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p73 in colorectal cancer
Linköping University, Department of Clinical and Experimental Medicine, Oncology . Linköping University, Faculty of Health Sciences.
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Colorectal cancer (CRC) is the third most common cancer in the world, with about 5000 new cases in Sweden every year. CRC is caused by mutation (inherited or acquired) in genes, by gene variants and changed expression of proteins. The primary way to achieve a curative result for CRC is to remove the tumor by surgery. To reduce risk of recurrence chemo- or radiotherapy are given as a complement to surgery. p73 is a structural and functional homologue of tumor suppressor p53. However, p73 is rarely mutated in tumors, but rather overexpressed as compared to normal tissue. There are two main isoforms of p73, the transactivation capable TAp73 and the truncated ΔNp73, which are involved in an autoregulatory loop with TAp73 and p53.

The aim of this study was to investigate the role of p73 and related proteins in the development and treatment of CRC. A G4C14-to-A4T14 polymorphism of p73 was studied in CRC patients and healthy controls (Paper I), and rectal cancer patients who were randomized to treatment with either surgery alone or preoperative radiotherapy and surgery (Paper II). The AT/AT genotype of the p73 polymorphism may increase risk of CRC development and CRC patients with the AT allele had a better prognosis. When dividing the cases into colon and rectal cancer it was seen that in colon cancer the AT allele tended to be more favorable for overall survival, while in rectal cancer the GC allele seemed to be more favorable. Rectal cancer patients, with a combination of GC/GC genotype, wild type p53 and weak survivin expression survived longer after preoperative radiotherapy. This was not observed in the patients only receiving surgery. The protein expression of p73 was further studied in the rectal cancer patients randomized to treatment with either surgery alone or preoperative radiotherapy and surgery (Paper III). p73 was expressed higher in tumor tissue than in normal mucosa. Patients with p73 negative tumors had a lower risk of local recurrence after radiotherapy, as opposed to patients that had p73 positive tumors or patients with p73 negative tumors that did not receive radiotherapy. Effects of γ-radiation was further studied in colon cancer cell lines KM12C, KM12SM and KM12L4a regarding cell cycle, survival fraction (clonogenicity), apoptosis and protein expression patterns of mutated p53, TAp73, ΔNp73, survivin and PRL-3 (Paper IV). KM12C displayed low survival fraction, low apoptosis, no cell cycle arrest and an upregulation of the antiapoptotic ΔNp73 after irradiation. KM12L4a showed a high survival fraction, but high apoptosis, arresting of the cell cycle and upregulation of the radio-resistance factor survivin. The effects of overexpression and knockdown of survivin on TAp73, ΔNp73 and p53 expression in colon cancer cell lines HCT-116p53+/+ and HCT-116p53-/- with and without γ-radiation were studied (Paper V). Overexpression of survivin decreased wild type p53, whilst downregulation of survivin lead to a simultaneous downregulation of TAp73 and ΔNp73, mRNA and protein, both with and without γ- radiation. Knockdown of survivin also demonstrated an increase in apoptosis.

In conclusion, we showed that the G4C14-to-A4T14 polymorphism of p73 and p73 protein expression may be involved in CRC development, radiotherapy response and survival. We further showed that TAp73, ΔNp73 and p53 were regulated by survivin in colon cancer cells.

Abstract [sv]

Cancer i tjocktarmen (kolon) och ändtarmen (rectum) är den tredje vanligaste cancerformen i världen och i Sverige drabbas varje år ca 5000 personer av tjockoch ändtarmscancer. Antalet drabbade individer är högre i de industrialiserade länderna än övriga världen, vilket tyder på att vissa omgivnings- och livsstilsfaktorer påverkar risken för tjock- och ändtarmscancer. Även vissa genetiska faktorer påverkar risken för insjuknande i dessa cancerformer.

En gen är mallen för ett eller flera proteiner och en förändring (mutation) i en gen kan innebära att motsvarande protein förändrar eller förlorar sin normala funktion. En del gener har också nedärvda naturliga variationer, så kallade polymorfier, vilka i sin tur kan leda till variation i funktionen hos motsvarande protein. Proteiner som förändrat eller förlorat sin normala funktion bidrar till de speciella egenskaper som finns hos cancerceller. Cancerceller till skillnad från normala celler delar sig okontrollerat, är motståndskraftiga mot den programmerade celldöd (apoptos) som normalt förstör skadade celler och kan sprida sig via blodbanan till andra organ (metastasera).

Tjock- och ändtarmscancer behandlas främst med kirurgi, cellgifter och strålning och tack vare förbättrade behandlingar har dödligheten minskat de senaste årtiondena. Genom att studera olika genetiska varianter, proteiner och förhållandet mellan proteiner i cancerceller får vi större insikt i vilka faktorer som ökar risken för tjock- och ändtarmscancer, men också vilka faktorer som påverkar hur effektiv en behandling är för varje individuell patient.

