CELLULAR SIGNALING IN CANCER

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It is generally accepted that cancers are caused by genetic and epigenetic alterations, which affect multiple oncoproteins and tumor suppressor proteins, resulting in deregulated cell signaling molecules, which override normal control mechanisms. These changes eventually lead to enhanced proliferation, evasion from apoptosis, and at the final stage cause invasion and metastasis. Crosstalk among signaling pathways is necessary for tumorigenesis. Each step of tumor progression results from cooperation of multiple pathways which interact with each other at distinct levels. Tissue specific tumor phenotype is contributed by crosstalk among unique signaling pathways. In addition, tumor-stromal interaction represents another level of crosstalk, which has been considered as an indispensable part of tumorigenesis. Similarly, tumor cells evade immune surveillance through interacting and regulating with immune system. Crosstalk provides cancer cells with redundant mechanisms to adapt to a versatile environment. Targeting one oncogene or tumor suppressor gene has been favored in the past decade to develop cancer therapy. However, this type of target therapy has not been very successful in most circumstances, largely due to signaling compensation after treatment. Previous experience has demonstrated that our current knowledge on cancer signaling remains limited and such complex crosstalk requires more detailed investigation. Uncovering the signaling crosstalk not only helps us understand tumor development and progression, but it can also provide guidance for developing novel cancer therapies.

CANCER GENETICS  

During the past several decades, molecular genetics has been providing answers concerning the mechanisms that are involved in the pathogenesis of human malignancies. Essentially two different mechanisms are involved ²³. One results in the activation of cellular proto-oncogenes. This activation can occur by activation of transcription, mutation, or gene fusion. One example of transcriptional activation of proto-oncogene is the deregulation of the c-myc oncogene by its juxtaposition to one of the human immunoglobulin loci in Burkitt's lymphomas. Oncogene overexpression may also be the result of oncogene amplification in both hematopoietic and solid malignancies. For example, the amplification of the c-myc oncogene is observed in a fraction of breast tumors and in many different types in malignancies; the N-myc gene is amplified in a fraction of neuroblastomas (12), and the erbB2 or neu gene is amplified in breast cancer ²⁴. Mutation of oncogenes has long been recognized as a major means to transform human cells. The ras family genes can be activated by a point mutation which affects about 30% of human cancers. Mutations of other oncogenes such as PI3K and BRAF are also critical for specific tumor type. The first example of transforming chimeric genes to be described was the result of the fusion of the BCR gene with the Abl proto-oncogene in chronic myelogenous leukemia. Recently, an additional example has been described in patients with acute promyelocytic leukemias carrying a t(15;17) chromosome translocation by which a retinoic acid receptor gene is activated ²³. It is becoming clear that this mechanism is very common in the pathogenesis of hematopoietic malignancies.

Another molecular mechanism involved in the development of human tumors is loss of function of a tumor suppressor gene ²⁵. As postulated by Knudson, loss of function of both alleles of a tumor suppressor gene may lead to malignancy ²⁵. Thus is the case of familial retinoblastoma where a copy of a faulty retinoblastoma gene is inherited, while the normal copy of the gene is either lost or mutated during tumorigenesis. Such mechanism has been shown to be involved in the pathogenesis of a variety of human tumors. Mutations and deletions of the p53 gene have been observed in a fraction of a large variety of human neoplasms. Loss of function for several cancer suppressor genes such as Rb and p53 and loss of heterozygosity at numerous loci have been observed in a variety of solid tumors. Thus, it seems likely that loss of function at several cancer suppressor loci may play a major role in the pathogenesis of some of the most common solid malignancies.

MicroRNAs (miRNAs) are a new class of RNAs that are not translated to proteins and regulate gene expression at posttranscriptional level. The miRNA regulates gene expression by translational repression, mRNA cleavage, and mRNA decay initiated by miRNA-guided rapid deadenylation. Recent studies show that some miRNAs regulate the cell proliferation and apoptosis processes that are important in cancer formation ²⁶. Increasing evidence showed that they play important roles in cancer development. miRNAs can be either oncogenes or tumor suppressors ²⁶. Overexpressed miRNAs in cancers, such as mir-17-92, may function as oncogenes and promote cancer development by negatively regulating tumor suppressor genes and/or genes that control cell differentiation or apoptosis. Underexpressed miRNAs in cancers, such as let-7, function as tumor suppressor genes and may inhibit cancers by regulating oncogenes and/or genes that control cell differentiation or apoptosis ²⁶. In addition, miRNA therapy could be a powerful tool for cancer prevention and therapeutics.

CANCER EPIGENETICS

Neoplasias have a distinct pattern of disrupted pathways, which are the result not only of genetic alterations but also of heritable patterns of disrupted gene expression ⁴. Epigenetics refers to these clonal changes in patterns of gene expression that are mediated by mechanisms that do not alter the primary DNA sequence.

