Drug/Target validation and identification in cancer

1. Drug/Target validation and identification in cancer Monday 12/December/2016

2. Cancer Biology- Briefly • Cancer is a disorder of cells, which involves disturbances in cell growth and division. • The appearance of tumor (mass of cells) is considered the end result of various series of changes, which may take years for development. Two main features that define the cancer cell: 1- Uncontrolled/abnormal cell growth and division (no response to normal growth controlling mechanism), and decreased cell death (apotosis) 2- Invading and colonize the territories of the neighboring cells/tissues Therefore, cell cycle plays an important role in tumor development Molecular biology of the cell , 5th edition

3. Types of Tumors: -Benign: non cancerous and not an immediate threat to life, even though treatment eventually may be required for health. -Malignant: tending to worsen and cause death, invasive and metastasis Characteristics of cancer cells: • Persistent cell proliferation • Invasive growth • Metastases (through blood vessels and lymphatic system and exosomes, circulating tumor cells) Etiology of Cancer: • Genetics • Viruses • Occupational and Environmental Carcinogens • Radiation

4. Genetic mutations are largely responsible for the generation of malignant cells. Two major categories of mutated genes are oncogenes and tumor suppressor genes. • 1-Oncogenes are mutated forms of their normal genes (proto-oncogenesare involved in controlling normal cell growth). These mutations may can lead to inappropriate stimulation of the cell cycle and excessive cell growth. e.g.: the ras gene encodes the Ras protein, which regulates cell division, by involving in cellular cell transduction. Mutations may result in the inappropriate activation of the Ras protein, leading to uncontrolled cell growth and division. 2-Tumor suppressor genes detect DNA damage and block DNA replication, to give time for the cell forDNA repair . If repair failed, the cell do apotosis critical for detecting inappropriate growth signals in cells. Tumor suppressor genes encodes proteins involved in checking , and repair DNA damage, in addition to induction of apotosis • Tumor protein p53 gene encodes, p53 protein, which prevents replication of damaged DNA in normal cells and promotes cell death (apoptosis) in cells with abnormal DNA. Inactive or altered p53 allows cells with abnormal DNA to survive and divide. The p53 gene is defective in many human cancers.

5. Viruses contribute to the pathogenesis of human malignancies through the • integration of viral genetic material into the host DNA. These new genes are expressed • by the host; they may affect cell growth or division, or disrupt normal host genes • required for control of cell growth and division. Alternatively, viral infection may result • in immune dysfunction, leading to decreased immune surveillance for early tumors. E.G.: - Epstein-Barr, nasopharyngeal carcinoma -Hepatitis B virus, hepatocellular carcinoma • Carcinogenesis can result from ionizing radiation and may develop from 2 different mechanisms: 1. Direct ionization – damages DNA and other molecules can cause direct somatic mutations 2. Secondary effectors such as oxygen radicals can be formed by interaction with ionizing radiation. Oxygen free radicals can damage and kill cells and also induce mutations.

6. Pathogenesis of cancer • Cancer development can begin with a brief exposure (hours or days) to a chemical into an activated form and the chemical need not be present ever again. • However, DNA is altered via mutagens including chemical carcinogens, viruses, and radiation. This mutations is inherited by at least one cell division (intiation). • This mutation mainly lead to activation of proto-oncogene into oncogene (leading to uncontrolled cell proliferation) and/or inactivation of tumor suppressor genes (leading to resistance to apoptosis.) • Upon exposure to other epigenetic factors (hormones, co- carcinogens, immunosuppressant…which themselves are non carcinogenic) tumor growth is promoted (promotion)

7. Pathogenesis of cancer Chemicals, viruses, irradiation, etc Inherited Mutations Acquired Mutations Protooncogenesoncogenes ↓ expression of tumor supressor genes (P53, Rb etc) Promoters, co-carcinogen, hormones Uncontrolled cell proliferation, dedifferentiation ↓ apoptosis, alterations in telomerase Development of primary tumor

9. Cell cycle • Cell cycle confirms: • Accurate duplication, separation and passing of the replicated genetic material into two daughter cells • Cell cycle has many check points to ensure various events in the cell cycle progression to occur accurately in correct order. • Control of cell cycle involves cyclins and cyclin dependent kinases (CDK) • There are at least 15 types of cyclins and 9 types of CDK, each has a role at different stages of cell cycle

