Doc seminar cervical cancer

Presented By:

Manali Baghel

Ph.D. Scholar

College of Biotechnology

DUVASU

Cervical cancer is a female-specific disease with ahigh incidence and mortality behind breast & lungcancer

A disease in which malignant cells form in thetissues of the cervix

It rises in 30–34 years of age and peaks at 55–65years

The worldwide incidence of cervical cancer is~510,000 new cases with ~288,000 deathsannually

Cervical cancer is a complex disease caused by theinteraction of viral, host, and environmental factors

Prevention, diagnosis, and treatment of female-specific diseases areincreasingly important issues due to lifestyle changes of women

Human pappilomavirus (HPV) has been detected in more than 90% ofcervical cancers and therefore implicated as the main cause of cervicalcancer

HPV infection alone is not sufficient to induce the malignanttransformation of HPV-infected cells

Other unidentified genetic alterations, such as microRNAs the masterswitches, are required.

MiRNAs are found to be associated in many cases of cervical cancer withchanged expressions.

Signs and Symptoms

The most common symptoms are:

Bleeding between periods

Bleeding after sexual intercourse

Bleeding in post-menopausal women

Discomfort during sexual intercourse

Smelly vaginal discharge

Vaginal discharge tinged with blood

Pelvic pain

Causes

Risk factors which are known to increase the risk ofdeveloping cervical cancer-

Human pappilomavirus (HPV)

Many sexual partners, becoming sexually active early

Smoking

Weakened immune system

Giving birth at a very young age

Several pregnancies

Contraceptive pill

Other sexually transmitted diseases (STD)

Chlamydia, gonorrhea

Genetic Alterations

Stages of Cervical Cancer

Mild Dysplasia

Moderate Dysplasia

Severe Dysplasia

Invasive Cervical Cancer

Stages of cervical cancer according to WHO classification-

Types of Cervical Cancer

Ectocervix-

Squamous Cell

Carcinoma

Endocervix-

Adenocarcinoma

Tests To diagnose Cervical Cancer

Phisical Exam & History

Pelvic Exam

Pap Test

HPV Test

Endocervical Curettage

Colposcopy

Biopsy

Cervical Cancer Vaccines

Two Vaccines are Licensed globally

which are also available in India:

A Quardivalent vaccine- Gardasil

(Merk)

HPV Serotypes 16, 18, 6 & 11

A bivalent vaccine- Cervarix

(Glaxo Smith Kline)

HPV serotypes HPV 16 & 18 Gardasil

Human Pappilomavirus (HPV)

One of the most common STIs

Papillomaviruses are small viruses approximately 52-55nm in size

HPVs are circular double stranded DNA viruses & belongs to family-Papillomaviridae

In 1981, Zur Hausen et al. reported the detection of HPV in cervicalneoplasia

In 1995 the WHO declared HPV as a known carcinogen for causingcervical cancer, because HPV DNA types could be detected in almost allcervical cancers

HPV Genome

Classification Of HPV

More than 100 types of human papilloma viruses (HPVs)

are known today, and they are generally classified according

to their potential to induce malignant transformation-

HPV Life Cycle and Infection

The HPV life cycle consists of initial infection, uncoating, genome

maintenance, genome amplification, and packaging to form new viral

particles

HPV Induced Dysregulation

Once viral DNA is incorporated into host DNA, oncogenic transformationis induced

Oncoproteins E6 & E7 inactivate tumor suppressor genes p53 & pRB.

