Survival Signalling

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Metabolic Regulation and Cancer

 

Cellular homeostasis relies on precise control over the myriad of metabolic processes occurring within an individual cell. The basis for coordinated cell metabolism relies on highly interconnected signaling pathways (1). These intricate molecular networks depend on the detection of fluxing metabolite reserves and execute appropriate physiological responses when changes in metabolic demands occur. Metabolism can be divided into two forms: anabolism and catabolism. Anabolic metabolism promotes biomass accumulation by upregulating genes for macromolecule synthesis (2). Conversely, catabolic metabolism promotes degradative processes for nutrient recycling, energy production, or cell death (2, 3). A typical cell will balance anabolic and catabolic processes when faced with growth or death signals. Conversely, a cancer cell will force metabolic processes to favour growth (4). In this section, we will explore the cell signaling pathways involved in metabolism and how cancer cells manipulate them to promote uncontrolled growth.

Anabolism and catabolism is the mechanism of building and breaking down substrates. Images retrieved from:  http://www.constructionweekonline.com/pictures/gallery/Arabian%20Business/stacking_blocks_art_large.jpg  http://canchild.ca/elearning/dcd_workshop/img/blocks.jpg

 

Metabolic Regulation by mTORC1:

 

The mammalian target of rapamycin complex 1, or mTORC1, is a major signaling hub involved in orchestrating responses to changing metabolic conditions (1). A large and dynamic protein complex, its important components include the Ser/Thr kinase mTOR, mLST8 (mammalian lethal with sec-13 protein 8), the positive regulator Raptor (regulatory-associated protein of mammalian target of rapamycin), and the negative regulators Deptor (DEP domain containing mTOR-interacting protein) and PRAS40 (proline-rich Akt substrate 40 kDa) (1). Activity of mTORC1 is maintained by RHEB, a GTPase which itself is negatively regulated by heterodimeric tuberous sclerosis complex, TSC1/2 (5). Many pathways regulating mTORC1 do so by affecting TSC1/2 functionality. Baseline mTORC1 activity is maintained by receptor signaling into Ras-ERK and PI3K-Akt pathways, but further exposure to growth factors such as insulin can potentiate the anabolism-promoting characteristics of mTORC1 (6, 7, 8). Growth factor signaling activates Akt and Raf, which phosphorylate and deactivate TSC1/2, releasing mTOR from inhibition (9).

 

The ultimate result of mTOR activation is stimulation of anabolic processes. This is mediated by:

1.    Increased protein production by stimulating synthesis of ribosomes and mRNA translation via activation of eukaryotic initiation factor 4B (eIF-4B) and 40S ribosomal protein S6 kinase (S6K). Both effector proteins promote translation, while S6K stimulates ribosome biogenesis (10)

2.    Lipid synthesis by activating PPARγ and SREBP1, which are transcription factors that upregulate lipid synthesis genes (11)

3.    Increased energy production by upregulating genes involved in oxidative phosphorylation and mitochondrial function (12).

4.    Suppression of the catabolic process, autophagy, by phosphorylating ULK1 and ATG13, which inhibits their autophagosome forming function (8).

 

Because mTORC1 regulates cell metabolism, it is critical that information regarding cellular nutrient reserves be signaled into the complex. Therefore pathways involved in nutrient sensing often integrate into mTORC1.

 

Amino Acid Sensing

Current knowledge suggests that the essential amino acid, leucine, maintains mTORC1 stimulation in a manner that is dependent on glutamine transport and human vacuolar protein-sorting-associated protein 34 (VPS34) (8, 13). Moreover, under amino acid rich conditions, mTORC1 complexes are localized via Raptor interactions with Rag proteins to RHEB-rich perinuclear regions in the cell, leading to sustained mTORC1 activation (14). Although a potent inhibitor of mTORC1, the manner in which amino acid deprivation restrains mTORC1 activity is still poorly understood (8). The observation that mTORC1 physically dissociates from RHEB during amino acid starvation may suggest destabilized Raptor-Rag protein interaction; however, the exact signaling pathway leading to this remains to be elucidated.

