinomas. Lapatinib is an ErbB1 and ErbB2 tyrosine kinase inhibitor that has been shown to have anti-proliferative effects in breast and lung cancer cells. Cells treated with Lapatinib undergo G1 phase arrest, followed by apoptosis. Lapatinib has been approved for clinical use, though patients have developed resistance to the drug, as seen previously with other EGFR inhibitors. Moreover, the therapeutic efficacy varies significantly within the patient population, and the mechanism of drug sensitivity is not fully understood. Expression levels of ErbB2 are used as a prognostic marker for Lapatinib response; however, even among breast tumor cell lines that express similar levels of ErbB2 there is marked difference in their proliferative responses to Lapatinib. To understand the mechanisms of acquired resistance, we established a cell line SkBr3-R that is resistant to Lapatinib, from a Lapatinib-sensitive breast tumor cell line, SkBr3. We have characterized the cell lines and demonstrated that Lapatinib resistance in our system is not facilitated by receptor-level activity or by previously known mutations in the ErbB receptors. Significant changes were observed in cell proliferation, cell migration, cell cycle and cell death between the Lapatinib resistant SkBr3-R and sensitive SkBr3 cell lines. Recent studies have suggested STAT3 is upregulated in Lapatinib resistant tumors in association with ErbB signaling. We investigated the role that STAT3 may play in Lapatinib resistance and discovered higher STAT3 activity in these resistant cells. In addition, transcriptional profiling indicated higher expression of STAT3 target genes, as well as of other genes that promote survival. The gene array data also revealed cell cycle regulators and cell adhesion/junction component genes as possible mediator of Lapatinib resistance. Altogether, this study has identified several possible mechanisms of Lapatinib resistance.

Dasatinib inhibits the growth of molecularly heterogeneous myeloid leukemias.

PURPOSE: Dasatinib is a dual Src/Abl inhibitor recently approved for Bcr-Abl+ leukemias with resistance or intolerance to prior therapy. Because Src kinases contribute to multiple blood cell functions by triggering a variety of signaling pathways, we hypothesized that their molecular targeting might lead to growth inhibition in acute myeloid leukemia (AML). EXPERIMENTAL DESIGN: We studied growth factor-dependent and growth factor-independent leukemic cell lines, including three cell lines expressing mutants of receptor tyrosine kinases (Flt3 or c-Kit) as well as primary AML blasts for responsiveness to dasatinib. RESULTS: Dasatinib resulted in the inhibition of Src family kinases in all cell lines and blast cells at approximately 1 x 10(-9) mol/L. It also inhibited mutant Flt3 or Kit tyrosine phosphorylation at approximately 1 x 10(-6) mol/L. Mo7e cells expressing the activating mutation (codon 816) of c-Kit were most sensitive to growth inhibition with a GI(50) of 5 x 10(-9) mol/L. Primary AML blast cells exhibited a growth inhibition of <1 x>10(-6) mol/L. Cell lines that showed growth inhibition at approximately 1 x 10(-6) mol/L showed a G(1) cell cycle arrest and correlated with accumulation of p21 and p27 protein. The addition of rapamycin or cytotoxic agents enhanced growth inhibition. Dasatinib also caused the apoptosis of Mo7e cells expressing oncogenic Kit. CONCLUSIONS: Although all of the precise targets for dasatinib are not known, this multikinase inhibitor causes either growth arrest or apoptosis in molecularly heterogeneous AML. The addition of cytotoxic or targeted agents can enhance its effects.

