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levels of type 1 cytokines these cells not only support the expansion of antigen specific cytotoxic T cells but also have the capability of directly modulating the tumor microenvironment to generate epitope spreading which would lead to tissue destruction.

Clinical strategies focused on eliciting tumor antigen specific Th1 are showing promise in terms of benefiting clinical outcomes in patients with breast cancer.

the cell's environment for the presence of growth factors and nutrients. If the cell needs additional resources, mTOR can increase nutrient uptake and promote angiogenesis. The signaling protein mTOR can also increase the activity of AKT, thus enhancing the other downstream effects of this protein. Because mTOR and its signaling partners are so powerful, the cell has mechanisms in place to regulate them. One important watchdog is phosphatase and tensin homolog (PTEN); a protein that, in humans, is encoded by the PTEN gene. Mutations of this gene are a step in the development of many cancers. PTEN orthologs have been identified in most mammals for which complete genome data are available. This gene was identified as a tumor suppressor that is mutated in a large number of cancers at high frequency. The protein encoded by this gene is a phosphatidylinositol-3,4,5-trisphosphate

PTEN removes the phosphate groups added to membrane phospholipids by PI3-kinase. This prevents activation of AKT and its downstream pathways (Nicholson and Anderson, 2002).

The signaling pathway that includes mTOR is highly active in many cancer cells. This can be the result of amplification or mutation of the PI3-kinase gene; amplification or mutation of the AKT gene; or loss of function of PTEN (Halilovic et al., 2010; She et al., 2010).

Increased activity of some growth factor receptors can also enhance the activity of the pathway. Activation of the mTOR pathway is associated with poor prognosis in many cancers, including breast cancer, and is linked to resistance to many types of therapy.

During the course of tumor progression, cancer cells acquire a number of characteristic alterations. These include the capacities to proliferate independently of exogenous growth-promoting or growth-inhibitory signals, to invade surrounding tissues and metastasize to distant sites, to elicit an angiogenic response, and to evade mechanisms that limit cell proliferation, such as apoptosis and replicative senescence (Martin, 2003). These properties reflect alterations in the cellular signaling pathways that in normal cells control cell proliferation, motility, and survival. Many of the proteins currently under investigation as possible targets for cancer therapy are signaling proteins that are components of these pathways.

Normal hematopoiesis is dependent on intricately regulated signaling cascades that are mediated by cytokines and their receptors. Orderly function of these pathways leads to the generation of appropriate constellation of hematopoietic cells, and their abnormal activation results in neoplastic transformation, impaired apoptosis, and uncontrolled proliferation

(Ravandi et al., 2003). Cytokines function in a redundant and pleiotropic manner; different cytokines can exert similar effects on the same cell type, and any particular cytokine can have several differing biological functions (Kishimoto et al., 1989). This complexity of function is a result of shared receptor subunits as well as overlapping downstream pathways culminating in activation of common transcription factors (Rane et al., 2002).

Insights into the cellular signaling pathways led to the discovery and the identification of a family of nonreceptor tyrosine kinases (TKs) (Rane et al., 2002) called Jaks and their target proteins ―Stats‖, which mediate gene transcription (Decker et al., 1999). The Jak-Stat pathways are commonly activated during cytokine signaling through phosphorylation of specific tyrosine residues (Rane et al., 2002). The interaction of a cytokine with its receptor induces its tyrosine phosphorylation and leads to activation of downstream protein; TKs including Jaks and Stats. Apart from their catalytic domain, protein TKs contain several other characteristic motifs including the SH2, SH3, and pleckstrin homology domain, which enable them to interact with other signaling molecules and propagate the message (Pawson et al., 1995).

The phosphorylation of serine and threonine residues is integral to the activation of numerous other intracellular proteins that mediate a number of other signaling pathways.

Cytokine receptors without intrinsic kinase activity transmit their signals primarily through activation of Jak kinases. It has been established that these pathways interact with serine/threonine kinase cascades such as the Ras/Raf/MEK/ERK (McCubrey et al., 2000).

