PT12 (8) Cell Growth and Metabolism

Metabolism

*Metabolism* = total chemical reactions The totality of an organism's chemical reactions. Metabolic pathways begin with a specific molecule series of steps product. Each stem of the path is catalyzed by a specific enzyme. Enzymes are tightly regulated.

*Metabolic adaptation: aerobic glycolysis: The Warburg Effect*

*Oncogenic Mutations* lead to increased uptake of nutrients, GLUCOSE! Cancer cells use glucose and make a lot of lactate, even though there is oxygen *Aerobic glycolysis - 4 mol of ATP are made from 1 glucose* Oncogenic mutations can result in increased uptake of nutrients, particularly glucose This realization has brought renewed attention to Warburg's observation. In 1924 Otto Warburg observed that cancer cells metabolize glucose in a manner that is distinct from that of cells in normal tissues. Warburg found that cancer cells metabolize glucose and produce large amounts of lactate regardless of the availability of oxygen. Thus, the metabolism of cancer cells is often referred to as "aerobic glycolysis." In aerobic glycolysis, 4 mol of ATP are produced from 1 mol glucose.

A mutation in RTK might result in : Check all that apply Increase in glucose metabolism Reduction of lipid synthesis Activation of p53 Activation of growth factor-independent MAPkinase pathway

Mutation in RTK = increase in glucose metabolism and activation of MAPkinase

The role of different signaling molecules in metabolic adaptation: *mTOR*

mTOR = PROTEIN SYNTHESIS AKT ACTIVATES AMPK INHIBITS *target of rapamycin* *TORC1 and 2* *Upstream regulation by LKB1-AMPK-TCS1/2* Downstream effects - mTOR enhances protein translation through inhibition of translational inhibitor 4E-BP1, promotes translation initiation by elF4F complex, and stimulation of ribosomal S6 kinase *BOTTOM LINE:* mTOR - protein synthesis and translation Stimulated by AKT and inhibited by AMPK Mammalian target of rapamycin TORC1 and TORC2 Upstream regulation by LKB1-AMPK-TSC1/2 Downstream effects: mTOR enhances protein translation through inhibition of the translational inhibitor 4E-BP1, which in turn promotes translation initiation by the eIF4F complex, and stimulation of ribosomal S6 kinase Bottom line: mTOR protein synthesis and translation Stimulated by AKT and inhibited by AMPK

Metabolic energy

*Energy stored in ATP bonds* ATP stores energy Free Energy and ATP During catabolism, energy is released and stored in the high energy chemical bonds of adenosine 5'-triphosphate (ATP) and other molecules. Break down of these bonds by specific enzymes yield free energy. So, ATP serves as a store of free energy, which is used to drive energy-requiring processes within cells.

Regulation of metabolism in normally proliferating and in non proliferating cells Multicellular organisms:

*Constant supply of nutrients* *No growth signals = no proliferation* constant supply of nutrients. Growth signals proliferative metabolism proliferation No growth signals quiescent cell metabolism no proliferation or differentiation. control systems to prevent aberrant individual cell proliferation when nutrient availability exceeds the levels required to support cell division. Uncontrolled proliferation is prohibited, as mammalian cells do not normally take up nutrients from their environment unless stimulated to do so by growth factors.

Anabolic Types of Metabolic Pathways

*Consume energy to build bigger molecules* Pathway that consumes energy to build complicated molecules from simpler ones Examples: Building proteins from amino acids Building polysaccharides and glycogen from glucose Building lipids from fatty acids.