E6 protein binds to E6-associated protein (E6-AP), a ubiquitin proteinligase , & forms E6/E6-AP complex & targets p53

E6 degrades p53, inhibits p53 binding to DNA & binds to p53 enhancers &suppressed p53 function

E6/E6-AP complex also activates telomerase & contributes to cellimmortalization

E7 protein contributes to carcinogenesis by degrading Rb family proteinsnecessary for cell cycle progression

HPV Determition

PCR-based methods are commonly used for HPV Detection

after DNA isolation

Most PCR assays utilize consensus primers, directed to a

conserved L1 gene, and hence are able to amplify most of the

mucosal HPV types

The other PCR type used to detect specific HPV is HPV type

specific PCR such as HPV 16 or 18

MicroRNA

MicroRNAs are small (~18-24 nt), non-coding RNAs that regulategene expression & associated with cancer

First miRNA, lin-4, was discovered in C. elegans in 1993, is foundin most eukaryotes, including humans

It is predicted that miRNA account for 1-5% of the human genomeand regulate at least 30% of protein-coding genes

To date, more than 17,000 miRNAs have been annotated in 142species, including over 1900 human miRNAs

MiRNA Biogenesis & Function

Classification of MiRNAs

MicroRNAs are classified in two group depending on their

origin-

Intergenic or Exonic miRNAs: located between the introns of

genes & transcribed by RNA pol II or pol III as a stem loop structure called

pri-miRNA

Interagenic or Intronic miRNAs: miRNAs located within an

intron of a protein coding gene & transcribed by RNA pol II as part of pre-

mRNA

MiRNA Time Line

(Lindow et al., 2012)

MiRNA Expression Profiling

Initially it was conducted on samples extracted from tissues, now stable

miRNAs are found in readily available body fluids including, serum ,

plasma, urine and saliva

MiRNA expressions are generally analyzed by microarray & qRT-PCR

using microRNA specific primers

U6 RNA, RNU44, and RNU48 is usually used as reference control & A.

thaliana miRNA as negative control

Quantification is done using the 2 delta Ct method, where fold change in

expression of a gene in an experimental sample is quantified relative to the

same gene in a reference sample

MiRNAs In Cervical Cancer

MiRNAs play a vital role in cancer regulating pathways, like controlling

cell proliferation, differentiation and survival

MiRNAs involved in carcinogenesis are classified into oncogenic miRNAs

(oncomiRs) and tumor suppressor miRNAs(TSG)

Involved in cancer pathogenesis by posttranscriptional regulation of gene

expression

50% of miRNA genes are localized in cancer-associated genomic regions

or in fragile sites or integration sites of high-risk HPVs

Expression patterns of miRNAs suggested that beyond HPV, microRNAs

play a major role in cervical cancer

Altered Expressions of MiRNAs in

Cervical Cancer

MicroRNAs expressions were analyzed for normal cervix and cervical

cancer tissues, by microarray in combination with RT-PCR verification &

found to be deregulated

On comparision, many miRNAs with cancer-specific upregulation or

downregulation have been found-

MiR-21 is overexpressed in cervical cancer and is a negative regulator of

expression of the tumor suppressor gene programmed cell death 4

(PDCD4)

MiR Let-7a was found to be downregulated by HPV & this

downregulation of miR let-7a leads to the aberrant expression of STAT3

(validate target of let-7a) developing CC

MiR- 218 is found to be underexpressed in CC tissues compared to the

normal cervix & leads to the decreased expression of LAMB3, which is

involved in cell migration and tumorigenicity

MiR-34a was identified as a direct transcriptional target of cellular

transcription factor p53, since HPV E6 oncoprotein destabilizes p53 during

virus infection, it causes down-regulation of miR-34a expression in most

CC tissues

Cont..