For example, after ligand binding, the βsubunit of IL-3 and GM-CSF receptors are phosphorylated and, through recruitment of adaptor proteins such as Shc, Grb2, and Sos, activate the Ras signaling pathway. The activation of Raf is followed by downstream activation of ERK1 and ERK2, and increased expression of transcription factors fos and c-jun.

The AKT/protein kinase B (PKB) is a cardinal node in diverse signaling cascades important in both normal cellular physiology and various disease states (Testa and Tsichlis, 2005).

The AKT signaling regulates cell proliferation and survival, cell growth (size), glucose metabolism, cell motility and angiogenesis (ibid). Aberrant regulation of these processes result in cellular perturbations considered hallmarks of cancer, and numerous studies testify to the frequent hyperactivation of AKT signaling in many human cancers. Various

oncoproteins and tumor suppressors intersect the AKT signal transduction pathway and are activated or inactivated, respectively, in cancer.

The AKT is a sreine/threonine kinase that mediates signaling downstream of tyrosine kinase receptors like Type1-insulin-like growth factor receptor (GF-IR) (Zou et al., 2011).

Mammary tumours induced by elevated expression of the IG-IR are associated with hyperactivation of AKT. Three mammalian isoforms of AKT (AKT1, AKT2, and AKT3) regulate distinct physiologic properties within cells. In lung cancer it has been observed that AKT1 or AKT2 significantly delay tumour onset and tumour growth rate but not significantly alter metastasis (ibid).

The AKT is required for Stat5 activation and mammary differentiation (Chen et al., 2010).

The authors observed in experimental animals that mice lacking AKT1 but not AKT2 exhibit a pronounced metabolic defects during late pregnancy and lactation that results from a failure to upregulate Glut1 as well as several lipid synthetic enzymes. Chen et al., (2010) demonstrated an unexpected requirement for AKT in Prlr-Jak-Stat5 signaling and established AKT as an essential central regulator of mammary epithelial differentiation and lactation. Hence therapeutic strategies targeting activation of individual AKT isoform will prove less effective than simultaneously inhibiting the activity of all three AKT isoforms for treatment of breast cancer (Watson and Moorehead, 2013)

The AKT1 and AKT2 play important role in the bcatenin/Tcf-4 signaling pathway promoting malignant transformation and a potential targets for brain glioma therapy;

effective way to prevent metabolism of gliomas (Zou et al., 2011).

The AKT signaling and activation has been associated with vascular maturation and angiogenesis (Somanath et al., 2008), cardiomyocyte survival and metabolism (Mushin, 2011), suppress tumour cell proliferation and neuroendocrine marker in GI carcinoid tumours (Pitt et al., 2009). Over activation of PI3K/AKT signaling facilitates tumour proliferation in several cancers. Therefore targeting AKT/PI3K pathway may enhance the management of carcinoid diseases.

Progress in the understanding of signaling through the Raf/MEK/ERK cascade has lead to the achievements in the inhibition of this signaling pathway (McCubrey et al., 2001). In many cases, however, activation of the Raf/MEK/ERK pathway alone is not responsible for oncogenic transformation rather evidence has suggested that the PI3K/Akt pathway is a viable target for novel antineoplastic drugs.

Protein-tyrosine kinases (PTKs) are important regulators of intracellular signal-transduction pathways mediating development and multicellular communication in metazoans (Blume-Jensen and Hunter, 2001). Their activity is normally tightly controlled and regulated.

Perturbation of PTK signaling by mutations and other genetic alterations results in deregulated kinase activity and malignant transformation. The lipid phosphoinositide 3-OH kinase (PI3K) and some of its downstream targets, such as the protein-serine/threonine kinases Akt and p70 S6 kinase (p70S6K), are crucial effectors in oncogenic PTK signaling.

The relation between dysregulated PI3K activities has been associated with different cancers. A mutated form of the p85 subunit of PI3K has been isolated in a Hodgkin's lymphoma-derived cell line (CO) (Jucker et al., 2002). The PI3K/Akt pathway has been shown to be the predominant growth-factor-activated pathway in lymph node carcinoma of prostate (LNCaP) human prostate carcinoma cells (Lin et al., 1999), breast cancer (Fry, 2001), lung cancer (Lin et al., 2001), melanomas (Krasilnikov et al., 1999), and leukemia (Martinez-Lorenzo et al., 2000) among others. Further evidence has shown that Akt (protein kinase B, PKB), a downstream kinase of PI3K, is also involved in malignant transformation (Nicholson and Anderson, 2002). Inhibition of this pathway is a promising approach for novel chemotherapeutic agents.