Målet med denna avhandling var att studera proteinet p73 och hur den påverkar risken för tjock- och ändtarmscancer och behandlingseffekten av sjukdomen. p73 tillhör samma proteinfamilj som “genomets väktare” p53 och båda har förmågan att skydda cellen genom att stoppa celldelning och inducera apoptos i skadade celler. Dock är p53 muterat i ungefär 50 % av alla tjock- och ändtarmscancrar.

Genom att studera en polymorfi i p73 hos tjock- och ändtarmscancerpatienter och friska blodgivare såg vi att de individer som har dubbel uppsättning av variantgenen har ökad risk att utveckla tjock- och ändtarmscancer och att de patienter som är bärare av variantgenen hade en längre överlevnad (Paper I). Ändtarmscancerpatienter som fått strålbehandling överlevde längre om de hade dubbel uppsättning av den ursprungliga p73-genen, icke-muterat p53 och lågt uttryck (mängd) av survivin. Survivin är ett protein som normalt hindrar cancerceller från att dö (Paper II). De ändtarmscancerpatienter som behandlats med strålning och som hade p73 negativa tumörer fick mycket färre återfall än de patienter som hade p73 negativa tumörer men inte fått strålbehandling, eller som hade p73 positiva tumörer (Paper III). För att närmare studera tjocktarmscancercellers motståndsmekanismer mot strålbehandling mättes bland annat olika typer av celldöd och uttrycket av två olika varianter av p73, kallade TAp73 och ΔNp73, survivin och PRL-3. PRL-3 är ett protein som uttrycks starkast i metastatiska tumörer och leder till ökat motstånd mot strålbehandling. Den cellinje som inte dog genom apoptos hade högt uttryck av ΔNp73, vilket är en variant av p73 som hindrar celler från att dö, medan den som trots strålning i hög grad fortsatte dela sig hade ett högt uttryck av survivin (Paper IV). Efter indikationer om att uttrycket av p73 proteinerna TAp73 och ΔNp73 är kopplade till survivin undersöktes ett eventuellt förhållande mellan dessa proteiner i tjocktarmscancerceller. I de fall där uttrycket av survivin minskades i cellerna minskade också uttrycket av TAp73 och ΔNp73. Detta fenomen sågs även i celler som behandlats med strålning. En minskning av survivin i cellerna ledde även till att fler celler dog genom apoptos (Paper V).

Sammanfattningsvis pekar våra resultat på att p73 spelar en viss roll i utvecklingen av tjock- och ändtarmscancer, men också avseende effekterna på strålbehandling av ändtarmscancer. Vi har också visat att TAp73, ΔNp73 och p53 regleras av survivin.

 

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press , 2009. , 99 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 1108
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-18432ISBN: 978-91-7393-677-4 (print)OAI: oai:DiVA.org:liu-18432DiVA: diva2:219167
Public defence
2009-05-29, Eken, Hälsouniversitet, ingång 65, Campus US, Linköpings Universitet, Linköping, 09:00 (English)
Opponent
Supervisors
Available from: 2009-05-26 Created: 2009-05-26 Last updated: 2009-08-21Bibliographically approved
List of papers
1. Polymorphism of the p73 gene in relation to colorectal cancer risk and survival
Open this publication in new window or tab >>Polymorphism of the p73 gene in relation to colorectal cancer risk and survival
2005 (English)In: Carcinogenesis, ISSN 0143-3334, E-ISSN 1460-2180, Vol. 26, no 1, 103-107 p.Article in journal (Refereed) Published
Abstract [en]