 

Glucose Sensing:

Glucose is indirectly sensed by gauging cellular energy pools via AMP kinase (AMPK), which detects the ADP:ATP and AMP:ATP ratios in cells (15). When glucose concentrations dwindle, these ratios increase and AMPK is activated, which will phosphorylate two targets, TSC1/2 and Raptor. Phosphorylation of TSC1/2 activates its repressive function towards mTORC1 and phosphorylation of Raptor inhibits its mTORC1 activating function (16, 17). When AMPK inhibits mTORC1, the cell's metabolism shifts to catabolic processes such as autophagy and glycolysis to promote nutrient turnover and rapid energy production (18, 19).

 

Oxygen Sensing:

Oxygen deprivation, or hypoxia, inhibits oxidative phosphorylation, resulting in an increased AMP:ATP ratio and induction of AMPK-mediated mTORC1 attenuation (15). Hypoxic conditions also activate regulation of DNA damage response 1, REDD1, which can release TSC1/2 from 14-3-3, a complex that suppresses TSC1/2, thereby leading to mTORC1 repression (20). The effects of hypoxia on tumor growth and survival will be elaborated on later in this section.

 

Metabolic Regulation by mTORC2:

The lesser characterized of the mTOR complexes, mTORC2 shares common functional components with mTORC1, including catalytic mTOR, mLST8, and Deptor (8). Unique to mTORC2 is Rictor (rapamycin-insensitive companion of mTOR), mSIN1 (mammalian stress-activated protein kinase interacting protein 1), and Protor-1 (protein observed with Rictor-1). As with mTORC1, mTORC2 is also negatively regulated by TSC1/2. Unlike mTORC1, the pathways that stimulate mTORC2 are not well defined, although growth factor signaling and amino acid sensing may be involved (8, 21). It is known, however, that mTORC2 is important for the activation of Akt, PKC, and SKG1, which are protein kinases important for controlling cell survival responses (22, 23). mTORC2 is also implicated in the orchestration of actin polymerization and focal adhesion complex dynamics (24).

 

Figure 14.4.1. Overview of mTORC1/2 Signaling. Figure from Meric-Bernstam & Gonzalez-Angulo (2009) (Ref. 32)

 

Manipulation of mTOR Complexes is a recurring feature in cancer cells:

Tumor cells hyperactivate biosynthetic processes to support their growth and expansion. Therefore mTORC stimulation creates ideal conditions for cancer development. To potentiate mTORC1 signaling, tumor cells can over-activate PI3K-Akt or Ras-ERK pathways, which directly stimulate mTORC1, or inhibit tumor suppressors such as PTEN or p53 that negatively regulate mTORC1 (25). Stimulation of mTORC1 can also result from increased expression of Slc1a5 (ASCT2), the transporter for the amino acid, glutamine, which is known to trigger mTORC1 signaling (13). Once constitutive mTORC1 signaling is instigated, deregulated protein synthesis occurs by increased S6K activation, which leads to upregulated ribosome production, and activation of eIF-4B, which specifically stimulates translation of cell cycle, growth, and survival proteins (26). Further, mTORC1 activation leads to increased lipid synthesis, a feature that strongly correlates with cancerous phenotypes (11). Lastly, constitutive activation of Akt by mTORC1/2 can lead to angiogenesis (27), and mTORC1/2 signaling is implicated in the cytoskeletal rearrangements involved in EMT and metastasis (28). In summation, mTOR signaling can support nearly every facet of tumorigenesis.