Contribution of Ectodomain Mutations in Epidermal Growth Factor Receptor to Signaling in Glioblastoma Multiforme

CONTRIBUTION OF ECTODOMAIN MUTATIONS IN EPIDERMAL GROWTH FACTOR RECEPTOR TO SIGNALING IN GLIOBLASTOMA MULTIFORME Publication No._________ Marta Rojas, M.S. Supervisory Professor: Oliver Bögler, Ph.D. The Cancer Genome Atlas (TCGA) has conducted a comprehensive analysis of a large tumor cohort and has cataloged genetic alterations involving primary sequence variations and copy number aberrations of genes involved in key signaling pathways in glioblastoma (GBM). This dataset revealed missense ectodomain point mutations in epidermal growth factor receptor (EGFR), but the biological and clinical significance of these mutations is not well defined in the context of gliomas. In our study, we focused on understanding and defining the molecular mechanisms underlying the functions of EGFR ectodomain mutants. Using proteomic approaches to broadly analyze cell signaling, including antibody array and mass spectrometry-based methods, we found a differential spectrum of tyrosine phosphorylation across the EGFR ectodomain mutations that enabled us to stratify them into three main groups that correlate with either wild type EGFR (EGFR) or the long-studied mutant, EGFRvIII. Interestingly, one mutant shared characteristics of both groups suggesting a continuum of behaviors along which different mutants fall. Surprisingly, no substantial differences were seen in activation of classical downstream signaling pathways such as Akt and S6 pathways between these classes of mutants. Importantly, we demonstrated that ectodomain mutations lead to differential tumor growth capabilities in both in vitro (anchorage independent colony formation) and in vivo conditions (xenografts). Our data from the biological characterization allowed us to categorize the mutants into three main groups: the first group typified by EGFRvIII are mutations with a more aggressive phenotype including R108K and A289T; a second group characterized by a less aggressive phenotype exemplified by EGFR and the T263P mutation; and a third group which shared characteristics from both groups and is exemplified by the mutation A289D. In addition, we treated cells overexpressing the mutants with various agents employed in the clinic including temozolomide, cisplatin and tarceva. We found that cells overexpressing the mutants in general displayed resistance to the treatments. Our findings yield insights that help with the molecular characterization of these mutants. In addition, our results from the drug studies might be valuable in explaining differential responses to specific treatments in GBM patients.

Inherited and noninherited sources of variation in metastasis and the growth rate of tumors: Implications for tumor evolution and therapy

I have undertaken measurements of the genetic (or inherited) and nongenetic (or noninherited) components of the variability of metastasis formation and tumor diameter doubling time in more than 100 metastatic lines from each of three murine tumors (sarcoma SANH, sarcoma SA4020, and hepatocarcinoma HCA-I) syngeneic to C3Hf/Kam mice. These lines were isolated twice from lung metastases and analysed immediately thereafter to obtain the variance to spontaneous lung metastasis and tumor diameter doubling time. Additional studies utilized cells obtained from within 4 passages of isolation. Under the assumption that no genetic differences in metastasis formation or diameter doubling time existed among the cells of a given line, the variance within a line would estimate nongenetic variation. The variability derived from differences between lines would represent genetic origin. The estimates of the genetic contribution to the variation of metastasis and tumor diameter doubling time were significantly greater than zero, but only in the metastatic lines of tumor SANH was genetic variation the major source of metastatic variability (contributing 53% of the variability). In the tumor cell lines of SA4020 and HCA-I, however, the contribution of nongenetic factors predominated over genetic factors in the variability of the number of metastasis and tumor diameter doubling time. A number of other parameters examined, such as DNA content, karyotype, and selection and variance analysis with passage in vivo, indicated that genetic differences existed within the cell lines and that these differences were probably created by genetic instability. The mean metastatic propensity of the lines may have increased somewhat during their isolation and isotransplantation, but the variance was only slightly affected, if at all. Analysis of the DNA profiles of the metastatic lines of SA4020 and HCA-I revealed differences between these lines and their primary parent tumors, but not among the SANH lines and their parent tumor. Furthermore, there was a direct correlation between the extent of genetic influence on metastasis formation and the ability of the tumor cells to develop resistance to cisplatinum. Thus although nongenetic factors might predominate in contributing to metastasis formation, it is probably genetic variation and genetic instability that cause the progression of tumor cells to a more metastatic phenotype and leads to the emergence of drug resistance. ^

Mechanisms of protein regulation in rat skeletal muscle during resistance exercise and training

