MMG175 mt2

Endogenous sources of DNA damage

Replication errors, ROS

Exogenous sources of DNA damage

Alkylating agents, UV, cisplatin, IR (ionizing radiation)

3 Classes of DNA damage

Errors in DNA replication, biochemically induced damage, physical damage

Errors in DNA replication

mismatched bases, indels, template slippage

Biochemically induced DNA damage

base damage, strand breakage, DNA adducts

Physical damage

Thymidine dimers, strand breaks

MutSa

Heterodimer of MSH6 and MSH2, scans for mismatch

MutLa

Heterodimer of MLH1 and PMS2, fixes mismatch

Deamination of DNA bases

Converts C -> T

What dimer is common in skin?

CC, from UV radiation

Common driver mutations in normal skin

Notch1 and p53, also BRAF and NRAS but less because they trigger apoptosis

Notch signaling

Cell surface receptor becomes TF through proteolysis, activates transcription

p53 signaling

p53 is a TF that induces p21 transcription, which blocks G1/S Cdk-cyclin kinase activity and prevents cells from entering S phase, allows DSB repair

3 mechanisms of biochemical protection from DNA damage

1. Enzymes reduce levels of harmful chemicals. ex/ Antioxidants like catalase and superoxide dismutase reduce ROS levels. 2. Enzymes reverse damage. ex/ MGMT reverses alkylation by removing methyl groups from modified bases. 3. Cell induces apoptosis by p53 activation

HR pathway

Homology directed repair: uses sister chromatid after DNA rep or homologous chromosome before (causes LOH). DSB sensed by MRN complex, DNA ends resected by EXO1, BLM, and CtIP to make long 3’ ends, RPA coats 3’ ends and Brca2 and Rad51 bind to prime for DNApol, DNApol and helicases synthesize DNA, ligases and resolvases resolve the 2 dsDNA molecules

What repair pathway is used for UV damage?

NER is used for bulky and distorting errors, DNA segment is removed and other strand used as template for repair

NHEJ pathway

Does not use template DNA. DSB sensed by MRN complex, Ku70/80 bind to DNA ends to prevent degradation and act as a scaffold to assemble NHEJ machinery, error-prone polymerases fill the gap, ligase 4 seals DNA ends tg

BCR-ABL translocation

constitutively active ABL kinase increases transcription

IGH-MYC translocation

IGH has strong promoter so MYC is overexpressed

KO Ku80 or Ligase4 mice experiment

Knockout NHEJ machinery but translocations still occur. Proves that even though NHEJ causes most, HR is also involved

VDJ recombination

Programmed DSBs in immune cells to generate variable antibodies. Endonuclease Rag cuts DNA and rearranges segments of heavy and light chains. Mistakes cause leukemia or lymphoma when strong promoter is placed next to oncogene

AID enzyme

Designed to cut within IgH for antibody class switching, but can induce breaks at IgH and Myc which triggers translocation and IgH-Myc, drives proliferation

Translocation risk factors

Factors that induce DSBs (ROS), AID action at IgH and off-target locations, replication stress and persistent replication fork stalling, defective NHEJ and HJ pathways

Replication stress

Increases DSB risk. ex/Repetitive DNA sequences, highly transcribed areas and loose chromatin, R-loops

MRN complex

Mre11, Rad50, Nbs1

Damage response cascade

Sensors (MRN, MutSa), transducers (kinases), effectors (Ku70/80, Brca1/2, p53)

Cancer stage vs grade

Grade is cell morphology. Stage measures spread, I-IV

Stages

Stage 0 is abnormal cells that has not spread to other tissue. Stage I is localized and contained within the organ of origin. Stage II and III are larger and may have spread to lymph nodes. Stage IV has metastasized to other organs. Tumorigenesis is process of cells becoming cancerous

Cancer detection methods

VIsual changes (ex/mole), symptoms, physical screens for masses, molecular screens

Cancer tests

Screening tests for people not suspected of having cancer to find it before symptoms develop. Diagnostic tests for people who are suspected of having cancer to determine if they have it, if it has metastasized, and to guide treatment. Monitoring tests for after diagnosis to monitor progress.