 

Cancer Therapeutics targeting mTOR:

 

As implied by the name, mTOR was uncovered as the primary target of rapamycin, an antifungal compound known to induce cell cycle arrest (29). Rapamycin, complexed with the cytosolic protein FKBP12, prevents mTOR interaction with Raptor, a critical scaffold protein that brings mTOR to a functional proximity with its substrates, S6K and 4E-BP (30). Although previous studies concluded that rapamycin only affected mTORC1, recently it has been appreciated that prolonged exposure to rapamycin can inhibit mTORC2 as well (31). In clinical trials, rapamycin and analogous compounds, or “rapalogs,” have been found particularly effective in renal cell carcinoma (32). Preliminary studies have shown that rapalog therapy may promote regression in lymphomas, soft-tissue sarcomas, and endometrial cancer (32, 33). However, rapalog therapy has limited success in treating melanoma, glioma, leukemia, lung or pancreatic cancers (33) Rapalogs can also be used in combination with chemotherapy or monoclonal antibodies to potentiate apoptotic effects (32).

 

Although generally well-tolerated, rapalog therapy can cause mild to moderate side effects, including nausea, diarrhea, lethargy, impaired clotting, and increased blood glucose, cholesterol, and triglycerides (32). These side-effects are reversible upon rapalog withdrawal (33). However, rapamycin also functions as an immunosuppressant and may make patients more vulnerable to infection. Treatment with rapalogs may lead to rapalog resistance by several mechanisms. Firstly, mTOR inhibition may be abrogated by accumulated mutations to FKBP12 or mTOR itself that prevent rapalog association with mTOR (34). Further, mutations to the downstream effectors of mTOR such as S6K or eIF-4B that leave them constitutively active will render rapalog therapy ineffective. Lastly, mTOR inhibition may lead to compensatory upregulation of pro-growth pathways such as PI3K-Akt or Ras-ERK signaling, which encourages tumor growth (34). In the future, personal oncogenomics may be used to evaluate whether the array of mutations present in tumor cells may influence the effectiveness of rapalog therapy.

 

 

Survival signalling in response to hypoxic stress via HIF

 

Hypoxia inducible factor 1 (HIF-1) is a master transcription factor involved in regulating genes in response to hypoxia (Fig. 14.4.2). HIF-1 is a heterodimer composed of an alpha and a beta subunit; the beta subunit is constitutively expressed, while the alpha subunit is tightly regulated (35). When the cell is in the presence of normal oxygen levels, the alpha subunit is degraded through the ubiquitin proteasome system (36). In the presence of oxygen, enzymes hydroxylate the alpha subunit (37). There are 3 prolyl hydroxylases (PHD 1, 2, 3) that hydroxylate proline residues 402 and 564 of HIF1a (35). This process is oxygen dependent. Hydroxylation of Pro402 and Pro564 increases the binding affinity of HIF1a for the Von Hipple Lindau (VHL) tumor suppressor gene (35). VHL is a component of the E3 ubiquitin ligase which is responsible for targeting HIF1a for proteasome degradation (35). Additionally, another hydroxylase, FIH-1 hydroxylates an asparagine residue of HIF1a, Asn803 (35). This hydroxylation prevents an interaction between HIF1a and its co-activators CBP/p300 (36).  When the cell is in the presence of low oxygen the alpha subunit is stabilized and transported to the nucleus where it can bind the beta subunit and together with co-activators CBP/p300, they can regulate gene transcription by binding hypoxia response elements (HRE) (36).  It has been reported that in a given cell line there may be over 500 genes that are transcriptional targets of HIF activation and the genes that are turned on by HIF-1 vary between cell type and conditions (37). 

Figure 14.4.2. (A) HIF1 signalling under hypoxic conditions.Figure 14.4.2. (B) HIF1 signalling under normoxic conditions.

