American Society of Cytopathology Core Curriculum in Molecular Biology

Transcription

1 American Society of Cytopathology Core Curriculum in Molecular Biology

2 American Society of Cytopathology Core Curriculum in Molecular Biology Chapter 1 The Molecular Basis of Cancer The Molecular Basis of Cancer: An Overview Marilee Means, PhD, SCT(ASCP) University of Kansas Medical Center Kansas City, Kansas

3 Objectives Discuss how various theories regarding the development of cancer have a common thread regarding genetic abnormalities.

4 Why should cytotechnologists learn about cancer from a molecular perspective? Knowledge about the root causes of cancer have exploded in recent years Much of this new knowledge is at the genetic and molecular level Many new tests and treatments are geared to utilizing this new knowledge Cytotechnologists will increasingly need to know how this information meshes with their traditional morphologic knowledge

5 What causes cancer? Many factors have been noted to cause an increase in cancer These include viruses, chemical exposure, UV light exposure, radiation, somatic mutations, inherited genetic abnormalities But exactly how do these factors cause a normal cell to become malignant?

6 Several theories were put forth Abnormal differentiation the cell s behavior changed, not its genes Human tumor viruses infected cells and changed them from normal to cancer Mutagens (chemicals, radiation, etc.) caused damage to the cell s DNA which led to CA

7 Which of these theories is correct? DNA defects Viral Mutagens Abnormal behavior of cells

8 List of CA causing agents/events Oncogenes activated, added to genome Tumor Suppressor Genes deactivated or deleted from genome Breakdown of DNA repair Immortalization Nongenetic promotors after initiation Ability to invade and metastasize

9 Discovery of oncogenes Proto-oncogene a gene with an inherent potential to become activated into a cancer-causing oncogene src (src gene) was found to carry cancercausing information in the viral genome of the Rous sarcoma virus (RSV) (a chicken virus)

10 How the src gene became transformed The src gene in RSV was found to have a closely related gene in the normal genome of the chicken This closely related gene was called c-src (cellular src) to differentiate it from the viral src gene (v-src) The virus copied the c-src into its own genome after infection of a cell and slightly remodeled it into v-src

11 Proto-oncogene vs. Oncogene Thus, the normal c-src gene is a protooncogene, harmless if normal but cancer causing if transformed into an Oncogene a cancer causing gene: v-src Many other proto-oncogenes and oncogenes have been found, including the ras family of oncogenes, as well as myc Virus infection can cause this transformation

12 Examples of viral induction of CA Human papilloma virus causes cervical CA Liver cancer is caused by HBV Epstein-Barr virus is associated with Burkitt s lymphoma

13 BUT nearly 80% of human CAs do not involve viruses Could mutagens such as radiation and chemicals also alter the proto-oncogenes into oncogenes? Yes! The normal H-ras gene varies by only ONE nucleotide from the tumor-associated H-ras gene (a point mutation) This small difference makes the harmless proto-oncogene into a virulent oncogene

14 Virus induced tumors do have genetic disturbances Myc oncogene is amplified (present in increased gene copy numbers) in some human hematopoietic tumors Myc oncogene is chromosomally translocated (moved) in other hematopoietic tumors to come under the control of immunoglobulin genes rather than its own control genes

15 myc genes visible as red immunofluorescent tags in CML

16 List of CA causing agents/events Oncogenes activated, added to genome Tumor Suppressor Genes deactivated or deleted from genome by o inheritance o somatic mutation o loss of heterozygosity (LOH) o DNA virus oncoproteins binding to TSGs Breakdown of DNA Repair Immortalization Nongenetic promotors after initiation Ability to invade and metastasize

17 Tumor Suppressor Genes Discovered by fusing (hybridizing) a tumor cell with a normal cell Hybrid cells were usually not cancer inducing when injected into mice The normal genes dominated over the cancer genes (i.e. the cancer genes were recessive) These normal genes which controlled the CA were named tumor suppressor genes: TSGs

18 Retinoblastoma (RB gene) Two types of the disease, familial and sporadic In familial type, cancer occurs early and is bilateral/multicentric In sporadic type, cancer occurs later and is unilateral Both types involve the loss of the RB gene (a TSG)

19 Retinoblastoma Cross section of eye with tumor. Cat s eye reflex is the appearance of a white, rather than black pupil. The white appearance is caused by a mass growing in the intraocular area of the eye. Histology of retinoblastoma.