Major epigenetic mechanisms include DNA cytosine methylation, histone modifications such as acetylation and methylation, and small non-coding RNA controlled pre- and posttranscriptional regulation of gene expression. Chromatin DNA can be covalently methylated at the C-5 position of cytosine, which is catalyse by DNA methyltransferases (DNMTs). In mammals, DNA methylation occurs primarily at symmetrical CpG dinucleotides. Physiologically, DNA methylation is involved in imprinting establishment, retrotransposons silencing and X-chromosome inactivation ^27,28. In cancer cells, DNA methylation pattern is altered, which includes global demethylation and promoter localized hypermethylation. Global hypomethylation is correlated to aging process and may lead to unwanted activation of oncogenes, loss of imprinting and chromosome instability which are important for tumorigenesis. Localized hypermethylation of tumor suppressor genes, such as p16, APC and p53, has also been demonstrated in cancer initiation and remains critical in tumor progression ²⁹.

Chromatin structure is thought to be inheritable through cell division and a major form of epigenetic regulation of gene expression ^30,31. Post-translational modifications of histones, including acetylation and methylation of conserved lysine residues on the amino terminal tail, are also dynamically regulated by chromatin modifying enzymes with opposing activities (3). Generally, lysine acetylation mediated by histone acetyltransferases (HATs) marks transcriptionally competent regions. In contrast, histone deacetylases (HDACs) catalyze lysine deacetylation which is usually associated with transcriptionally inactive chromatin structures ³². Several groups of histone modifying proteins involved in the control of gene expression have been identified. For example, the trithorax group (TrxG) and the polycomb group (PcG) proteins appear to have opposing roles in the regulation of gene expression. Methylation of histone H3 on lysine residue 4 (H3K4) by some members of the TrxG is most often associated with positive regulation of gene expression, whereas methylation of histone H3 on lysine residue 27 (H3K27) by PcG members represses gene expression ³³. Abnormal TrxG or PcG function often results in aberrant gene expression that can lead to tumor development in model systems, suggesting that deregulation of these epigenetic programs can initiate tumor formation ^34,35. Translocations that involve the mixed lineage leukaemia (MLL) gene identify a unique group of acute leukaemias that have a poor prognosis. The MLL gene encodes a DNA-binding protein that methylates H3K4, and positively regulates gene expression. The MLL protein can transform haematopoietic cells into leukaemia stem cells, which further demonstrates that epigenetic alteration is an important part of MLL development ³⁶.

Epigenetic silencing of tumor suppressor genes by DNA methylation and histone modification is considered to be an early event in tumorigenesis. Silenced genes can be used as an indicator for early epigenetic alterations and present novel diagnostic and therapeutic cancer targets. For example, to identify very early epigenetic events that occur in breast cancer, microarray-based screening has been performed to identify gene pathways that were suppressed in immortalization of human mammary epithelia cells ³⁷. Down regulation of multiple TGF-beta family members in the immortalized mammary epithelial cells is associated with a decrease in histone H3 lysine 27 trimethylation and an increase in histone H3 lysine 9 dimethylation and deacetylation, indicating that the TGF-beta signaling pathway is a novel target for gene activation by epigenetic therapy ³⁷.

CANCER SIGNALING PATHWAYS

Transformation of a normal cell into a cancer cell requires the acquisition of several essential alterations in cell physiology, including self-sufficiency in growth signals, insensitivity to antigrowth signals, evasion of apoptosis, limitless replicative potential, sustained angiogenesis, and tissue invasion and metastasis ⁴.

The molecular alterations can affect any step of a signaling pathway that is involved in cell proliferation and survival. The defects may be at the levels of ligands, receptors, transcriptional factors or signal transducers. For example, epithelial growth factor receptor (EGFR) and its respective ligands are overexpressed in various tumors, and this over-expression correlates with poor prognosis in selected cancers. Crosstalk with other signaling pathways could further augment the constitutiveness of cancer growth. For example, the large family of G-protein-coupled receptors (GPCRs) has been reported to transactivate EGFR via both ligand-dependent and independent mechanisms ³⁸. This form of receptor crosstalk may contribute to the modest clinical responses to EGFR-targeted therapies, since GPCR is variously expressed in tumors. The progression of colon, lung, breast, head and neck, prostate and ovarian cancers have all been reported to be mediated, at least in part, by GPCR-EGFR crosstalk. Increased understanding of the specific signaling pathways involved in this type of crosstalk will facilitate the identification of new biomarkers and therapeutic targets ³⁸.

Crosstalk with other signaling pathways could also result in different or even opposite cellular effects. For example, transforming growth factor-β (TGF-β) family members show either dual tumor suppressive or oncogenic effects depending on their cellular context ³⁹.  In addition to its classic signaling through Smads, TGF-β activates mitogen-activated protein kinase (MAPK) signaling pathways, which crosstalk with Smad signaling and regulate growth, survival and motility of cells. MAPK can act as either positive or negative regulators of Smad signaling. The c-Jun N-terminal kinases (JNK) and p38 MAPKs phosphorylated Smad2 and Smad3 and promote their nuclear translocation ^40-42. On the other hand, the extracellular signal-regulated kinases (ERK1/2) phosphorylate Smads and prevent them from translocation to the nucleus, thus leading to repression of Smad-dependent gene transcription ^43,44. During tumorigenesis, cells often lose the response to the tumor suppressive effects of TGF-β, which then acts as an autocrine tumor promoting factor by enhancing cancer invasion and metastasis.