The cellular mechanisms through which adult rat skeletal muscle protein is regulated during resistance exercise and training was investigated. A model of non-voluntary resistance exercise was described which involves the electrically-stimulated contraction of the lower leg muscles of anesthetized rats against a weighted pulley-bar. Muscle protein synthesis rates were measured by in vivo constant infusion of $\sp3$H-leucine following a single bout of resistance exercise. Specific messenger RNA levels were determined by dot-blot hybridization analysis using $\sp{32}$P-labelled DNA probes after a single bout and multiple bouts of phasic training. The effects of phasic training on increasing skeletal muscle mass was assessed. Between 12 and 36 hours following a single resistance exercise bout (24-192 contractions), total mixed and myofibril protein synthesis rates were significantly increase (32%-65%) after concentric (gastrocnemius m.) and eccentric (tibialis anterior m.) contractions. Eccentric contractions had greater effects on myofibril synthesis with more prolonged increases in synthesis rates. Lower numbers of eccentric than concentric contractions were required to increase synthesis. Cellular RNA was increased after exercise but the relative levels of skeletal $\alpha$-actin and cytochrome c mRNAs were unchanged. Since increases in synthesis rates exceeded increases in RNA, post-transcriptional mechanisms may be primarily responsible for increased protein synthesis after a resistance exercise bout. After 10-22 weeks of phasic eccentric resistance training, muscle enlargement (16%-30%) was produced in the tibialis anterior m. after all training paradigms examined. In contrast, gastrocnemius m. enlargement after phasic concentric training occurred after moderate (24/bout) but not after high (192/bout) repetition training. The absence of muscle growth in the gastrocnemius m. after high repetition training despite increased synthesis rates after the initial bout and RNA and possibly mRNA accumulation during training suggests a role for post-translational mechanisms (protein degradation) in the control of muscle growth in the gastrocnemius m. It is concluded that muscle protein during resistance exercise and training is regulated at several cellular levels. The particular response may be influenced by the exercise intensity and duration, the training frequency and the type of contractile work (eccentric vs. concentric) performed. ^

The regulation of JAB1 and its role in herceptin resistance

The Jun activation domain-binding protein (JAB1) is a c-Jun co-activator and a member of the COP9 signalosome. Additionally, it has recently been named a key negative regulator of the cyclin-dependent kinase inhibitor, p27. JAB1 overexpression has been observed in breast cancer and correlates with low p27 levels as well as poor prognosis, yet the mechanism of JAB1 deregulation is unknown. Data from our laboratory suggest that constitutive transcriptional activation of the jab1 gene is responsible for JAB1 protein overexpression. Therefore, we hypothesized that overexpression of JAB1 in breast cancer can be attributed to increased transcriptional activity. To identify potential positive regulators of JAB1, we characterized the promoter and found a 128 bp region that was critical for jab1 transcriptional activation. Our studies show that two oncogenic transcription factors, C/EBPβ and STAT3, play an important role in modulating jab1 transcription. Further, we have identified jab1 as a direct target gene of the SRC/STAT3 pathway. These studies provide insight to the mechanism of JAB1 overexpression in breast cancer and open up possibilities for therapies to inhibit its expression. ^ The development of the humanized monoclonal antibody, Herceptin (trastuzumab) targeting the HER2 (ErbB2) receptor has provided promising treatment to patients with aggressive HER2 positive breast cancer. However, many patients are resistant to Herceptin and additional therapies are needed to overcome resistance. Recent findings indicate that one mechanism of resistance involves AKT phosphorylation and subsequent mislocalization of the cyclin dependent kinase inhibitor, p27. We examined whether JAB1 facilitated degradation of p27 may be another mechanism of resistance to Herceptin. Our studies show that overexpression of JAB1 inhibited Herceptin induced G1-arrest and p27 accumulation. Interestingly, increased JAB1 levels were observed in two BT-474 Herceptin resistant clones. Targeted silencing of JAB1 increased p27 protein levels, reinstated a G1 checkpoint, and reduced cellular proliferation in the resistant clones. Our studies have demonstrated that inhibition of JAB1 sensitizes Herceptin resistant cells to treatment. Therefore, inhibition of JAB1 could provide a novel method of sensitizing resistant tumors to Herceptin-induced tumor growth arrest. ^