HIF-1 and cancer

 

Many different types of solid tumors often contain hypoxic regions; hypoxia is considered a marker of tumor aggressiveness and is typically correlated with poor prognosis (36). Although there is some conflicting evidence regarding the role of HIF signaling in cancer, immunohistochemical staining has revealed HIF1a overexpression in many human tumors (35). In many cases, HIF1a overexpression correlates with poor survival, but there have been some isolated studies showing that HIF1a overexpression actually correlates with better patient survival (35). Increased HIF expression and activity in tumors may result from regions of intratumoral hypoxia and genetic mutations in both oncogenes and tumor suppressor genes (35). For example, mutations in VHL can prevent degradation of HIF1a and mutations in PTEN can promote translation of the HIF alpha subunits (37). Oncogenic mutations to ras, myc, and src can also lead to dysregulation of HIF (36). Of the many HIF regulatory targets identified, there are four groups of genes that are relevant to cancer growth and survival: genes that contribute to metabolic adaptation, genes that help the cell resist apoptosis, angiogenesis factors, and genes that are involved in invasion and metastasis (35). Angiogenesis and metastasis are discussed in detail in chapters 10 and 12, respectively. Although genes in all four of these groups can provide benefit to the tumor, anti-apoptotic factors such as ADM, EPO, IGF2, TGFa and VEGF are considered important survival factors (35). Additionally, expression of the HIF protein, and consequently HIF activity, can be increased in response to activation of other signaling pathways such as the previously discussed mTOR pathway (35). Growth factors binding to receptor tyrosine kinases activate the PI3K and MAPK pathways resulting in downstream increases in protein translation; HIF1a mRNA is one such mRNA affected by this binding (35). HIF is also involved in autocrine signaling in cancer cells to promote proliferation and survival, since growth factors that are targets of HIF1 activation can themselves activate signal transduction pathways that lead to increased HIF1 expression (35). In essence, this provides a positive feedback loop that promotes cancer progression. Fig. 14.4.3 provides an overview of the role of HIF-1 in cancer progression. There is also evidence that HIF1 is involved in the creation of the Warburg effect, whereby cancer cells primarily use glycolysis to produce ATP, rather than oxidative phosphorylation, even in the presence of oxygen (38). One of the results of increased glycolysis is a decrease in pH and consequently, cancer cells need to manage the export of protons in order to maintain cellular homeostasis (38). 

Figure 14.4.3. Role of HIF signalling in cancer.

HIF-1 would be considered a secondary target for cancer therapy, whereas oncogenes and tumor suppressor genes are considered primary targets (35). There are different therapies suggested for downregulating HIF expression in cancers. These include expressing a dominant negative form of HIF1a and blocking the coactivators of HIF in order to block HIF-dependent transcription (35). A compound, echinomycin, that inhibits HIF1, has been shown to suppress leukemia cell growth and decrease expression of HIF1 target genes (39). HIF1 may also be targeting using siRNA technology; chemoresistant adenocarcinoma cells transfected with siRNA for multiple genes including HIF1 became more susceptible to chemotherapy following siRNA treatment (40). 

 

The Role of Hormones:

 

Estrogen

 

           In normal breast tissue, estrogen plays a vital role in tissue morphogenesis and regulation of the cell cycle (41). In addition, it plays important roles in many other systems as well, including the immune, skeletal, cardiovascular, and central nervous systems (41). The main biologically active estrogen compound is a hormone molecule called 17β-estradiol (normally refered to as E2) (41). E2 is able to diffuse into cells, where it binds to one of the classical estrogen receptors (ERα or ERβ) or to the G-protein receptor GPR30 (42). From there, it is able to exert its effects either through direct activation of genes containing a estrogen response elements (EREs), which is caused by dimerization of the receptor and subsequent binding to DNA, or indirectly through the activation of other transcription factors such as AP-1, NF-kB, and SP-1 (43). Binding of these elements to DNA results in increased transcription of the target genes, causing a variety of cellular activity including increased cellular proliferation and differentiation (43). In addition, ER bound to the plasma membrane can exert non-genomic effects by activating cellular signalling pathways involved in proliferation and cell cycle control through direct phosphorylation, including the MAPK and PI3K signalling pathways (43). This incredibly complex set of possible signalling events is further complicated by the presence of other cellular regulatory proteins that play a role in the integration of signals from ERα (often activating) and ERβ (often inhibitory) as well as in the suppression or activation of these signals (43).