Regulation of miRNAs by HPV

Oncoproteins

Deletions or mutations in miRNA genes, as well as

aberrant expression of oncogenic or tumor-

suppressive miRNAs, are common in human

cancers

Deregulation of oncogenic and tumor suppressive

miRNAs in human cervical cancer is associated

with HR-HPV integration

Cervical cancer represents a unique tumor model

for understanding how viral E6 and E7

oncoproteins deregulate the expression of the

microRNA

MiRNAs

HPV

HPV-oncoproteins regulated miRNAs and factors

involved in malignant transformation.

miRNA Chromosome Putative Function

hsa-miR-210 11 Oncogenic (og)

hsa-miR-182 07 Og/tumeor suppressor(tsg)

hsa-miR-183 08 Og/tsg

hsa-miR-200c 12 Tumor suppressor


hsa-miR-193b 16 oncogenic

hsa-miR-34a 01 tsg

hsa-miR-31 11 og/tsg


hsa-miR-27a 19 Og/tsg

hsa-miR-503 X Og/tsg

hsa-miR-27b 09 Og/tsg

hsa-miR-127 14 og/tsg

MiRNAs Underxpressed in Cervical

Cancer Cell Lines

MiRNA Chromosome Putative Function







hsa-miR-1 01 Og/tsg




hsa-miR-223 X Og/tsg


HPV-Oncoproteins are able to regulate the

expression of miRNAs

HPV Proteins MiRNAs Up/Downregulated Target Gene

E5 mir-146a Up-regulated ZNF813

E5 mir-324-5p Down-regulated CDH2, CTNNB1

E5 mir-203 Down-regulated p63

E6 mir-34a Down-regulated p18Ink4c, CDK4,CDK6, Cyclin E2

E6 mir-218 Down-regulated LAMB3

E6 mir-23b Down-regulated uPA

E6/E7 mir-29 Down-regulated YY1 and CDK6

E7 mir-15b Down-regulated CCNA2, CCNB1,CCNB2 MSH6

E7miR-15a/miR-16-1 andmiR-203

Down-regulated c-Myc, c-Myb, PPAR

Diagnosis & Treatment of Cervical Cancer

Using miRNAs

Many studies have examined the use ofmiRNAs as cancer diagnostic marker and asanticancer therapy.

Diagnosis of Cervical Cancer Using

miRNAs in Serum

MiRNAs with expression changes in cancer have the potential to be

diagnostic biomarkers based on plasma & serum tests

miR-21 and miR-126 are found to be overexpressed in serum and are

associated with cervical cancer.

Overexpression of other miRs including miR-27a, miR-34, miR-34a, miR-

146a, miR-155, miR-196a, miR-203, and miR-221 was detected in the

serum of CC samples

These results indicate that miRNA levels in serum can be used for

diagnosis of cervical cancer

Diagnosis of Lymph Node Metastasis

Using miRNAs in Serum

Several miRNAs in serum have been identified as candidate markers for

lymph node metastasis in CC

Zhao et al. analyzed expression of miR-20a and miR-203 in serum

collected before surgery and treatment in 80 patients:

The miR-20a level in serum of patients with CC was markedly higher than

that in healthy volunteers and was overexpressed in patients with lymph

node metastasis

The expression level of miR-203 in patients with CC was higher in

comparison with healthy volunteers, however, lymph node metastasis was

found only when miR-203 expression was suppressed

MiRNA Therapeutic Approaches

MiRNA

Inhibition therapy

when the target miRNA

is overexpressed

MiRNA

Supplementation

therapy when the

miRNA is repressed

Treatment with miRNA

Supplementation

Anticancer treatment may be achieved by regulating the expression

level of miRNAs

The function of tumor suppressor miRs with reduced levels may be

recovered by supplementation of the miRNA itself

Atelocollagen, is being examined as a potential delivery system for

nucleic-acid-based drugs

This protein is extracted from calf dermis and then digested with

protease to reduce antigenicity & can be transferred in to tissues or

cells

Supplementary agents can be classified as:

o Hairpin single-stranded pre-miRNA

o Double-stranded RNA

Liu et al. introduced miR-143 into HeLa CC cells and showed that cell

growth was inhibited and apoptosis was enhanced with increased miR-143

expression & maintained Bcl2 (oncogene) expression

o Therefore, miR-143 has an association with Bcl2 and treatment targeting

this pathway may be possible

Similarly, supplementing anti-let7a miR in HPV16 positive CC cases can

increase the expression of down-regulated let-7a miR & maintain the

expression of STAT3

Cont..