Analysis of Mycobacterium tuberculosis in the state of Texas for rifampin resistance using molecular beacons

Mycobacterium tuberculosis, a bacillus known to cause disease in humans since ancient times, is the etiological agent of tuberculosis (TB). The infection is primarily pulmonary, although other organs may also be affected. The prevalence of pulmonary TB disease in the US is highest along the US-Mexico border, and of the four US states bordering Mexico, Texas had the second highest percentage of cases of TB disease among Mexico-born individuals in 1999 (CDC, 2001). Between the years of 1993 and 1998, the prevalence of drug-resistant (DR) TB was 9.1% among Mexican-born individuals and 4.4% among US-born individuals (CDC, 2001). In the same time period, the prevalence of multi-drug resistant (MDR) TB was 1.4% among Mexican-born individuals and 0.6% among US-born individuals (CDC, 2001). There is a renewed urgency in the quest for faster and more effective screening, diagnosis, and treatment methods for TB due to the resurgence of tuberculosis in the US during the mid-1980s and early 1990s (CDC, 2007a), and the emergence of drug-resistant, multidrug-resistant, and extremely drug-resistant tuberculosis worldwide. Failure to identify DR and MDR-TB quickly leads to poorer treatment outcomes (CDC, 2007b). The recent rise in TB/HIV comorbidity further complicates TB control efforts. The gold standard for identification of DR-TB requires mycobacterial growth in culture, a technique taking up to three weeks, during which time DR/MDR-TB individuals harboring resistant organisms may be receiving inappropriate treatment. The goal of this study was to determine the sensitivity and specificity of real-time quantitative polymerase chain reaction (qPCR) using molecular beacons in the Texas population. qPCR using molecular beacons is a novel approach to detect mycobacterial mutations conferring drug resistance. This technique is time-efficient and has been shown to have high sensitivity and specificity in several populations worldwide. Rifampin (RIF) susceptibility was chosen as the test parameter because strains of M. tuberculosis which are resistant to RIF are likely to also be MDR. Due to its status as a point of entry for many immigrants into the US, control efforts against TB and drug-resistant TB in Texas is a vital component of prevention efforts in the US as a whole. We show that qPCR using molecular beacons has high sensitivity and specificity when compared with culture (94% and 87%, respectively) and DNA sequencing (90% and 96%, respectively). We also used receiver operator curve analysis to calculate cutoff values for the objective determination of results obtained by qPCR using molecular beacons. ^

The transcriptional repressor Delta EF1 controls resistance to the EGFR inhibitor erlotinib in human HNSCC lines

Epidermal Growth Factor Receptor (EGFR) overexpression occurs in about 90% of Head and Neck Squamous Cell Carcinoma (HNSCC) cases. Aberrant EGFR signaling has been implicated in the malignant features of HNSCC. Thus, EGFR appears to be a logical therapeutic target with increased tumor specificity for the treatment of HNSCC. Erlotinib, a small molecule tyrosine kinase inhibitor, specifically inhibits aberrant EGFR signaling in HNSCC. Only a minority of HNSCC patients were able to derive a substantial clinical benefit from erlotinib. ^ This dissertation identifies Epithelial to Mesenchymal Transition (EMT) as the biological marker that distinguishes EGFR-dependent (erlotinib-sensitive) tumors from the EGFR-independent (erlotinib-resistant) tumors. This will allow us to prospectively identify the patients who are most likely to benefit from EGFR-directed therapy. More importantly, our data identifies the transcriptional repressor DeltaEF1 as the mesenchymal marker that controls EMT phenotype and resistance to erlotinib in human HNSCC lines. si-RNA mediated knockdown of DeltaEF1 in the erlotinib-resistant lines resulted in reversal of the mesenchymal phenotype to an epithelial phenotype and significant increase in sensitivity to erlotinib. ^ DeltaEF1 represses the expression of the epithelial markers by recruiting HDACs to chromatin. This observation allows us to translate our findings into clinical application. To test whether the transcriptional repression by DeltaEF1 underlines the mechanism responsible for erlotinib resistance, erlotinib-resistant lines were treated with an HDAC inhibitor (SAHA) followed by erlotinib. This resulted in a synergistic effect and substantial increase in sensitivity to erlotinib in the resistant cell lines. Thus, combining an HDAC inhibitor with erlotinib represents a novel promising pharmacologic strategy for reversing resistance to erlotinib in HNSCC patients. ^