20 Retinoblastoma genetics Familial type Sporadic type Defective Rb Normal Rb First somatic mutation Loss of BOTH normal Rb genes leads to disease; if present, normal Rb acts as TSG Second somatic mutation Bilateral or multicentric disease Unilateral disease

21 How likely is the second somatic mutation to occur in sporadic type? Usually a rare event BUT The evolving premalignant cells with one inactivated Rb gene tend to discard the second, still intact Rb gene and replace it with a duplicated version of the abnormal Rb gene This is an example of LOH loss of heterozygosity Areas of the genome of tumor cells which suffer LOH are thought to contain TSGs

22 Most CAs have more inactivation of TSGs Most tumors have only one oncogene but have multiple inactivated TSGs TSGs are inactivated more frequently than oncogenes are activated in many tumors TSGs play as important a role in carcinogenesis as oncogenes

23 DNA viruses may target TSGs DNA viruses such as HPV and SV40 may produce oncoproteins which bind and sequester TSGs such as p53 and prb. This inactivates these tumor suppressor genes and leads to tumor progression just like the somatic mutation TSG inactivation seen in retinoblastoma

24 List of CA causing agents/events Oncogenes activated, added to genome Tumor Suppressor Genes deactivated or deleted from genome Breakdown of DNA Repair by o somatic mutation o DNA methylation (epigenetic mechanisms) Immortalization Nongenetic promotors after initiation Ability to invade and metastasize

25 Breakdown of DNA repair Several familial cancers, such as HNPCC (hereditary nonpolyposis colon cancer) and Xeroderma pigmentosum (XP) are associated with defective DNA repair mechanisms These usually detect, delete, and correct defective DNA copies before they reproduce as defective clones

26 Accelerates the mutation process These diseases have defects in repair genes Resultant genetic instability leads to more possibilities for damage to TSGs and/or activation of oncogenes Mutant versions of BRCA1 and BRCA2 are associated with familial breast cancer Normally thought to repair ds DNA breaks Mutant forms not found in sporadic CA

27 Epigenetics Switching on and off (activation and deactivation) of genes WITHOUT a change in the genetic makeup of the cell Can be inherited by progeny cells Includes deactivation by DNA methylation If affects TSGs and DNA repair genes, will promote development of CA

28 DNA methylation The addition of a methyl group to cytidine in CpG dinucleotide sequences Results in chromatin configuration shifts and shutdown of expression of nearby genes transcriptional repression Daughter cells have methyl groups added as well by DNA maintainance methlyase This results in the inheritance of methylation and subsequent repression of nearby genes in progeny cells

29 DNA methylation enetics/control/ enetics/rats/ Nurturing behavior in rats Rat pups who receive high or low nurturing from their mothers develop epigenetic differences that affect their response to stress later in life. When the female pups become mothers themselves, the ones that received high quality care become high nurturing mothers. And the ones that received low quality care become low nurturing mothers. The nurturing behavior itself transmits epigenetic information onto the pups DNA, without passing through egg or sperm.