The Hallmarks of Cancer (Hallman and Weinberg, 2000)

CANCER CELLS = PROLIFERATE IGNORE SUPPRESSORS INVADE AND METASTASIZE IMMORTAL ANGIOGENESIS RESIST APOPTOSIS These provide a logical framework for understanding the remarkable diversity of neoplastic diseases As normal cells progressively evolve to a neoplastic state, they acquire these hallmark capabilities

Impact of the tumor-anabolic pathways Cancer Cachexia

Proinflammatory environment in the body Increased liver gluconeogenesis to supply the tumor cells with energy hyperglycemia and insulin resistance. Hyperglycemia is an early marker for several cancers, e.g. pancreatic cancer Catabolism: fat mass loss and muscle wasting Acidemia weight loss Reduced physical Activity Browning of the white fat increased UCP and thermogenesis Anorexia

Clinical applications for altered metabolism

AMPK activation (Metformin in type 2 diabetes and cancer) Glycolysis inhibition Glutaminase inhibitors mTOR inhibitors? FAS inhibitors PI3K and AKT inhibitors (kinase inhibitors/side effects) Imaging: uptake of 18 F-deoxy glucose- Pet imaging FDG-PET to monitor progression/regression of tumors after therapy (tyrosine kinase blockers)

Anaerobic glycolysis

*Glycolysis = anaerobic breakdown of glucose private then lactic acid* *DIFFERENTIATED CELLS MAKE A LOT OF LACTATE AND LITTLE ATP* Our current understanding of metabolic pathways is based largely on studies of non proliferating cells in differentiated tissues. Glycolysis is a cytosolic process that involves the anaerobic breakdown of glucose pyruvate then to lactic acid. Differentiated cells produce large amounts of lactate and limited amounts of ATP. Here, 2 mol of ATP are produced from each mol of glucose.

Catabolic / Anabolic Relationship : Homeostasis

*Increase in metabolism* = more efficienct Overall, a cell is either working to secrete needed chemicals or metabolizes materials. An increase in metabolism makes cells more efficient at carrying out these tasks. Energy released from catabolic reactions can be stored and then used to drive the anabolic pathways.

In cancer cells, metabolic reprogramming and adaptation happen to meet the demands of cell proliferation in absence of growth factor signals

*Increased glycolysis despite adequate O2* *Aerobic glycolysis - Warburg effect* - cancer cells continue to make lactate in presence of oxygen. Due to ineffective mitochondria Early 20th century Observed that in cancer cells there are increased rates of glycolysis despite the availability of adequate O2 levels Aerobic glycolysis Warburg Effect (Cancer cells continue to ferment lactate in the presence of oxygen) Enhanced fermentation is the signature metabolic malady of all cancer cells These changes must be due to defective mitochondria

Regulation of metabolism in normally proliferating and in non proliferating cells Unicellular organism:

*Lots of nutrients is good for metabolism. Scarce nutrients = metabolism starves, cells stop growing* Abundant nutrients -> proliferative metabolism = carbon, nitrogen, and free energy are efficiently utilized into generating the building blocks needed to produce a new cell. Scarce nutrients starvation metabolism = cells stop growing and adapt their metabolism to extract the maximum free energy from available resources to survive the starvation period. So, there are tight regulatory mechanisms that control cellular metabolism in proliferating versus non- proliferating cells.

What is the metabolic profile in proliferating cells?

*Proliferation* - cell must increase biomass and replicate genome before dividing into daughter cells The decision to proliferate presents a significant bioenergetic challenge for a cell. *During proliferation a cell must increase its biomass and replicate its genome prior to dividing to create two daughter cells.* For normal proliferating tissues, such as in the developing embryo, signals from growth factors allow cells to utilize nutrients for growth.

Why would a less efficient metabolism (in terms of ATP production) be selected in proliferating cells?

*Ribose 5P and Acetyl CoA* Glucose from gluconeogenesis liver Cancer cells hog glucose All energy utilized by cancer cells

Oxidative metabolism

*With O2 tissues make glucose to CO2 and H2O in TCA. Makes NADH which fuels oxidative phosphorylation* 36 mol of ATP made for 1 mol glucose In the presence of O2 (aerobic conditions), most cells in differentiated tissues metabolize glucose to CO2 and H2O in the mitochondrial tricarboxylic acid (TCA) cycle. This reaction produces NADH [nicotinamide adenine dinucleotide (NAD+), reduced], which then fuels oxidative phosphorylation. As a result of the oxidative metabolism, ~36 mol of ATP are produced for 1 mol of glucose. So, oxidative metabolism produces much more energy than is obtained from glycolysis.