 

Figure showing the different types of signalling events initiated by the estrogen receptor (ER). Free estrogen bound to the ER can initiate ranscripioon either through direct binding of ER-E2 to DNA (1), or through interaction with and activation of a cellular transcription factor (2). Additionally, membrane-bound ER can activate a variety of cellular pathways (3) (original figure, with information from (43)).  

 

           In breast cancer, aberrant expression of the ER defines a broad molecular subtype known as ER positive cancers that accounts for nearly 70% of breast cancers (44). Constitutive activation of this receptor aids tumourigenesis by deregulating the process of cellular proliferation and growth (41). Thus, treatment of these tumours is based on the use of anti-estrogens such as tamoxifen that bind ER and prevent signalling (41). Unfortunately, resistance to tamoxifen and other anti-estrogens often develops due to slight alterations in the ER that block binding or upregulation of estrogen-independent growth pathways (41). The latter can often be overcome by treatment with a secondary anti-estrogen that binds ER at a different location (41). In addition, complete blockade of the estrogen synthetic pathway can be achieved by treating patients with aromatase inhibitors or other suppressive mechanisms that target production of estrogen by the ovaries (44). Alternatively, treatment with agents such as fulvestrant can cause degradation of the ER to target estrogen-independent cancers that activate the ER signalling pathway in the absence of estrogen (44).

 

Androgen/Testosterone

 

            Similarly to estrogen in breast tissue, androgen plays a vital role in the normal cellular processes that occur within the tissue of the prostate (45). Of particular importance to the prostate is the androgen testosterone (45). Free testosterone enters the cell through a membrane pore, where it is converted into its biologically active form DHT by the enzyme SRD5A1 (45). It then binds to the inactive androgen receptor (AR) complex, which dimerizes and enters the nucleus where it initiates transcription of many genes required for proliferation and differentiation of prostate cells (45).

 

            In prostate cancer, deregulation on non-AR processes is actually responsible for the onset of most cancers (46). However, the tumours are often still dependent on AR signalling for growth and proliferation and thus treatment with anti-androgens or other molecules that inhibit either downstream of upstream signalling events (such as the conversion of circulating testosterone to DHT or the dimerization of the AR complex) have proven effective in treating the disease (46). Unfortunately, many prostate cancers are able to relapse as castration-resistant prostate cancer (CRPC), most often through reactivation of the AR signalling axis (46). The mechanisms are varied, including upregulation of DHT production to outcompete anti-androgens for AR binding, overexpression of AR to induce androgen hypersensitivity, and mutation of the AR gene to produce AR that show either ligand promiscuity or constitutive activity in the absence if ligand (46). These are often treated with novel therapeutics that are designed to more effectively inhibit AR signalling, such as abiraterone and enzalutamide, and new drugs are currently being developed to inhibit chaperone proteins and upstream signalling molecules such as HSP27 and CYP17, respectively (46). 

 

Figure showing the androgen receptor signalling pathway, with particular focus on the tenants of the pathway that are alterred in castration resistant prostate cancer. Additionally shown are novel therapuetics currently undergoing clinical trials designed to interfere with the androgen signalling axis (taken from (46)). 

 

Discussion Questions

1) Rapamycin blocks mTOR signaling by preventing the binding of mTOR to a critical scaffold protein, Raptor. What other facets of mTOR regulation can be manipulated by therapeutics to interfere with mTOR signaling?

 

2) Regarding the Hallmarks of Cancer:

a) how does mTORC1 signaling contribute to the Hallmarks of Cancer?

b) how does mTORC2 signaling contribute to the Hallmarks of Cancer?

c) how does HIF-1 signaling contribute to the Hallmarks of Cancer? 

 

3. Discuss the benefits and limitations of targeting HIF-1 therapeutically. 

 


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