THE BACON CHOW STUDY: THE RELATIONSHIP BETWEEN INFECTIOUS ILLNESS AND GROWTH OF INFANTS

Multiple dietary deficiencies and high rates of infectious illness are major health problems leading to malnutrition and limitation of growth of children in developing countries. Longitudinal studies which provide information on illness incidence and growth velocity are needed in order to untangle the complex interrelationship between nutrition, illness and growth. From 1967 to 1973, researchers led by Dr. Bacon Chow of the Johns Hopkins University School of Hygiene undertook a quasi-experimental prospective study in Suilin Township, Taiwan to determine the effects of a nutritional supplement to the diets of pregnant and lactating women on the growth, development and resistance to disease of their offspring. This dissertation presents results from the analysis of infant morbidity and postnatal growth.^ Maternal nutritional supplementation has no apparent effect on the postnatal growth or morbidity of infants. Significant sex differences exist in growth response to illness and in illness susceptibility. Male infants have more diarrhea and upper respiratory illness. Respiratory illness is positively associated with growth rate in weight in the first semester of life. Diarrhea is significantly negatively associated with growth in length in the second semester. Small-for-date infants are more susceptible to illness in general and have a different pattern of growth response than large-for-date infants.^ Principal components analysis of illness data is shown to be an effective technique for making more precise use of ambiguous morbidity data. Multiple regression with component scores is an accurate method for estimating variance in growth rate predicted by indepenent illness variables. A model is advanced in which initial postnatal growth rate determines subsequent susceptibility to nutritional stress and infection. Initial growth rate is a function of prenatal nutrition, but is not significantly affected by maternal supplementation during gestation or lactation. Critical evaluation is made of nutritional supplementation programs which do not afford disease control.^

Platelets and Anti-Angiogenic Resistance in Ovarian Carcinoma

Background: Resistance to targeted anti-angiogenic therapy is a growing clinical concern given the disappointing clinical impact of anti-angiogenic. Platelets represent a component of the tumor microenvironment that are implicated in metastasis and represent a significant reservoir of angiogenic regulators. Thrombocytosis has been shown to be caused by malignancy and associated with adverse clinical outcomes, however the causal connections between these associations remain to be identified. Materials and Methods: Following IRB approval, patient data were collected on patients from four U.S. centers and platelet levels through and after therapy were considered as indicators of recurrence of disease. In vitro effects of platelets on cancer cell proliferation, apoptosis, and migration were examined. RNA interference was used to query signaling pathways mediating these effects. The necessity of platelet activation for in vitro effect was analyzed. In vivo orthotopic models were used to query the impact of thrombocytosis and thrombocytopenia on the efficacy of cytotoxic chemotherapy, the effect of aspirin on thrombocytosis and cancer, and platelet effect on anti-angiogenic therapy. Results: Platelets were found to increase at the time of diagnosis of ovarian cancer recurrence in a pattern comparable to CA-125. Platelet co-culture increased proliferation, increased migration, and decreased apoptosis in all cell lines tested. RNA interference implicated platelet derived growth factor alpha (PDGFRA) and transforming growth factor beta-receptor 1 (TGFBR1) signaling. Biodistribution studies suggested minimal platelet sequestration of taxanes. Blockade of platelet activation blocked in vitro effects. In vivo, thrombocytosis blocked chemotherapeutic efficacy, thrombocytopenia increased chemotherapeutic efficacy, and aspirin therapy partially blocked the effects of thrombocytosis. In vivo, withdrawal of anti-angiogenic therapy caused loss of therapeutic benefit with evidence of accelerated disease growth. This effect was blocked by use of a small-molecule inhibitor of Focal Adhesion Kinase. Anti-angiogenic therapy was also associated with increased platelet infiltration into tumor that was not seen to the same degree in the control or FAK-inhibitor-treated mice. Conclusions: Platelets are active participants in the growth and metastasis of tumor, both directly and via facilitation of angiogenesis. Blocking platelets, blocking platelet activation, and blocking platelet trafficking into tumor are novel therapeutic avenues supported by this data. Copyright © 2012 Justin Neal Bottsford-Miller, all rights reserved.