30 Epigenetics explains how organisms functionally adapt to change Does not rely on random mutations which might provide a benefit in a new environment or not Turns off or on genes by methylation and acetylation which may provide better survival Does not require a change in the DNA May be inherited by progeny

31 Higher rates of methylation Loss of DNA repair and TSGs is more commonly caused by methylation than by somatic mutations Thus cancer is a disorder of genes and gene function, but does not always depend on genetic alterations p.11 The Molecular Basis of Cancer

32 List of CA causing agents/events Oncogenes activated, added to genome Tumor Suppressor Genes deactivated or deleted from genome Breakdown of DNA Repair Immortalization by o Activating expression of telomerase enzyme Nongenetic promotors after initiation Ability to invade and metastasize

33 Telomere shortening Normally, telomeres at the end of each chromosome shorten after each replication Results in a pre-programmed number of replications When telomere length becomes too short, cells enter a crisis stage and die Telomerase restores and maintains telomeres, resulting in immortality for cells

34 Telomerase expression Cancer cells induce expression of telomerase by an array of transcriptional regulators Telomerase is detectable in over 90% of human tumors but at low or undetectable levels in normal tissue

35 List of CA causing agents/events Oncogenes activated, added to genome Tumor Suppressor Genes deactivated or deleted from genome Breakdown of DNA Repair Immortalization Nongenetic promotors after initiation o These assist reproduction of abnormal clones Ability to invade and metastasize

36 Cancer development - multistep First step usually involves genetic damage Subsequent steps are usually non-genetic and include such factors as diet, environment, chronic inflammation leading to release of growth-stimulating factors, etc.

37 List of CA causing agents/events Oncogenes activated, added to genome Tumor Suppressor Genes deactivated or deleted from genome Breakdown of DNA Repair Immortalization Nongenetic promotors after initiation Ability to invade and metastasize o Invasion-metastasis cascade o Epithelial-mesenchymaltransition (EMT)

38 Invasion-Metastasis Cascade Invade adjacent tissue Enter blood and lymphatic systems Travel to distant sites Escape into tissue Establish small tumors (micromets.) Colonize macroscopic metastases 90% of cancer deaths are caused by mets.

39 Liver metastases gross and histology Liver with white appearing metastases present throughout. Histology of metastatic breast CA stained with a brown marker for b1.1.

40 EMT occurs normally in embryonic morphogenesis Transcription factors (Snail, Slug, etc.) can program epithelial cells to behave like mesenchymal cells during early embryonic development Carcinoma cells may exploit these early genes to perform the first 5 of the 6 steps in the I-M cascade

41 Activation of EMT Certain genomes of CA cells receive signals from the tumor microenvironment and stroma, which then induce EMT For example, TGF-β elicits expression of several transcription factors capable of beginning an EMT EMT cannot however, help in colonization; this mechanism remains unclear

42 Summary Oncogenes (bad) Tumor Suppressor Genes (bad if lost) DNA repair mechanisms (bad if lost) Immortalization (bad) Nongenetic promotors (bad) Ability to invade and metastasize (bad)

43 Flowchart of Molecular Basis of Cancer Acquired (environmental) DNA damage Normal Cell DNA Repair DNA Damage Failure of DNA Repair Mutations in the genome of somatic cells Inherited mutations in genes for DNA repair Genes affecting cell growth or apoptosis Activation of oncogenes Inactivation of TSGs Alterations in genes for apoptosis Unregulated cell proliferation Decreased apoptosis Angiogenesis Escape from immunity Clonal expansion Tumor progression Additional mutations Invasion and metastasis

44 Who was right? Everyone in a way Many tumors do have elements of genetic abnormality such as familial tumors Carcinogens play a role in causing DNA abnormalities Viruses may either carry in an oncogene or produce an oncoprotein which inactivates one or more TSGs

45 Who was right? Cell s behavior did change due to signaling from tumor environment causing EMT event and by immortalization (telomerase) Promotor (non-genetic) and epigenetic (DNA methylation) events are important as well

46 Summary Cancer is almost always a multistep process with a combination of genetic, epigenetic, environmental, and infectious factors These are just a few of the known mechanisms Seems to be related to loss or gain of function of genes through a complex process We have much more to learn Reference: The Molecular Basis of Cancer, Mendelsohn J, et al. 3rd ed. Saunders, 2008.