The PI3K associates with the β chain of IL-3 receptor, recruits PKB/AKT by phosphorylation of its serine residues, and transmits cellular survival signals (Tilton et al., 1997). Protein-tyrosine kinases (PTKs) are important regulators of intracellular signal-transduction pathways mediating development and multicellular communication in metazoans. Their activity is normally tightly controlled and regulated. Perturbation of PTK signaling by mutations and other genetic alterations results in deregulated kinase activity and malignant transformation. The lipid kinase phosphoinositide 3-OH kinase (PI3K) and some of its downstream targets, such as the protein-serine/threonine kinases Akt and p70 S6 kinase (p70S6K), are crucial effectors in oncogenic PTK signaling.

A number of oncogenes with constitutive kinase activity derived from genes including c-ABL, c-FMS, FLT3, c-KIT, and PDGFRβ, are normally involved in the regulation of hematopoiesis (Djordjevic and Driscoll, 2002). The kinase activity of the oncogene is constitutively activated by mutations that remove inhibitory domains of the molecule or induce the kinase domain to adopt an activated configuration. As a result of such

constitutive activation a number of signaling cascades such as the Jak-Stat pathway, the Ras/Raf/MAPK pathway, and the PI3K pathway are activated.

RAS is another signaling molecule that has been associated with tumourigenesis.The importance of the interaction of mutant RAS with endogenous p110α in tumor development was established by the findings that mice with mutations in the RAS binding domain (RBD) of p110α were highly resistant to mutant Kras-induced lung cancer formation and mutant Hras-induced skin cancer formation (Pylayeva-Gupta et al., 2011; Castellano et al., 2013).

It has been speculated that the signaling pathway requirements for tumor cells to survive (tumor maintenance) may be considerably relaxed relative to the signaling pathway activity needed to convert a normal cell into a tumor cell (tumor development). As tumor maintenance is more relevant than tumor initiation to the treatment of preexisting human cancers, the question of whether blocking the interaction of RAS with p110α will affect the maintenance of an existing RAS mutant tumor is of considerable importance.

Activating point mutations in the genes encoding the RAS subfamily of small GTP binding proteins drive the formation of a large proportion of human tumors. RAS proteins control cell growth through several direct effector enzyme families, the best studied of which are RAF kinases, type I phosphoinositide (PI)3-kinases, and RAL-guanine nucleotide exchange factors (RAL-GEFs) (Pylayeva-Gupta et al., 2011). Of these, BRAF and PIK3CA, the genes encoding BRAF and the p110α PI3-kinase catalytic subunit, respectively, are frequently activated by somatic mutation in human cancer, with overall mutation frequencies around 10% for each. PI3-kinase activity is further implicated in carcinogenesis by the frequent inactivation of the inositol lipid 3′phosphatase PTEN.

Inhibition of PI3-kinase activity has been reported to have a significant impact on RAS-induced tumors in some settings, especially when combined with inhibition of the RAF/mitogen-activated protein kinase extracellular signal-regulated kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway (Sos et al., 2009; Halilovic et al., 2010; She et al., 2010). However, it has been unclear whether PI3-kinase signaling is more important for RAS mutant tumors compared to tumors with other oncogenic drivers or to normal cells. Also, where such PI3-kinase dependency exists, it is not known whether this is because of acute RAS-induced activation of PI3-kinase through direct interaction with the RBD of p110 or more indirect and long-term mechanisms, such as transcriptional upregulation of ligands of growth factor receptor tyrosine kinases.

The work of Castellano et al., (2013) among other novel discoveries observed that removal of RAS interaction with PI3-Kinase p110α in early-stage tumors reduces tumor burden, in established tumors induces partial tumor regression as well as inhibits tumor progression.

Also they discovered that combined PI3-Kinase pathway inhibition with MEK inhibition causes enhanced tumor regression.