10. Defects in any of these checkpoints will cause normal cell cycle control leading to: malignant transformation

11. Cell cycle and drugs G1 – L- Asparginase S – Methotrexate 6-Mercaptopurine 5-Fluorouracil Mitomycin C Hydroxyurea Doxorubicin G2 – Bleomycin Etoposide, Topotecan Daunorubicin M – Vincristine Vinblastine Paclitaxel, Docetaxel

12. Classification of chemotherapeutic agents CLASSIFICATION - I: CELL CYCLE NON SPECIFIC (CCNS) : Kills resting cells & dividing cells • Cyclophosphamide • Chlorambucil • Cisplatin • Actinomycin-D • L-asparaginase CELL CYCLE SPECIFIC (CCS): Kills actively dividing cells • G1 – Vinblastine • S – Methotrexate 6-Mercaptopurine 5-Fluorouracil • G2 –Bleomycin Etoposide, Topotecan Daunorubicin • M – Vincristine Vinblastine Paclitaxel,Docetaxel

13. CLASSIFICATION - II:Depending on mechanism at cellular level • Directly acting cytotoxic drugs: • Alkylating agents: Meclorethamine, Melphalan, Chlorambucil, cyclophosphamide, ifosfamide , Thiotepa..etc • Antimetabolites: folate antagonists as methotraxate, Purine antagonists as: 6-mercaptopurine, and Pyrimidine antagonist as: 5-fluoruracil • Natural products • Antibiotics: Doxorubicin, Bleomycin..etc • Vinca alkaloids: Vincristine, Vinblastine • Taxanes: Paclitaxel, docetaxel • Epipodophyllotoxins: etoposide, tenoposide • Camptothecin analogs : irinotecan • Enzymes : L-Asparginase 4. Miscellaneous: Cisplastin, carboplatin, Hydroxurea, Procarbazine, Mitotane , Imatinib

15. I-Alkylating Agents (CCNS) Mechanism of Alkylating Agents • These drugs work by alkylation with nucleophilic substitution . They alkylate a variety of cellular constituents, such as cell membranes, proteins, and most importantly DNA. More specifically, the nitrogenous bases of DNA are what get alkylated. • The drugs start off as pro-drugs that become activated when a chlorine atom is extracted. A carbonium ion is thus formed. This “carbonium ion” is very electrophilic and will then attack any free pair of electrons (i.e. on the N7 of guanine). This electrophilic attack results in a bond being formed between the drug and the guanine of DNA. As a result of this “alkylation”, there are a few consequences: 1) Miscoding (In transcription) 2) Cross linking this occurs only if the drug is bifunctional (cross DNA) The net result is cancer cell undergo apoptosis.

17. II-Antimetabolites (CCS) • An antimetabolite is a chemical with a similar structure to a metabolite required for normal biochemical reactions, yet different enough to interfere with the normal functions of cells, including cell division. • All antimetabolites are used in cancer treatment, as they interfere with DNA production and therefore cell division and the growth of tumors (mainly in S-phase specific). • They are classified into: 1- Folic acid analogues 2- Purine analogues 3- Pyrimidine analogues Uses • leukemia. • non-Hodgkin's lymphoma • inflammatory bowel disease such as Crohn's Disease and ulcerative colitis • It is widely used as immunosuppressant in transplantations to control rejection reactions.

18. 1-Folic acidanalogues Methotrexate: -A folic acid analogue, prevents the formation of tetrahydrofolate, essential for purine and pyrimidine synthesis, by inhibiting dihydrofolate reductase. This leads to inhibition of production of DNA, RNA and proteins (as tetrahydrofolate is also involved in the synthesis of amino acids as serine and methionine). • It is actively taken up into the cells by the same transport system for folate • The most common toxicity is nepherotoxicity (pptn of the drug in renal tubule.)