Catabolic Types of metabolic pathways

*breakdown of molecules* Ex. glucose to CO2 and H2O Energy is released to do work Result in breakdown of complex molecules into simpler compounds Examples: Glucose is broken down into CO2 and H2O Energy is released and becomes available to do work for the cell.

Mitochondrial reprogramming: Glutaminolysis

*cMyc induces glutamine transporters at membrane and increases glutaminase* *Glutaminolysis makes purines and pyrimidines for DNA and RNA synthesis* makes *NADPH and malate* for *FA synthesis* *Glutamine* serves as *source of energy in cancer cells* Glutaminase inhibitors reduce cancer cell growth, transformation, and tumorigenesis Transaminase inhibitors as anticancer agents c-Myc induces the expression of glutamine transporters at the plasma membrane and acts indirectly to increase the expression of glutaminase (GLS). Glutaminolysis results in the production of purines and pyrimidines needed for DNA and RNA synthesis Glutamine contributes to several biosynthetic pathways, including the production of NADPH and Malate that are needed for fatty acid synthesis Glutamine serves as another source of energy in cancer cells Glutaminase inhibitors reduce cancer cell growth, transformation, and tumorigenesis. Transaminase inhibitors have also been suggested as anticancer agents because

Cancer

A group of diseases in which *cells divide uncontrollably, forming malignant tumors, and invade* nearby parts of the body

The role of different signaling molecules in metabolic adaptation: *AMPK, the master switch!*

AMPK 1) SENSES AMP:ATP 2) INHIBITS ANABOLISM AND ACTIVATES CATABOLISM 3) When ATP is low = ACTIVATES (when nutrient or oxygen deprived) 4) LKB1 activates INHIBITS PROTEIN SYNTH BY INHIBITING MTOR INHIBITS LIPID SYNTH BY INHIBITING ACC *Protein kinase* *Sensitive to changes in AMP/ATP ratio* *Activated when ATP levels are reduced ex. nutrient deprivation and hypoxia* *Upstream activator* - LKB1, a tumor suppressor PJ syndrome Actions - general inhibition of anabolism and activation of catabolism - Inhibition of lipid synthesis through inhibition of ACC - Inhibition of protein synthesis through inhibition of mTOR - Stimulation of p53 - cell cycle arrest and inhibition of glycolysis Heterotrimeric protein kinase Sensitive to changes in AMP/ATP ratio. Activated when ATP levels are reduced e.g. nutrient deprivation and hypoxia. Upstream activator: LKB1, a tumor suppressor PJ syndrome. Actions: General inhibition of anabolism and activation of catabolism. Inhibition of lipid synthesis through inhibition of ACC Inhibition of protein synthesis through inhibition of mTOR Stimulation of p53 cell cycle arrest and inhibition of glycolysis

Metabolic adaptation through hypoxia-induced angiogenesis

CANCER CELLS ALTER RTK SO CAN CONTINUE TO GROW *Cancer cells overcome growth factor dependence by acquiring genetic mutations that alter RTK - cells continue to grow* *Excessive cell growth - hypoxia in the middle of tumor - stress and activation of vascular endothelial growth factor VEGF - binding to RTKs on endothelial cells sprouting of new blood vessels = angiogenesis* Cancer cells overcome growth factor dependence by acquiring genetic mutations that functionally alter RTK pathways cells continue to grow Excessive cell growth hypoxia in the middle of the tumor stress and activation of hypoxia-inducible factor-1 alpha (transcription factor/ HIF-1𝛂) activates the transcription of vascular endothelial growth factor (VEGF) biding to RTKs on endothelial cells sprouting of new blood vessels = angiogenesis