One of the major mediators of EGFR signaling is Ras protein, which is a proto-oncogene and mutated in about 30% of human cancers. Being a small GTPase, Ras act as a molecular switch cycling between GDP and GTP binding states ⁴⁵.  Mutations of Ras impair its intrinsic GTPase activity and lead to preferential binding to GTP which confers a constituitive stimulation of downstream signaling pathways. Three isoforms of Ras genes exist in mammalian cells, but they are not evenly distributed in various malignant tissues.  KRAS mutation is the most common one in human cancer and are prevalent in pancreatic, colorectal, endometrial, biliary tract, lung, cervical cancers and myeloid malignancies ⁴⁶. GTP bound Ras can interact with more than 20 effectors, including Raf, phosphatidylinositol 3-kinase (PI3K) and Ral guanine nucleotide-dissociation stimulator (RalGDS), thus regulate various cellular responses including proliferation, survival and differentiation (Fig.1.1) ⁴⁷. Despite its importance in many aspects of tumor phenotype establishment, how mutated Ras protein plays its role in tumorigenesis is still not well understood. Particularly, classic effectors of Ras may not account for all effects of Ras mutation. Crosstalk with other signaling pathways, which are either oncogenic or tumor suppressive, provides a potential efficient way for Ras mutation to modulate cell behavior. Hedgehog (Hh) pathway may be one of such pathways that interact with Ras and mediate Ras tumorigenic property.

Hh signaling is a developmental pathway and components of it were first identified and characterized in drosophila ⁴⁸. Hh is a secreted molecule functioning through binding to its transmembrane receptor Patched (PTCH), which then lead to activation of another transmembrane protein Smoothened (SMO). Gli is a transcriptional factor and mediates the intracellular effects of Hh/PTCH/SMO activation (Fig.1.1). Hh pathway is regulated by different mechanism at different cellular context. One of the major regulation points is Gli protein expression and activation. Gli protein can be phosphorylated sequentially by protein kinase A (PKA), glycogen synthase kinase 3β (GSK3β) and casin kinase 1 (CK1) and directed to proteasome dependent degradation (Huangfu 3). Therefore, stabilization of Gli protein could lead to activation of Hh pathway. In normal adult cells, Hh pathway is not activated. Reactivation of Hh pathway activity by mutation or expression/activity changes of pathway members plays critical roles in many human cancers such as pancreatic cancer and skin cancer. In pancreatic cancer, Hh pathway is activated in the early stage of tumorigenesis and its activity is essential for cancer development, but little is known about the mechanism of Hh pathway activation. Cooperation of Hh pathway with other major oncogenic pathway has not been extensively investigated despite its significance in the cancer formation process. Recent report that MAPK could promote Hh target gene transcription sheds light on potential collaboration between Hh pathway and other intracellular signaling events ⁴⁹. Since KRAS is also mutated in the early stage of pancreatic cancer formation and MAPK is a major downstream molecule of Ras signaling, it’s conceivable that there’s a potential crosstalk between KRAS and Hh pathway. This hypothesis is discussed and demonstrated in Chapter 2.

Cyclic AMP (cAMP) signaling is a classic cell signaling pathway and can be important for cancer growth. Increased intracellular cAMP can either inhibit or promote apoptosis, depending upon the specific cellular context ⁵⁰. cAMP synergizes strongly with the Glucorcorticoid (GC) signaling in inducing lymphoid-cell apoptosis ^51-53. The mechanism underlying this synergy is poorly understood, although induction of BIM (Bcl-2–interacting mediator of cell death) has been suggested to play a role in the eventual apoptotic effect ^53-55. There are two intracellular receptors for cAMP, PKA and exchange protein activated by cAMP (Epac) (fig.1.1) ⁵⁶. As discussed above, PKA can phosphorylate Gli and promote Gli degradation by ubiquitin-proteasome pathway. In addition, phosphorylation of Gli1 by PKA also prevents Gli1 translocating from cytoplasm to nucleus in COS7 cells ⁵⁷. Therefore, PKA is considered as a negative regulator of Hh signaling. Hh signaling regulates cell proliferation and differentiation in the early phase of lymphoid development, but is inactivated in mature lymphoid cells⁵⁸. We hypothesize that Hh pathway may be reactivated and contribute to the lymphoid malignant cell growth and survival and that cAMP may exert its synergism with GC through regulating Hh signaling by PKA. In Chapters 3 and 4, we test this hypothesis in the CEM acute lymphoblastic leukemia cells.