Role of cancer screening

Disease must be common with high morbidity or mortality, treatment must be available, must be safe, inexpensive, and ethical

Common cancer screening modalities

CT, PET, MRI, ultrasound. Often multiple used tg

CT scan

Computed tomography scan creates 3D model with images from different angles. Not good for soft tissues

MRI

Magnetic resonance imaging. Similar to CT but useful for soft tissues

PET scan

Positron emission tomography. Uses radioactively labeled glucose to highlight cancer, which takes up a lot of glucose (brain and bladder too)

Ultrasound

High frequency sound waves bounce off tissues, distinguish between solid and fluid filled masses (often benign)

Mammogram

Low dose x-ray of breast, identifies even small tumors. Diagnosis increased but cancer rate unchanged

Decide to treat tumor based on its class

1. indolent with low invasive potential (prostate cancer), 2. aggressive and probably already metastasized by diagnosis (pancreatic cancer), 3. intermediate grade with potential to disseminate but can be excised or treated before metastasis (breast cancer)

Colonoscopy study

Invited lots of ppl to get colonoscopy and did 10 year follow up, result showed it is not very effective at reducing prostate cancer. But only 42% of people invited actually got a colonoscopy, so result was better, still not very high. Also prostate cancer is indolent and grows slowly so 10 years is not a long time for it to develop

PSA test

Prostate specific antigen blood test, increased PSA indicates prostate growth and potential cancer, but leads to overdiagnosis because detects indolent tumors that are not very harmful

AFP test

AFP protein is found in liver cancer patient blood. Many patients with early liver cancer have normal AFP and elevated AFP can be caused by other stuff, so kind of low sensitivity and specificity. Use in combination with other tests

Sensitivity and specificity

Sensitivity is the ability to correctly identify those with disease (idenfity 95 positive results, 95% sensitivity). Specificity is the ability to correctly identify those without (3 false positives, 97% specificity)

CA19-9

Tumor marker for pancreatic cancer, not very specific or sensitive

Biopsy

Remove a small tissue sample for analysis, invasive but accurate.

Pancreatic tumor subtypes

Subtype histology looks very different. diff subtype diff prognosis diff treatment. PNET rare and high survival, PDAC common and low survival

Immunohistochemistry IHC

Primary antibody detects protein of interest and secondary antibody is labeled and can be stained. Used in breast cancer to label ER, PR, and HER2. triple negative is difficult to treat. Herceptin treats HER2+

Molecular Pathology

Use molecular data to classify cancers: proteins, DNA, RNA, expression. Gene expression profiling technique studies RNA conc to see what genes are highly transcribed DNA -> RNA, identify “poor signature” and genes associated with metastases

Transcriptome

Complete set of RNA in a cell.

DNA and RNA seq

RNAseq identifies which genes are active using tissue from biopsy. DNAseq detects driver mutations (ex/ identifies KRAS G12C mutation, treat with specific KRAS G12C inhibitor)

Basket vs umbrella trial

Basket trial groups patients by shared mutation, tests one drug. Umbrella trial groups patients by origin tissue, tests different drugs depending on subtype

Liquid biopsy

Blood test that analyzes circulating tumor cells (CTCs, rare, more CTCs worse prognosis) and cell free DNA (cfDNA, released when cells die, increased level in cancer patient, helps identify mutations), and protein markers. Effectiveness depends on cancer type

ctDNA usage post surgery

Test for ctDNA (circulating tumor DNA) after surgery to see if chemo is necessary, early marker of residual disease.

Therapeutic window

Range of drug concentrations (doses) where desired response is achieved but side effects are acceptable, balances efficacy and toxicity

Therapeutic index

Larger index safer drug. TI=TD50/ED50, toxic dose causing unmanageable side effects in 50% of individuals/effective dose causing desired response in 50%

Chemotherapy classes

Alkylating agents, crosslinking agents, topoisomerase inhibitors, antimetabolites, microtubule inhibitors, nucleoside analogs, drugs that interfere with nucleoside biosynthesis

Alkylating agents

MMS, nitrogen mustards

Crosslinking agents

Cisplatin and carboplatin, Induce apoptosis via p53 especially in testicular and ovarian cancers

Topoisomerase inhibitors

Camptothecin (DNA topoisomerase I), doxorubicin (II). Topoisomerase function is to cut DNA to relieve stress, but inhibitors prevent it from resealing DNA, disrupts checkpoint control so cell enters mitotic catastrophe and eventually dies

Anti-metabolites

Small molecules that interfere with normal metabolite function. Methotrexate binds DHFR enzyme better than its natural substrate and inhibits DNA synth. Taxol prevents microtubule depolymetization (microtubule stabilizer) so cell can’t divide

Microtubule stabilizer

Taxol

Microtubule destabilizer

Vinblastine

Synthetic lethality

Loss of both genes is much worse than loss of either alone, ex/ PARP and HR (BRCA1/2) inhibition removes both repair pathways

Induced differentiation

Some muts block differentiation, ex/ PML-RARa so induce differentiation by adding ATRA (retinoic acid derivative) to inactivate fusion protein