20. 2-Purine analogues Mercaptopurine (6−mercaptopurine, or 6−MP) : -It is immunosuppressive cytotoxic substance. It is widely used in transplantations to control rejection reactions. -It is acts as a purine analogue and once enter the cell, it is converted to 6-MP-ribosephophate and can be incorporated into RNA&DNA resulting in non functioning RNA & DNA &finally inducing cell cycle arrest and apoptosis. -It also inhibits purring ring biosynthesis Azathioprine: It is one of the main immunosuppressive cytotoxic substance. It is widely used in transplantations to control rejection reactions. It is non-enzymatically cleaved to 6 - M P that acts as a purine analogue and inhibits DNA synthesis

21. 3-Pyrimidine analogues 5-flurouracil (5-FU) • It act as a uracil analogue, it is transformed inside the cell into 5-FU deoxynucleotide which compete with deoxyuridinemonophosphate DUMP for thymidylate synthase leading to inhibition of deoxythymidinemonophosphate DTMP synthesis inhibition of DNA synthesis (Not RNA or protien) • Also it is incorporated into DNA , resulting in non functioning DNA . • finally inducing cell cycle arrest and apoptosis by inhibiting the cell's ability to synthesize DNA. • It is an S-phase specific drug • 5−FU may be used in combination with other chemotherapy agents to treat cancers of the breast, stomach, colon, rectum, and pancreas. .

23. Cytarabine • It is analogue to 2-deoxycytidine and in the body it is converted into cytosine triphosphate and inhibit DNA polymerase thus inhibiting DNA synthesis

24. III-Antitumor antibiotics (CCNS) : 1-Dactinomycin • is isolated from soil bacteria of the genus Streptomyces. • It was the first antibiotic shown to have anti-cancer activity and used in treatment of a variety of cancers. • It inhibits transcription by binding to DNA at the transcription initiation complex and preventing elongation by RNA polymerase. • As it can bind DNA duplexes, it can also interfere with DNA replication 2-Doxorubicin (adriamycin) Mechanism of action • Doxorubicin is anthracyclin antibiotic interferes with the cells' production of DNA and RNA by inserting itself between adjacent base pair causing local uncoiling thus blocking DNA and RNA synthesis. • Also its antitumor effect is related to its inhibition of Topoisomerase II enzyme (responsipole for DNA repair). • CYTP 450 catalyzes the conversion of Dox. into semiquinone free radicals which produce superoxide ion & H2O2 that mediate single strand scission in DNA

25. 3-Mitomycin−C Mitomycin−C is an antitumor antibiotic. Mechanistically however, it belongs to DNA alkylating agents. Upon bioactivation inside the cell ,it preferentially alkylates O6 of guanine base in DNA leading to cross linking of DNA. It also degrade DNA through formation of free radicals 4-Bleomycin It is cytotoxic in any phase of the cycle even on G0 phase Bleomycin degrade performed DNA causing chain fragmentation and release of free bases through the formation of free radicals (superoxide and hydroxyl radicals). 5-Procarbazine Procarbazine is an anticancer agent inhibits DNA and RNA synthesis in cells interfering with mitosis

26. IV-Plant alkaloids (Phase specific) 1-The vinca alkaloids Vincristine & vinblastine (M-phase) Mechanism of action Tubulin is a structural protein which polymerizes to form microtubules. The cell cytoskeleton and mitotic spindle, amongst other things, are made of microtubules. Vincristine binds to tubulin inhibiting polymerization of microtubule structures. Disruption of the microtubules arrests mitosis in metaphase. The vinca alkaloids therefore affect all rapidly dividing cell types including cancer cells, but also intestinal epithelium and bone marrow.

27. 2-Taxanes Paclitaxel & docetaxel it is used for treatment of lung, ovarian and breast cancer. Mechanism of action • paclitaxel hyper-stabilizes microtubule structure (freez them). Paclitaxel binds to the β subunit of tubulin ,the resulting microtubule/paclitaxel complex does not have the ability to disassemble. This adversely affects cell function because the shortening and lengthening of microtubules is necessary for their function. • Further research has indicated that paclitaxel inducesprogrammed cell death (apoptosis) in cancer cells by binding to an apoptosis stopping protein called Bcl-2 (B-cell leukemia 2) and thus arresting its function.

28. 3-Etoposide • Chemically it is deriven from podophyllotoxin, a toxin found in the mandrake root. • An inhibitor of the enzyme topoisomerase II. cause breaks in the DNA inside the cancer cells and prevent them from further dividing and multiplying. Then the cells die.