The results regarding a GC/AT polymorphism in the p73 gene in relation to cancer risk are inconsistent, and the significance of loss of heterozygosity (LOH) of the gene is unclear. In the present study, we investigated whether this polymorphism was related to the risk of colorectal cancer, and whether there were relationships between the polymorphism and LOH, protein expression or clinicopathological variables. 179 patients with colorectal cancer and 260 healthy controls were genotyped for the polymorphism by PCR-restriction fragment length polymorphism (RFLP). Fifty informative cases were examined for LOH in tumours. Immunohistochemistry was performed on distant (n = 42) and adjacent normal mucosa (n = 33), primary tumour (n = 6 9) and lymph node metastasis (n = 12). The frequencies of the genotypes were 63% for wild-type (GC/GC), 30% for heterozygotes (GC/AT) and 7% for variants (AT/AT) in patients, and 62, 36 and 2% in controls, respectively. The frequencies of the genotypes in the patients and controls were significantly different (P = 0.02). The patients carrying the AT allele had a better prognosis than those with the GC/GC genotype (OR = 0.42, 95% CI = 1.15-5.02, P = 0.02). No LOH was observed at the p73 locus. Expression of p73 protein was increased from normal mucosa to primary tumours (P = 0.02), but was not significantly changed between primary tumours and metastases (P = 1.0). In conclusion, the AT/AT homozygotes may have a greater risk of developing colorectal cancer, while the patients who carried the AT allele had a better prognosis.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-18428 (URN)10.1093/carcin/bgh305 (DOI)15485994 (PubMedID)
Available from: 2009-05-26 Created: 2009-05-26 Last updated: 2017-12-13Bibliographically approved
2. Significance of an exon 2 G4C14-to-A4T14 polymorphism in the p73 gene on survival in rectal cancer patients with or without preoperative radiotherapy
Open this publication in new window or tab >>Significance of an exon 2 G4C14-to-A4T14 polymorphism in the p73 gene on survival in rectal cancer patients with or without preoperative radiotherapy
2009 (English)In: Radiotherapy and Oncology, ISSN 0167-8140, E-ISSN 1879-0887, Vol. 92, no 2, 215-220 p.Article in journal (Refereed) Published
Abstract [en]

Background and Purpose: An exon 2 G4C14→A4T14 polymorphism in the p73 gene was shown to be related to survival in several types of cancers, including colorectal cancer. The purpose was to investigate if this polymorphism was related to survival in rectal cancer patients with or without preoperative radiotherapy.

Material and Methods: DNA extracted from tissue of 138 rectal cancer patients that received preoperative radiotherapy or had surgery alone was typed for the polymorphism by PCR using confronting two-pair primers.

Results: Among patients, 69% had GC/GC genotype, 27% GC/AT and 4% AT/AT. In the radiotherapy group, patients carrying the AT (GC/AT+AT/AT) allele had stronger expression of p53 (p=0.001) and survivin protein (p=0.03) than those carrying the GC/GC genotype. Further, among patients receiving preoperative radiotherapy the GC/GC genotype tended to be related to better survival (p=0.20). Patients with GC/GC genotype, along with negative p53 and weak survivin expression showed better survival than the other patients (p=0.03), even after adjusting for TNM stage and tumor differentiation (p=0.01, RR, 7.63, 95% CI, 1.50-38.74). In the non-radiotherapy group, the polymorphism was not related to survival (p=0.74).

Conclusions: Results suggest that the p73 G4C14→A4T14 polymorphism could be one factor influencing outcome of preoperative radiotherapy in rectal cancer patients.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-18429 (URN)10.1016/j.radonc.2009.06.007 (DOI)
Note
Original Publication: Jasmine Lööf, Daniella Pfeifer, Gunnar Adell and Xiao-Feng Sun, Significance of an exon 2 G4C14-to-A4T14 polymorphism in the p73 gene on survival in rectal cancer patients with or without preoperative radiotherapy, 2009, Radiotherapy and Oncology, (92), 2, 215-220. http://dx.doi.org/10.1016/j.radonc.2009.06.007 Copyright: Elsevier Science B.V., Amsterdam. http://www.elsevier.com/ Available from: 2009-09-09 Created: 2009-05-26 Last updated: 2017-12-13Bibliographically approved
3. Expression of the p73 protein in rectal cancers with or without preoperative radiotherapy
Open this publication in new window or tab >>Expression of the p73 protein in rectal cancers with or without preoperative radiotherapy
2006 (English)In: International Journal of Radiation Oncology, Biology, Physics, ISSN 0360-3016, E-ISSN 1879-355X, Vol. 65, no 4, 1143-1148 p.Article in journal (Refereed) Published
Abstract [en]

Purpose: To investigate p73 expression in normal mucosa, primary tumor, and metastasis in relation to radiotherapy (RT) response and clinicopathologic/biologic variables in rectal cancers.

Methods and Materials: p73 was immunohistochemically examined on biopsies (unirradiated, n = 102), distant (from the large bowel, n = 82), and adjacent (adjacent to primary tumor, n = 89) normal mucosa samples, primary tumors (n = 131), and lymph node metastasis (n = 32) from rectal cancer patients participating in a clinical trial of preoperative RT. Seventy-four patients received surgery alone and 57 received additional RT.

Results: Cytoplasmic p73 was increased in the primary tumor compared with the distant or adjacent mucosa (p ≤ 0.0001). Nuclear (p = 0.02) and cytoplasmic (p = 0.003) p73 was higher in irradiated distant mucosa samples than in unirradiated ones, and nuclear p73 tended to be increased in irradiated primary tumors compared with unirradiated ones (p = 0.06). p73 was positively related to cyclooxygenase-2 expression in irradiated tumors (p = 0.03). p73-negative tumors tended to have a lower local recurrence after RT compared with unirradiated cases (p = 0.06).