Druggable targets

Proteins with a cavity, catalytic cleft. Yes kinase (ex/ Gleevec inhibits Bcr-Abl in CML, Velcade leads to IKB accumulation which inhibits NFkB in multiple myeloma), Yes oncogene when cell is addicted (ex/ Iressa and Tarveca inactivate EGFRs), no TFs, no G protein

Metaplastic conversion

Normal-looking cells in the wrong location, ex/ metaplastic Barrett’s epithelium, precursor stomach cancer cells start looking like normal esophageal cells

Colorectal cancer stepwise progression

Series of events are needed for cancer to develop and the order matters. LOH in Chr. 5q occurs initially, then activation of K-ras, etc etc, metastasis. PanIN is types of benign tumors that eventually become cancer, each mutation pushes it into the next stage. Diff mutations same results

Cells in G0 or replicating cells are more susceptible to becoming cancerous?

Replicating cells, ex/ at wound sites

Why does inflammation increase cancer risk?

Immune cells are recruited to inflammation sites and they produce cytokines, which act as growth factors. Experiment where they injected Rous sarcoma virus and control, both sides grew tumor bc inflammation. ex/ Hep virus infection doesn’t cause cancer but does cause inflammation. ex/ exp where helicobacter induction causing inflammation and immune system defect caused death, but NHEJ intact to repair immune system caused no death

TPA pathway

Tumor promoter, does not cause cancer on its own. DMBA causes initial mutation but TPA promotes it by induces NF-kB activation (promotes growth) and TNFa (inflammation-like signal), remove promoter and cell regresses to normal. TPA ->PKC ->>IKK ->NFkB->TNF->inflammation

Hyperplasia

A growth caused by cells replicating faster than normal

Senescene protective mech

Overexpression of oncogene induces senescence

Simultaneous oncogene activation and loss of tumor suppressor gene

Myc and Ras, V-Erb A and B (erythroid leukemia). trigger apoptosis when expressed alone, but together form foci (transformed clump of cells with no contact inhibition) which eventually can become cancer

How do cancer cells prevent apoptosis protective mech?

Overexpress survival pathways (BCL2, Myc) or inactivate apoptosis pathways (p53)

Proteins that lead to apoptosis

Caspase, p53, p14ARF, Bax, PTEN

Proteins that promote survival

ERK, RAF, PI3K, AKT, NFkB, MDM2, BCL2

EMT

Epithelial-mesenchymal transition. During normal embryogenesis, cells derived from the ectoderm migrate to form the mesoderm, triggered by HGF binding MET RTK. Cancer cells use this pathway for mobility to metastasize. MET is reverse pathway to help them settle at new tissue. (different MET acronyms)

Seed and soil hypothesis

Certain cancers preferentially metastasize to certain organs that provide supportive conditions

Galen theory 160 AD

4 humors, blood phlegm, yellow bile, black bile. Cancer is from black bile. Vesalius in 1500s discovered no black bile, led to rise of surgery for “movable” tumors

Surgery history

1840s anesthesia and hand washing, 1860 disinfectants, 1890s Halsted removed gallbladder to treat gallstones and introduced radical surgery concept, 1940s critics of radical surgery questioned how patient still relapsed, 1971 radical masectomy study, 1989 compares masectomy and with vs without radiation

Neoadjuvant vs adjuvant therapy

Neroadjuvant is before, Adjuvant is After surgery to kill residual cells

TNM staging

Tumor, lymph Nodes, distant Metastasis. ex/T1N0M0 is Stage I, small tumor with no lymph node involvement and no metastasis. T4N3M1 is Stage IV with invasive tumor and extensive lymph node involvement and metastasis present

Surgical margin

Negative margin resection = removed all tumor

BBB

Chemo drugs cannot cross blood-brain barrier so surgery is the only treatment option, but challenging

Gliolan

AA that accumulates in glioma tumor cells and labels them fluorescently, used for several other cancer types. Probes light up nerves so surgeon can avoid them

Why is radiation so effective?

Cancer cells cannot tolerate damage because their repair pathways are often mutated, also creates multiple types of DNA damage. Targets specific area with a beam. Effective but toxic to normal cells too

Internal radiation treatments

Pill that contains iodine is placed surgically, decays over time and slowly releases radiation

Radioactive antibodies

Conjugate iodine to antibodies specific for tumor antigens to target tumor cells

Bone marrow transplant

Blood cancer patients are treated with intense chemo/radiation which kills cancer cells but also normal blood cells, replace blood stem cells in marrow with transplant from donor (allogeneic). Autologous transplant in genetic disease where you fix your own DNA with crispr/cas and reinsert