29. Hormones and hormone antagonist 1-Corticosteroids Corticosteroids have broad use in cancer treatment. Some are used to treat adult leukemias, adult lymphomas, and acute childhood leukemia. Immunosuppressive mechanism • Glucocorticoids suppress the cell-mediated immunity. They act by inhibiting genes that code for the cytokines interlukin and TNF-γ, the most important of which is the IL-2. The inhibition of cytokine production reduces the T cell proliferation. • Glucocorticoids also suppress the expansion and antibody synthesis. Side effects • Hyperglycemia due to increased gluconeogenesis, insulin resistance caution in those with diabetes mellitus • reduced bone density (osteoporosis, higher fracture risk, slower fracture repair) • weight gain due to increased visceral and truncal fat deposition (central obesity) and appetite stimulation • adrenal insufficiency (if used for long time and stopped suddenly without a taper) • muscle breakdown (proteolysis), weakness; reduced muscle mass and repair • growth failure, pubertal delay • Increased urea formation; negative nitrogen balance The most common corticosteroids used in cancer treatment are: • · dexamethasone (Decadron) • · hydrocortisone • · methylprednisolone (Medrol)

30. 2-Estrogens & Progestons Mainly used in androgen dependent prostatic tumors 3-Gonadotropin−releasing hormone analogues Goserelin Acetate· Goserelin acetate is a synthetic hormone that acts similarly to the naturally occurring gonadotropin−releasing hormone (GnRH). In men, this results in decreased blood levels of the male hormone testosterone. In women, it decreases blood levels of the female hormone estrogen. • It is used for treatment of breast and prostatic cancer Side effects • sweating ,hot flashes, impotence (erectile dysfunction),sterility & gyncomestia • depression or other mood changes • Other common side effects in women include: light, irregularvaginal bleeding & no menstrual period

31. Hormone antagonists Tamoxifen • Tamoxifen selectively inhibits the effects of estrogen on breast tissue, while selectively mimicking the effects of estrogen on bone (by increasing bone mineral density) and uterine tissues. These qualities make tamoxifen an excellent therapeutic agent against breast cancer. it is known to compete with estrogen by binding to estrogen receptors on the membrane of target cells, thus limiting the effects of estrogen on breast tissue. • Tamoxifen may also has other anti−tumor activities :affecting oncogene expression& promotion of apoptosis (cancer cell death) Adverse Effects • CNS: Depression, light headedness, dizziness, headache, decreased visual acuity &retinopathy • GI: Nausea, vomiting • Hematological: Hypercalcemia • GU: Vaginal bleeding, vaginal discharge & menstrual irregularities • Dermatologic: Hot flashes, skin rash

32. Immuno-modulators Immunosuppressant Immunostimulants 1- Monoclonal antibodies 2- Biological response modifiers 3- Angiogenesis inhibitors • 1 Glucocorticoids • 2 Cytotoxic • a- Alkylating agents • b- Antimetabolites • 1 Methotrexate • 2.Azathioprine and Mercaptopurine

33. 1-Monoclonal Antibodies Monoclonal antibodies are proteins produced in the laboratory from a single clone of a B−cell, the type of cells of the immune system that make antibodies. When used as a treatment for cancer, there are three general strategies with monoclonal antibodies: • 1. One uses the ability of the antibodies to bind to the cancer cells having the tumor antigens on their surface. The immune system will see the cancer cells marked with bound antibodies as foreign and destroy them. • 2. A second strategy is to use the antibodies to block the binding of cytokines or other proteins that are needed by the cancerous cells to maintain their uncontrolled growth. Monoclonal antibodies designed to work like this bind to the cytokine receptors that are on the tumor cell surface. • 3. A final strategy involves special antibodies that are linked (conjugated) to a substance that is deadly to the cancer cells. E.G. radioactive isotopes, have been successfully conjugated to antibodies. The antibodies are then used to specifically destroy he tumor cells with the radioactivity or toxic substance. Trastuzumab • Trastuzumab is a humanized monoclonal antibody produced by recombinant DNA technology that binds specifically to the human epidermal growth factor receptor 2 protein (also known as HER2) that is found on the cell surface of some cancer tumors, most notably breast cancer(25−30% of breast malignancies) and also targets it for destruction by the natural killer cells of immune system.

34. 2-Biological response modifiers Researchers have been working on stimulating the immune cells during cancer with substances broadly classified as biological response modifiers. Cytokines are one such substance. These are proteins that are predominantly released by immune cells upon activation or stimulation.