Conclusions: Normal epithelial cells seem more sensitive to RT than tumor cells regarding p73 expression. Patients with p73-negative rectal tumors may have a lower risk of local recurrence after RT.

Place, publisher, year, edition, pages
Elsevier, 2006
Keyword
p73; Radiotherapy; Local recurrence; Rectal cancer; Immunohistochemistry
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-18430 (URN)10.1016/j.ijrobp.2006.02.028 (DOI)16750334 (PubMedID)
Available from: 2009-05-26 Created: 2009-05-26 Last updated: 2017-12-13Bibliographically approved
4. Protein expression following gamma-irradiation relevant to growth arrest and apoptosis in colon cancer cells with mutant p53
Open this publication in new window or tab >>Protein expression following gamma-irradiation relevant to growth arrest and apoptosis in colon cancer cells with mutant p53
2009 (English)In: Journal of Cancer Research and Clinical Oncology, ISSN 0171-5216, E-ISSN 1432-1335, Vol. 135, no 11, 1583-1592 p.Article in journal (Refereed) Published
Abstract [en]

We previously found that p53, p73, survivin and PRL were implicated in the outcome of radiotherapy in rectal cancer patients. In the present study, we tried to understand mechanisms of colon cancer cell line response to radiation based on protein expression related to proliferation and apoptosis. KM12C, KM12SM and KM12L4a, cell lines with one origin, were radiated with 0, 10 or 15 Gy γ-radiation. Radiosensitivity was determined with cell cycle and apoptosis analysis, and protein expression of TAp73, ΔNp73, mutated p53, survivin and PRL-3 was determined by Western blot. KM12C showed transient G2-arrest, low apoptosis and up-regulation of resistance factors such as PRL-3. In KM12C expression of ΔNp73 increased after 10Gy, but not after 15Gy. KM12SM had permanent G2-arrest, low apoptosis and showed up-regulation of the anti-apoptotic survivin and down-regulation of the pro-apoptotic TAp73 and the radioresistance factor PRL-3 was down-regulated. KM12L4a, the most radiosensitive cell line, showed up-regulation of TAp73 and down-regulation/no up-regulation of resistance factors such as ΔNp73, survivin and PRL-3 after radiation. In conclusion, the KM12C cell line was more radioresistant than KM12L4a regarding apoptosis and certain apoptotic proteins. The radiosensitivity of KM12L4a might partly depend on the lack of up-regulation of proteins negative for the outcome of radiotherapy.

 

National Category
Cancer and Oncology
Identifiers
urn:nbn:se:liu:diva-14786 (URN)10.1007/s00432-009-0606-4 (DOI)
Available from: 2008-09-24 Created: 2008-09-24 Last updated: 2017-12-13Bibliographically approved
5. Survivin regulates expression of p73 isoforms TAp73 and ΔNp73 in colon cancer cell lines with and without γ-radiation
Open this publication in new window or tab >>Survivin regulates expression of p73 isoforms TAp73 and ΔNp73 in colon cancer cell lines with and without γ-radiation
(English)Manuscript (Other academic)
Abstract [en]

Wild type p53 represses expression of survivin, however little is known if survivin regulates p53 and the two isoforms of the p53 homologue p73, TAp73 and ΔNp73. The aim of our study was to investigate a possible connection between survivin and TAp73 and ΔNp73 in colon cancer cell lines HCT-116p53+/+ and HCT- 116p53-/- with and without γ-irradiation. HCT-116p53+/+ and HCT-116p53-/- were transfected with either survivin cDNA or siRNA against survivin. Cells transfected with siRNA were irradiated with 0Gy or 4Gy γ-irradiation to induce DNA-damage. Expression levels of survivin, TAp73, ΔNp73, mRNA and protein, and p53 protein was measured by RT-PCR and Western blot. Apoptosis was measured by means of M30- Apoptosense™ Elisa. Over-expression of survivin did not significantly change TAp73 or ΔNp73 mRNA and protein, whilst wild type p53 decreased. When survivin was knocked down, mRNA and protein of TAp73 and ΔNp73 decreased, both with and without γ-irradiation. Knockdown of survivin increased apoptosis in both HCT-116 cell lines with and without γ-irradiation. Knockdown of survivin decreased levels of TAp73 and ΔNp73 in both HCT-116p53+/+ and HCT-116p53-/-, and caused an increase in apoptosis, especially noticeable in HCT-116p53-/- after irradiation. Survivin down-regulation did not seem to affect the levels of wild type p53, whilst an up-regulation of survivin down-regulated wild type p53.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-18431 (URN)
Available from: 2009-05-26 Created: 2009-05-26 Last updated: 2010-01-14Bibliographically approved

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