It has been documented in experimental animal (mice) that lung tumors driven by activated Kras, and the direct binding of RAS to p110α is a critical component of the regulation of this PI3-kinase isoform. However, ablating only the RAS interaction with p110α has less systemic toxicity for the mice than total p110α loss, as exemplified by less disruption of glucose homeostasis and also better general health status (ibid). The authors noticed however, that ubiquitous loss of p110α in adult mice does not lead to very major effects on mouse viability when compared with p110α loss or inactivation in developing embryos (Foukas et al., 2006), possibly reflecting some redundancy between PI3-kinase isoforms, as well as inevitably less than 100% deletion of Pik3ca on Cre activation. Pharmacological strategy that target RAS interaction with p110α has fewer adverse effects than targeting p110α lipid kinase activity, but it still is effective in opposing tumor progression.

Recently, some progress has been made in identifying small molecules that bind directly to RAS (Sun et al., 2012; Shima et al., 2013), and these might eventually lead to drugs that would be able to block the interaction of RAS with downstream effector enzymes.

However, the relatively low potency of the compounds identified, which is low μM at best, has precluded assessment of their efficacy in the Kras lung cancer mouse model.

Another likely advantage of direct RAS effector interaction site targeted drugs would be that they would also be expected to inhibit other RAS effector pathways such as RAF/MEK/ERK, which are activated from the same region of RAS. It has long been shown that inhibition of MEK and PI3-kinase together promotes death of RAS mutant cancer cells (Shima et al., 2013), although this approach has significant toxicities and may not show enhanced selectivity for the RAS mutant phenotype relative to cells with wild-type RAS.

Combining pharmacological inhibition of MEK together with genetic blockade of RAS interaction with p110α also considerably improved tumor clearance in the model system used, as did drug-mediated inhibition of both MEK and type I PI3-kinases. Use of a p110α isoform-specific PI3-kinase inhibitor had similar effects to a pan type I PI3-kinase inhibitor, reinforcing the view that p110α is the critical PI3-kinase isoform. The authors opined that

RAS does not control p110β through direct interaction (ibid), and hence it is unlikely that inhibition of this ubiquitous isoform will be helpful in RAS-driven cancer.

E-cadherin- adhesion molecule is another signaling protein in adherens junction (Zhou et al., 2011). The cytoplasmic region of E-cad directly interacts with β –catenin (β-cat), which in turn interacts with actin cytoskeleton. Loss of E-cad leads to cell-cell dissociation and acquisition of a migratory phenotype during development, tissue remodeling or carcinogenesis. In addition to E-cad, other molecular constituents of the adherens junction have important roles in maintaining cell-cell adhesion within the epithelium and preventing tumour invasiveness during carcinogenesis (ibid). Among them α-cat is essential for coordinating actin dynamics and inversely linking cell adhesion with proliferation.

Another important signaling molecule is the Slit family. Slit family of guidance cues binds to roundabout (Robo) receptors and modulates cell migration (Zhou et al., 2011). Slit-Robo signaling regulates the repulsion or attraction of projecting axons and migrating neurons during development of nervous system. Slit2 secreted by vascular endothelial cells binds to Robo1 on leucocytes and acts as endogenous inhibitors of leucocyte chemotaxis.

Additionally slit2 mediates directional migration of malignant cells. Slit2 protein secreted by solid tumours binds Robo1 expressed on vascular and lymphatic endothelial cells to stimulate angiogenesis and lymphangiogenesis.

Slit 2 has been associated in human colorectal carcinoma tissue and liver. Slit-Robo signaling enhances tumour growth and liver metastasis, recruits Kakai and ubiqitinates E-cad for lysosomal localization (Palacia et al., 2005), degrades E-E-cad through Hakai (Lu et al., 2003), fails to suppress E-cad transcription and induces transformation of MEK293 cells. Expression of Slit2 and Robo1 have been found to correlate with enhanced metastasis and shortened survival (Zhou et al., 2011).