35. Aldesleukin • Aldesleukin is interleukin, that is used to treat metastasis renal cell carcinoma (a form of kidney cancer) and metastasis melanoma. • Aldesleukin is also known as interleukin−2 • When renal cell carcinoma and metastasis melanoma (cancer of the skin that arises in the pigmented cells of the skin or eyes) do not respond to other therapies, they are candidates for treatment with aldesleukin. • Aldesleukin is a biological response modifier (BRM). It promotes the development of T cells, or the cells in the lymphatic system that can fight cancer cells. Side effect • Flu-like symptoms (chills, fever, fatigue) • Loss of appetite • Skin problems such as a rash, itchiness, scaling • Cardiac arrhythmias • Gastrointestinal disturbance, such as nausea and vomiting • Neurological effects, such as depression and poor concentration

36. 3-Angiogenesis inhibitors • Angiogenesis is the normal process by which the human body • forms new blood vessels. Angiogenesis is important in the • development of cancer because tumors require blood vessels • to grow and spread to nearby tissue. Once a tumor reaches a • certain size it needs to develop a blood supply in order to • grow. • Cancer cells will secrete certain chemicals to promote • angiogenesis. • Angiogenesis inhibitors are drugs that can stop this process. • These anti-angiogenesis agents are being investigated as • potential cancer therapies.

37. Thalidomide • It interferes with the growth of rapidly dividing cells. It interferes with the formation of blood vessels. It is called an antiangiogenic drug • It is used to treat several types of cancers, including kidney, ovarian, and breast cancer. Side effects : • Hypotension • Thalidomide may cause peripheral neuropathy (numbness, tingling, pain, or burning sensations in the feet or hands). • Thalidomide may cause severe birth defects or fetal death if taken by pregnant women (phocomelia). • Rash • Lack of bowel movements

38. Exosomes • Exosomes are one of the three extracellular vesicles released by the cells: • 1- Microvesicles /extracellular vesicles/ ectosomes (100-1000 nm) • 2- apoptotic blebs (bodies) (1 um- 5um). Taken by phagocytic cells, and recycle their components. • 3- exosomes (20- 100/150 nm): They are of endosomal origin • Maturation of early endosomes into late endosome results in the formation of intraluminal vesicles that forms what is known Multivesicular bodies (MVBs). • MVBs may fuse with lysosome for degradation of their cargo, or fuse with the plasma membrane and released in the form of exosomes by exocytosis • Exosomes formed from the inward budding of late endosome, while MV are formed by outward budding of plasma membrane • Released in the extracellular medium by almost all types of cells upon fusion of multi-vesicular bodies (MVB) with plasma membrane. • They contain various biomolecules: lipids, proteins, mRNA, non-coding RNA, DNA • They play a role in cell- cell communication, and metastasis • Therefore, exosomes probably contain most of the cell machinery biomolecules, used for cancer progression in the recipient cells ( target cells) • They are found in all body fluids (urine, blood, breast milk, CSF, saliva…etc) • Their number in cancer patient are higher than normal

39. EmanuleCocucci & jacopo M. Trends in cell biology, 2015

40. PM Gross, Nat CellBiol 2012 Baietti, Nat CellBiol 2012 Early Endosome ESCRT Tetraspanins ESCRT Ceramide Ceramide Tetraspanins Perez-Fernandez, JBC 2013 Abrami, Cell reports 2013

42. Applications of exosomes • Due to their protein and nucleic acids contents, which closely reflects the nature and state of their parental cells, exosomes are considered full of essential genetic information. • All of the molecules found in exosomes are potentially useful for diagnostic purposes • screening testing • confirmatory diagnostics • monitoring of treatments • Drug/Target validation by: • Transmission Electron microscope (quantifying their number (increased in malignant), analysis of their components (mRNA, non coding RNA, DNA using PCR). Proteins using ELISA, mass spec., chromatography)

44. Validation of anti-cancer drugs 1- Tumor markers 2- RNA, DNA, proteins tools depending on the drug target 3- Transgenic animals 4- Biochemical assays /in-vitro assays depending on the target 5- Microscopic techniques as confocal microscopy

