p53

p53 and p53 and

chemotherapychemotherapyBlossom Sabi

p53 and chemotherapyp53 and chemotherapy

1.Introduction 1.Introduction

2. Relationship between p53 and chemotherapy 2. Relationship between p53 and chemotherapy

3. Summary 3. Summary

HistoryHistory

p53 Introduction

p53 Introduction

RegulationRegulation

FunctionFunction

ActivationActivation

Introduction

p53 introduction

• A 53 kD nuclear phosphoprotein • Encoded by TP53 gene • Short arm of chromosome 17• Regulates cell growth and proliferation• Prevents unrestrained cell division after

chromosomal damage• The absence of p53 increases the risk of developing

cancers

History

Function

• Apoptosis

• Cell circle arrest

• Senescence

• DNA repair

• Metabolic homeostasis

• Antioxidant defence

Activation

Stabilization

Antirepression Promoter-specific activation

NextNext

Kruse JP, et al. Cell 2009; 137; 609-22

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Regulation

• Phosphorylation of N-terminal domain

• Protein kinases targeting p53

MAPK family (JNK1-3, ERK1-2, p38 MAPK)

ATR, ATM, CHK1 and CHK2, DNA-PK, CAK

Oncogenes p14ARF

• Mdm2 More

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P53 Chemotherapy Prognosis

Controversy

• p53 is favourable to chemotherapy Pros

• p53 is unfavourable to chemotherapy Cons

• p53 is irrelevant with chemotherapy Neutral

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Relationship between p53 and chemotherapy

Pros

p53 is favourable to chemotherapy

Yamasaki M, et al. Ann Surg Oncol 2010, 17, 634-42

p53 is favourable to chemotherapy

Patients with mutations in p53 showed significantly poorer prognosis than those without mutant p53.

p53 IHC staining did not correlate with prognosis.

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p53 is unfavourable to chemotherapy

TP53 status was strongly associated with response

Bertheau P, et al. PLoS Med 2007, 4, 585-92

p53 is unfavourable to chemotherapy

·TP53-mutant patients have a more favorable event-free survival· Patients with wild-type TP53 tumors had favorable overall survival, despite the lack of complete response.· Patients with TP53-mutant tumors who did not reach complete response had a significantly shorter overall survival Bac

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p53 is irrelevant with chemotherapy

· p53 protein overexpression is a significant marker of prognosis (B)

· Patients with p53 positive tumors deri-ved significant benefit from chemo-therapy(C).

· Patients with p53 negative tumors had no survival benefit from chemotherapy (D).

p53 IHC results, observation

p53 IHC positive p53 IHC negative

Tsao MS, et al. J Clin Oncol 2007, 25,5240-7

p53 IHC results

p53 is irrelevant with chemotherapy

· p53 mutation was not prognostic for survival in the ob-servation arm(B)

· Patients with p53 mutations did not derive significant survival benefit from chemotherapy (C).

· Chemotherapy significantly prolonged survival in patients with wild type p53 compared with observation (D),

p53 mutation results, observation

wild-type p53mutant p53

Tsao MS, et al. J Clin Oncol 2007, 25,5240-7 Back

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Basic researchClinical data

p53 and chemotherapy

Induce cell death in response to drug-induced

DNA damagesInduce early cell

cycle arrest, protecting tumor

cells from further damages

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Cyclophosphamide Inhibits Tumor Proliferation and Stimulates Apoptosis In Vivo

· In response to CPM, Ki-67 labeling decreased dramatically even by 24 hours (C).· Control tumors had negligible staining for the apoptosis marker cleaved caspase-3 (D),· High levels of cleaved caspase-3 were observed 3 and 6 hours after treatment with CPM (E F )· A wave of apoptosis throughout the tumor was demonstrated at low power (H–J ), with maximalstaining visible at 6 hours after treatment

Chesler L ,et al. Neoplasia 2008, 10, 1268-74

Apoptosis in Tumors Treated with Cytotoxic Chemotherapy Is Driven by p53

· Activation of the p53 pathway and caspase-3 cleavage were minimal in vehicle-treated tumors, consistent with the low-level caspase-3 staining

· Rapid induction of p53 was observed at 3 hours after treatment with CPM, with a peak at 6 hours after treatment

· Cleaved caspase-3 and -9 were maximal at 6 hours after treatment and were sustained during a 12-hour period.


CPM Treatment of Murine NeuroblastomaActivates Apoptosis

· Bax, a downstream target of PUMA and a critical effector of myc-induced mitochondrial apoptosis, was strongly expressed and PARP occurred concurrently, indicating high levels of apoptosis.

· The proapoptotic effect of CPM proceeds through the intrinsic p53 regulatory pathway of BH3 proteins, implicating PUMA, Bax, and Bim as key effectors of p53 function in primary murine neuroblastoma tumors.