Treatment by Inhibition of

miRNA Function

One strategy for overexpressed miRNA in cancer is to inhibit the miRNA

function using agents with complementary binding to the miRNA

Antisense miRNA oligonucleotides (AMOs) or ‘antagomirs’ are the most

common miRNA inhibitors based on Antisense technology

• For miR-21, an oncomiR in cervical cancer, anti-miR-21 was developed as

a modified 2’-O-methoxyethyl (2’-O-MOE) phosphorothioate antisense

agent

• Wang et al. found, miR-21 expression was downregulated and tumor

growth was markedly suppressed by the AMO in comparison with a control

group

• MiR-21 inhibition may be achieved with other approaches,

including miRNA sponges, miRNA erasers, and tough decoys

• Tough decoys may have particularly potent inhibitory activity

• Haraguchi et al. inhibited miR-21 using a tough decoy and

recovered expression of PDCD4, a target gene of miR-21

Cont..

Difference MiRNA Based Drug &

Traditional Drug

Miravirsen, first miR based drug, made for silencing

miR-122 in HCV infected patients.

Anti-miR Treatment In Other Deseases

miRNAs Associated with Therapeutic

Resistance for Cervical Cancer

Expression of various miRNAs is up- or downregulated in cervical cancer

and these expression levels can increase or decrease sensitivity to

chemotherapy and radiotherapy

Phuah et al. showed that the expression patterns of 25 miRNAs, including

miR-138, miR-210, and miR-744, altered the sensitivity to 1’S-1’-

acetoxychavicol (ACA) and cisplatin

Lei et al. found that miR-155 negatively regulates the EGF-induced

epithelial-mesenchymal transition(EMT), inhibits proliferation, metastasis,

invasion, and increases sensitivity to cisplatin

Thus, miRNAs may have an important role in the response to

chemotherapy

Summary & Conclusion

HPV

Cervical

Cancer

Progression

Cervical cancer remains as a leading cause of

morbidity and mortality for women worldwide

HPV Integration may alter miRNA expression via

deletion, amplification, or genomic rearrangement

which may have implications for their expression in

cervical cancer

Depending on the nature of their targets, miRNAs

can function as either tumor suppressor miRs or

oncogenic miRs.

These findings suggest many approaches to miRNA-

specific personalized treatment and molecular targeted

therapy

Therefore, miRNAs are likely to be important in

diagnosis and treatment of cervical cancer.

Target

MiRNA

References

• Walboomers JM, Jacobs MV, Manos MM, Bosch FX, Kummer JA, et al.(1999) Human papillomavirus is a necessary cause of invasive cervicalcancer worldwide. J Pathol 189: 12-19.

• de Villiers EM, Fauquet C, Broker TR, Bernard HU, zur Hausen H (2004)Classification of papillomaviruses. Virology 324: 17-27.

• Ho GY, Bierman R, Beardsley L, Chang CJ, Burk RD (1998) Natural history ofcervicovaginal papillomavirus infection in young women. N Engl J Med338: 423-428.

• zur Hausen H (2002) Papillomaviruses and cancer: from basic studies toclinical application. Nat Rev Cancer 2: 342-350.

• Klingelhutz AJ, Foster SA, McDougall JK (1996) Telomerase activation bythe E6 gene product of human papillomavirus type 16. Nature 380: 79-82.

• Scheffner M, Werness BA, Huibregtse JM, Levine AJ, Howley PM (1990)The E6 oncoprotein encoded by human papillomavirus types 16 and 18promotes the degradation of p53. Cell 63: 1129-1136.

• Werness BA, Levine AJ, Howley PM (1990) Association of humanpapillomavirus types 16 and 18 E6 proteins with p53. Science 248: 76-79.