45. CLASSIFICATION OF TUMOR MARKERS 1- Enzymes and iso-enzymes: • a)Acid Phosphatase (ACP) : not reliable test for prostate cancer because it may elevated in benign cases, and has many isoenzymes. • b)Prostatic Isoenzyme (PAP) : ACP isoenzymes (elevated in early prostatic carcinoma and in benign prostatic hyperplasia) • c) Prostatic Specific Antigen (PSA) : more specific for screening or for detection of early cancer as it is found in prostate tissue with large quantities. It is classified as enzyme or antigens. • d) Alkaline Phosphatase (ALP) • Raise in primary bone and hepatic cancer and metastases to other organs. • ALP isoenzymes : bone, liver, kidney, small intestine, and placenta. • e) Placental ALP (isoenzyme): elevated in a variety of malignancies, including ovarian, lung, gastrointestinal cancers. • Therefore, It is necessary to identify which ALP iso-enzyme is elevated before taking the decision of the tissue source of its elevation. - Enzymes and isoenzymes - Hormones- Receptors- Proteins (Tumor Antigens)- Genetic Markers

46. f) Neuron specific enolase (NSE): isoenzyme of the glycolysis enolase • enzyme (converts 2- phosphoglycerate into phosphophenol Pyruvate) • NSE gamma iso-enzyme is present only in brain and peripheral neuro-endocrine cells (cells that receive neurotransmitters released by nerve cells or neuro-secretory cells and, release hormones in blood) such as lungs and is useful marker for lung cancer, and neuroblastoma.

47. 2- Hormones: • eutopic production : Secreted bye endocrine glands normally responsible for their production • ectopic production: Hormones produced by organs that normally do not produce these hormone • Hormones are elevated in benign neoplasm as tumors • B-hCG (Beta-human Chorionic Gonadotropin): synthesized in placenta • It is used in conjunction with AFP in the classification of germ cell tumors of the ovary and testes • Used as a marker in placental carcinoma ( B-subunit). • - Calcitonin: used in early diagnosis of thyroid gland carcinoma. • - Vanillylmandelic acid (VMA) : catecholamine metabolites • They are found in small amount in blood because they are metabolized quickly so, detected in urine • It is used for testing Pheochromocytomastumors in urinary samples. (tumor of • adrenal or extra adrenal medulla of the adrenal gland). • -hydroxindole-3-acetic acid (5-HIAA) • Serotonin metabolite. Serotonin is produced in the brain and the enterochromaffin cells in the GI tract : epithelial cells lining the lumen of the GI-tract that release serotonin • GIT and brain tumor markers

48. - Hormones receptors • Receptors: Recognition, initiation a biological response, regulation of the cells. • Tumors change in receptors function and amount so, can be used as tumor markers. • estrogen (ER) and progesterone receptors : used for breast cancer response to treatment • 3- Tumor Antigens: • a)Oncofetal antigens : found in fetal and cancer, but not in healthy • They are tumor associated but not tumor specific antigens • -Alfa feto protein: liver and germ cell tumors • - Carcinoembryionic antigen: colon, pancrease, ovary, lung and breast cancer • b)Carbohydrate Antigens (CA) • glycoproteins defined by their antigenic properties. • cell surface antigens or secreted antigens that are expressed by the tumor cells. • mucins CA15-3 ( monitor breast cancer patients), CA125 : ovarian cancer, • CA19-9 ( pancreatic cancer and colon cancer), CA72-4 ( ovary, breast, colon, lung, pancreatic ) cancers.

49. 4- Genetic markers: • -detection of mutations and/or alterations of genes • Prognosis • Predict or evaluate treatment response • Oncogenes: is a mutant form of a normal gene (proto-oncogene) involved in • cell growth or division • Derived from normal cellular genes which are involved in • cell growth, proliferation, differentiation and apoptosis • Alteration in these genes is a main cause for cancer development. • suppressor genes (Anti-oncogenes) • They are genes that protect a cell from being cancer cell by promoting • apotosis, 2) repressive effect on cell cycle growth in case of DNA damage • 3) inhibition of metastasis • The loss of these genes and consequently their protein products, • allows continuous transcription of growth, proliferation, and metastasis.

50. Four classes of genes are implicated in development of cancer 1- Proto Oncogenes which are responsible for normal cell growth and differentiation 2- Tumor suppressor genes which are involved in recognition &damaged DNA repair 3- Apoptosis-related genes, that is responsible for regulation of apoptosis 4- DNA repair genes: as Topoisomerases, Tyrosyl phosphodiesterases enzyme Alterations on these genes develop cancer cells • HER-2/neu • It codes for a transmembrane receptor. • Overexpressed in breast and ovarian cancer, sometimes in GI cancer