Insulin-like growth factor (IGF); another important signaling protein, contributes to malignant transformation of haemopoietic progenitor by MLL-AF9 onco-protein (Jenkins et al., 2012). Malignant transformation of normal haemopoietic progenitor is a multiple step process that requires interaction between collaborating oncogenic signals and critical junction. MLL to AF-9 fusion oncogene contribute to myeloid leukemigenesis by driving a haemopoietic stem cell-like self-renewal gene expression signature in committed myeloid progenitor.

Notch signaling plays a key role in the normal development of many tissues and cell types, through diverse effects on differentiation, survival, and/or proliferation that are highly dependent on signal strength and cellular context (Allenspach et al., 2002). Because perturbations in the regulation of differentiation, survival, and/or proliferation underlie malignant transformation, pathophysiologic Notch signals potentially contribute to cancer development in several different ways. Notch signaling was first linked to tumorigenesis through identification of a recurrent t(7;9)(q34;q34.3) chromosomal translocation involving the human Notch1 gene that is found in a small subset of human pre-T-cell acute lymphoblastic leukemias (T-ALL) (Ellisen et al., 1991). Since this discovery, aberrant Notch signaling has been suggested to be involved in a wide variety of human neoplasms.

Notch genes, named after the notched wing phenotype of mutant Drosophila, encode highly conserved cell surface receptors. The Notch signaling pathway, in which almost all elements are conserved from Drosophila to humans, consists of Notch receptors, ligands, negative and positive modifiers, and transcription factors.

Notch activation is associated in lymphoblastic leukemia/lymphomas, other lymphoproliferative disorders, and mammary gland tumors (Allenspach et al., 2002).Other findings suggested that certain epithelial tumors arise through downregulation of Notch signaling. Notch potentially functions as an oncoprotein or a tumor suppressor in certain disease conditions and during normal development (Al-Hussaini et al., 2011).

Transforming growth factor beta (TGF-β) is a protein that controls proliferation, cellular differentiation and other functions in most cells. It is a type of cytokine which plays a role in immunity, cancer, bronchial asthma, heart disease, diabetes, hereditary hemorrhagic telangiectasia, Marfan syndrome, Vascular Ehlers-Danlos syndrome, Loeys–Dietz syndrome, Parkinson's disease and AIDS (Herpin, 2004).TGF-beta is secreted by many cell types, including macrophages, in a latent form in which it is complexed with two other polypeptides, latent TGF-beta binding protein (LTBP) and latency-associated peptide (LAP). Serum proteinases such as plasmin catalyze the release of active TGF-beta from the complex. This often occurs on the surface of macrophages where the latent TGF-beta complex is bound to CD36 via its ligand, thrombospondin-1 (TSP-1). Inflammatory stimuli that activate macrophages enhance the release of active TGF-beta by promoting the activation of plasmin. Macrophages can also endocytose IgG-bound latent TGF-beta complexes that are secreted by plasma cells and then release active TGF-beta into the extracellular fluid (ibid).

TGF-beta acts as an antiproliferative factor in normal epithelial cells and at early stages of oncogenesis. Some cells that secrete TGF-β also have receptors for TGF-β. This is known as autocrine signaling. Cancerous cells increase their production of TGF-β, which also acts on surrounding cells.

Signaling from transforming growth factor-beta (TGF-beta) through its unique transmembrane receptor serine-threonine kinases plays a complex role in carcinogenesis, having both tumor suppressor and oncogenic activities (de Caestecker, 2000; Matise et al., 2012). Tumor cells often escape from the antiproliferative effects of TGF-beta by mutational inactivation or dysregulated expression of components in its signaling pathway.

Decreased receptor function and altered ratios of the TGF-beta type I and type II receptors found in many tumor cells compromise the tumor suppressor activities of TGF-beta and enable its oncogenic functions. Recent identification of a family of intracellular mediators, the Smads, has provided new paradigms for understanding mechanisms of subversion of TGF-beta signaling by tumor cells (de Caestecker, 2000). In addition, several proteins recently have been identified that can modulate the Smad-signaling pathway and may also be targets for mutation in cancer.

The transforming growth factor beta (TGF-β) signaling pathway is known to control human breast cancer invasion and metastasis (Nagaraj and Datta, 2010; Drabsch et al., 2013).