• Huibregtse JM, Scheffner M, Howley PM (1991) A cellular proteinmediates association of p53 with the E6 oncoprotein of humanpapillomavirus types 16 or 18. EMBO J 10: 4129-4135.

• Dyson N, Howley PM, Munger K, Harlow E (1989) The human papillomavirus-16 E7 oncoprotein is able to bind to the retinoblastoma geneproduct. Science 243: 934-937.

• Munger K, Werness BA, Dyson N, Phelps WC, Harlow E, et al. (1989)Complex formation of human papillomavirus E7 proteins with theretinoblastoma tumor suppressor gene product. EMBO J 8: 4099-4105.

• Farh KK, Grimson A, Jan C, Lewis BP, Johnston WK, et al. (2005) Thewidespread impact of mammalian MicroRNAs on mRNA repression andevolution. Science 310: 1817-1821.

• Volinia S, Calin GA, Liu CG, Ambs S, Cimmino A, et al. (2006) AmicroRNA expression signature of human solid tumors defines cancer genetargets. Proc Natl Acad Sci U S A 103: 2257-2261.

• He, L., He X., Lim, L.P. et al., (2007). A microRNA component of the p53tumour suppressor network. Nature. 447, 1130–1134.

• H. L. Howie, R. A. Katzenellenbogen, and D. A. Galloway, “PapillomavirusE6 proteins,” Virology, vol. 384, no. 2, pp. 324–334, 2009.

• T. Kiyono, A. Hiraiwa,M. Fujita, Y. Hayashi, T. Akiyama, and M. Ishibashi,“Binding of high-risk human papillomavirus E6 oncoproteins to the humanhomologue of the Drosophila discs large tumor suppressor protein,”Proceedings of the National Academy of Sciences of the United States ofAmerica, vol. 94, no.21, pp. 11612–11616, 1997.

• Kozomara A, Griffiths-Jones S. MiRBase: integrating microRNA annotation and deep-sequencing data. Nucleic Acids Res 2011;39:D152–7.

• Q. Yao, H. Xu, Q.-Q. Zhang, H. Zhou, and L.-H. Qu, “MicroRNA-21 promotescell proliferation and down-regulates the expression of programmed celldeath 4 (PDCD4) in HeLa cervical carcinoma cells,” Biochemical andBiophysical Research Communications, vol. 388, no. 3, pp. 539–542, 2009.

• S. Gilad, E. Meiri, Y. Yogev et al., “Serum microRNAs are promising novelbiomarkers,” PLoS One, vol. 3, no. 9,Article ID e3148, 2008.

• P. S. Mitchell, R. K. Parkin, E. M. Kroh et al., “Circulating microRNAs as stableblood-based markers for cancer detection,” Proceedings of the NationalAcademy of Sciences of the United States of America, vol. 105, no. 30, pp.10513–10518, 2008.

• C. H. Lawrie, S. Gal, H. M. Dunlop et al., “Detection of elevated levels oftumour-associated microRNAs in serum of patients with diffuse large B-celllymphoma,” British Journal of Haematology, vol. 141, no. 5, pp. 672–675,2008.

• S. M. Wilting, R. A. van Boerdonk, F. E. Henken et al., “Methylation-mediatedsilencing and tumour suppressive function of hsa-miR-124 in cervical cancer,”Molecular Cancer, vol. 9, article 167, 2010.

• X. M. Wang, J. Xu, Z. Q. Cheng et al., “Study on effects of microRNA-21antisense oligonucleotide in vivo and in vitro on bionomics of human cervicalsquamous carcinoma cell lines SiHa,” Chinese Journal of Pathology, vol. 41, no.4, pp. 254–259, 2012.

• T. Haraguchi, Y. Ozaki, and H. Iba, “Vectors expressing efficient RNA decoysachieve the long-term suppression of specific microRNAactivity inmammaliancells,”Nucleic Acids Research, vol. 37, no. 6, article e43, 2009.