Summary

Cross-talk between signaling pathways and metabolic pathways Increased glycolysis in cancer cells (Warburg effect): essential for cancer cell survival. Genetic mutations leading to loss of function in tumor suppressors or gain of function of oncogenes render the cells unresponsive to growth factor signaling, which lead to cell cycle changes as well as metabolic changes. Metabolic changes: aerobic glycolysis, glutaminolysis and lipogenesis Role of some factors: AMPK: AMP/LKB1 stimulated, inhibits cell growth by reducing anabolic reactions PI3K/AKT: majority of cancer mutations, inhibited by PTEN (tumor suppressor), increases cell growth by inducing mTOR and increasing nutrient transporters and their metabolic enzymes mTOR: activated by PI3K/AKT and inhibited by AMPK, increases cell growth by increasing protein synthesis: inhibition of 4E-BP1 and consequent activation of eIF4F complex. P53: inhibits cell growth by: known mechanisms (cell cycle arrest and apoptosis) and novel: inhibition of glycolysis through TIGAR. Clinical applications: dieting, calorie/glucose restriction, metformin (AMPK), and imaging to monitor glucose uptake.

Neoplastic Lipogenesis

FAS IS A TUMOR ONCOGENE - SUPPLIES LIPIDS FOR CANCER CELLS FAS IS TIGHTLY REGULATED LKB1 TUMOR SUPPRESSOR GENE inhibits FAS SCRBP-1 a transcription actor activates FAS AMPK senses AMP:ATP, activated by LKB - then phosphorylates and inactivates ACC, then FAS activity decreases *WORSE PROGNOSIS*= HIGH FAS (increases metastasis and tumor survival) *citrate leaves mitochondria, converted to acetyl CoA by ATP citrate lyase/ACLY* Acetyl CoA is converted to malonyl CoA, makes FA by enzyme FAS with NADPH *FAS = tumor oncogene, provides constant supply of lipids and precursors to fuel membrane production* FAS is tightly regulated - LKB1 tumor suppressor gene inhibits FAS while SREBP-1 transcription factor, activates it *AMPK senses energy of cell via AMP/ATP ratio* gets phosphorylated and activated by LKB, phosphorylates and inactivates ACC Patients with high levels of tumor FAS show worse prognosis since FAS has been implicated in cell proliferation and angiogenesis - increasing metastatis and makes more radio resistant Citrate leaves the mitochondria converted to Acetyl CoA (by ATP citrate lyase/ACLY) Acetyl CoA is converted to malonyl CoA (by acetyl CoA carboxylase/ACC/ACACA) production of long-chain fatty acids by the enzyme fatty acid synthase (FAS), in presence of NADPH. FAS, which is also a tumor oncogene, provides a constant supply of lipids and lipid precursors to fuel membrane production and lipid-based post-translational modification of proteins in the highly proliferating cancer cell population. FAS is tightly regulated: The LKB1 (tumor suppressor genes) inhibits FAS while SREBP-1, a transcription factor, activates it. AMPK protein senses the energy status of a cell (by sensing the AMP:ATP ratio) and if it gets phosphorylated and activated by LKB, it proceeds to phosphorylate and inactivate ACC. When ACC is inhibited like this, the expression and activity of FAS decreases. Patients with high levels of tumor FAS show worse prognosis since FAS has also been implicated in endothelial cell proliferation and angiogenesis increasing rates of metastasis, and as a survival factor for tumor cells rendering them more radio resistant.