Pathways leading to apoptosis resistance in a murine B -cell lymphoma cell system

The aberrant activation of signal transduction pathways has long been linked to uncontrolled cell proliferation and the development of cancer. The activity of one such signaling module, the Mitogen-Activated Protein Kinase (MAPK) pathway, has been implicated in several cancer types including pancreatic, breast, colon, and lymphoid malignancies. Interestingly, the activation of MAP-Kinase-Kinase-Kinase proteins often leads to the additional activation of NF-κB, a transcription factor that acts as a cell survival signal through its control of antiapoptotic genes. We have investigated the role of a specific dimer form of the NF-κB transcription factor family, NF-κB1 (p50) homodimers, in its control of the proto-oncogene, Bcl-2, and we have identified the MEK/ERK (MAPK) signaling cascade as a mediator of NF-κB1 activity. ^ Two murine B cell lymphoma cell lines were used for these studies: LY-as, an apoptosis proficient line with low Bcl-2 protein expression and no nuclear NF-κB activity, and LY-ar, a nonapoptotic line with constitutive p50 homodimer activity and 30 times more Bcl-2 protein expression than LY-as. Experiments modulating p50 activity correlated the activation of p50 homodimers with Bcl-2 expression and additional gel shift experiments demonstrated that the Bcl-2 P1 promoter had NF-κB sites with which recombinant p50 was able to interact. In vitro transcription revealed that p50 enhanced the production of transcripts derived from the Bcl-2 P1 promoter. These data strongly suggest that Bcl-2 is a target gene for p50-mediated transcription and suggest that the activation of p50 homodimers contributes to the expression of Bcl-2 observed in LY-ar cells. ^ Studies of upstream MAPK pathways that could influence NF-κB activity demonstrated that LY-ar cells had phosphorylated ERK proteins while LY-as cells did not. Treatment of LY-ar cells with the MEK inhibitors PD 98059, U0126, and PD 184352 led to a loss of phosphorylated ERK, a reversal of nuclear p50 homodimer DNA binding, and a decrease in the amount of Bcl-2 protein expression. Similarly, the activation of the MEK/ERK pathway in LY-as cells by phorbol ester led to Bcl-2 expression that could be blocked by PD 98059. Furthermore, treatment of LY-ar cells with TNFα, an IKK activator, did not change the suppressive effect of PD 98059 on p50 homodimer activity, suggesting an IKK-independent pathway for p50 homodimer activation. Lastly, all three MEK inhibitors sensitized LY-ar cells to radiation-induced apoptosis. ^ These data indicate that the activation of the MEK/ERK MAP-Kinase signaling pathway acts upstream of p50 homodimer activation and Bcl-2 expression in this B cell lymphoma cell system and suggest that the activation of MEK/ERK may be a key step in the progression of lymphoma to advanced-staged disease. Other researchers have used MEK inhibitors to inhibit cell growth and sensitize a number of tumors to chemotherapies. In light of our data, MEK inhibitors may additionally be useful clinically to radiosensitize cancers of lymphoid origin. ^