Therapeutic Targeting

Glutaminase inhibitors FAS inhibitors Telomerase inhibitors Anti-CTLA4 mAb activation

*The role of lactate and the creation of acidic microenvironment around cancer cells*

LACTATE STIMULATES M2 - IMMUNOSUPPRESSION PROMOTES ANGIOGENESIS, MMPS METASTASIS *Lactate* 1) Attenuate dendrite and T cells 2) Stimulates polarization of resident macrophages - M2 state, which plays a role in immunosuppression (tumor associated macrophages TAMs) 3) Promotes angiogenesis through stabilizing HIF1alpha and activating NFKB and PI-3 kinase - MMPs - metastasize VEGF angiogenesis Hyaluronic acid production by fibroblasts - tumor invasiveness Lactate attenuate dendritic and T cells immune-permissive microenvironment lactate stimulates the polarization of resident macrophages M2 state, which plays a role in immunosuppression (tumor associated macrophages TAMs). Lactate promotion of angiogenesis through stabilization of HIF1α and activation NF-κB and PI-3 kinase signaling in endothelial cells inflammatory profile and transcription of proinflammatory cytokines and matrix metalloproteinases (MMPs) metastasis Lactate secretion of a pro-angiogenic factor VEGF from tumor-associated stromal cells angiogenesis Lactate hyaluronic acid production by fibroblasts tumor invasiveness

Normal cells vs. Cancer cells

NORMAL: tight control, identical, stop reproducing, stick together, self destruct CANCER: can't stop reproducing/proliferating, ignore signals, spread, not specialized *Normal cells:* Reproduce under *tight control* and make *identical copies* of themselves *Stop reproducing* at the right time *Stick together* in the right place *Self destruct* if they are damaged *Cancer cells:* *Don't stop reproducing (Chronic proliferation)* *Don't obey signals* from other cells *Don't stick together (Become metastatic)* *Don't specialize (differentiate)*, but stay immature

The role of different signaling molecules in metabolic adaptation: *PI3K/AKT:*

PI3K/AKT 1) REGULATES PIP3 2) ACTIVATES mTOR 3) AA and glucose transport *central regular in metabolism* *regulates levels of PIP3* *regulated by PTEN* *Activation of PI3K leads to* -AA and glucose transport -gene transcription -mTOr activation -protein synthesis A central regulator of metabolism in Regulates the levels of phosphorylated phosphatidylinositol (PIP3) at the plasma membrane. Regulated by PTEN. Growth factor-dependent activation of PI3K leads to: AA and glucose transport Gene transcription mTOR activation Protein synthesis

??? Two more hallmarks were introduced:

REPROGRAM METABOLISM - *Tumor cells use glucose inefficiently and make small ATP and lots of lactate into tumor environment* EVADE DESTRUCTION *Immune Editing* - immune competent eliminate cancer cells leaving weak ones to become tumors? 7. *Reprogramming Energy Metabolism:* -Tumor cells prefer to utilize glucose inefficiently to produce small amount of ATP and large amounts of lactates that are secreted into the tumor microenvironment. - A subpopulation of cancer cells utilize lactate as a source of energy - The metabolic adaptation in cancer cells is not functionally independent from other core hallmarks, as many of the involved proteins are common. 8. *Evading immune destruction*: - Highly immunogenic cancer cell clones are routinely eliminated in immunocompetent hosts - a process called "immune editing"- leaving behind only weakly immunogenic variants to grow and generate solid tumors

The role of different signaling molecules in metabolic adaptation: *p53*

p53 1) ACTIVATES CELL CYCLE ARREST AND APOPTOSIS *DNA damage, hypoxia, oxidative stress - post translational modification - stabilization and activation - p53 activates cell cycle arrest, senescence, and apoptosis genes* Activation of AMPK p53 by stress triggers catabolism including inhibition of glycolysis through stimulation of gene TIGAR (TP53 induced glycolysis and apoptosis regulator) DNA damage, hypoxia, and oxidative stress post-translational modification stabilization and activation p53 activates cell cycle arrest, senescence, and apoptosis genes. Activation of the AMPK-p53 pathway by metabolic stress triggers multiple effects on catabolic metabolism, including inhibition of glycolysis through stimulation of the gene TIGAR (TP53-induced glycolysis and apoptosis regulator).