Conclusion

• The p53 pathway plays a significant role in opposing MYCN-driven oncogenesis in a mouse model of neuroblastoma and that basal inactivation of the pathway is achieved in progressing tumors.

• Chemotoxic agents induce p53-dependent apoptosis in such tumors. It is consistent with clinical observations that therapy-associated mutations in p53 are a likely contributor to the biology of tumors at relapse and secondarily mediate resistance to therapy.

Chesler L ,et al. Neoplasia 2008, 10, 1268-74 Back

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· In the TP53 wild-type tumorKi67 immunostainings were not statistically different before and aftertreatment .· SA-b-gal staining was negative before treatment in all tumors. After treatment, 5-10% tumor cells in TP53wt showed SA-b-gal cytoplasmic staining, as soon as Day 1 (Figs. d–2).· p21 immunostained cells increased starting at Day 3 with a mean number of stained cells over 50%(Figs. g–i).

Treatment-induced senescence-like phenotype is dependent on TP53 status

Treatment-induced mitotic catastrophe and apoptosis is dependent on TP53 status

· The TP53 mutant tumors remained negative for SA-b-gal at all time points after treatment (Figs. d–f).

· No significant change for p21 immunostaining was observed in the TP53 mutant tumors (Figs. g–i).

· The number of abnormal mitoses on semi-thin sections significantly increased between Day 3 and Day 5 (Figs. p–r).


The results were simi-lary to TP53mut1 tumor


· No significant change in Bax mRNA was found in TP53mut1 and TP53mut2 (Figs. e and h).

· Similarly PUMA showed a strong mRNA overexpression at D3and D5 in TP53wt, but no change in mutant tumors (Figs. c, f and i)

Conclusion

• Treatment-induced senescence-like phenotype is

dependent on TP53 status.• Treatment-induced mitotic catastrophe and apoptosis

is dependent on TP53 status• The lack of response in TP53 wild-type tumors may

be due to the induction of cell cycle arrest, allowing tumor cells to reinitiate proliferation at the end of chemotherapy

Varna M, et al. Int J Cancer 2009, 124, 991-7

Other agents

P53- and p21-deficient cells undergo apoptosis after treatment with DNA-damaging agents

Cells with wild type p53 were quite sensitive to 5-FU, and a large proportion underwent apoptosis.

Cells with p21 deletions were as sensitive to 5-FU as p53 wild-type cells.

Bunz F, el at. J Clin Invest 1999, 104,263-9

Cells with targeted p53 deletion are resistant to apoptosis induced by 5-FU

· Apoptosis was not observed in p53-deficient cells with 5-FU treatment.

· Tight control of 5-FU sensitivity by p53.

· p21 does not play a role in the ability of p53 to modulate the response to 5-FU.


Cell cycle distribution of drug-treated cells with wild-type p53 and disrupted p53 genes

· Adriamycin: p53-deficient cells accumulated in a single peak with 4N DNA content.

· 5-FU: regardless of p53 genotype, cellsaccumulated in a single peak that spanned the G1/S phase boundary.

· Tomudex: both p53-proficient and p53-deficient cells exhibited an S-phase block.

· Methotrexate: induced identical responses in all cells, with an increase in the S-phase fraction


· Both adriamycin and 5-FU caused increases in p53 protein levels over a similar time course.

· The stabilization of p53 was associated with increased levels of p21

· 5-FU treatment led to the dramatic induction of death in cells with intact p53, a relatively small proportion of cells apparently survived and gave rise to colonies upon replating.

Characteristics of 5-FU–induced apoptosis

Growth of xenograft tumors


Tumors with intact p53 genes regressed during the treatment, whereas the tumors with deleted p53 genes continued to grow.

Conclusion

• p53 had profound effects on drug responses, and

these effects varied dramatically depending on the drug.

• The p53-deficient cells were sensitized to the

effects of DNA-damaging agents as a result of the

failure to induce expression of the cyclin-dependent kinase inhibitor p21.

• p53 disruption rendered cells strikingly resistant to

the effects of the 5-FU.


PFTA potentiates the antitumor effect of cyclophosphamide

PFTa greatly potentiated the antitumor effect of cyclophosphamide, due to the effect of the p53 inhibitor on stromal cells.

Burdelya LG ,et al. Cancer Res 2006, 66, 9356-61

Experimental design and model

· The levels of GSE56 expression were similar between virus-producing and nonproducing populations (B)

· No EYFP expression detected in the endothelium of tumors formed by the cells transduced with retroviral vector lacking the packaging signal as judged by immunofluorescent staining of tumor sections by antibodies against mouse endothelium marker CD31 (C, bottom).

· Both cells producing and not producing GSE56 virus were equally sensitive to treatment withseveral chemotherapeutic drugs in vitro (D).

Dependence of tumor chemosensitivity and radiosensitivity on the p53 status of stroma

· No differences in the growth rate of tumors were formed (A).

· Tumors formed by the cells that released the p53-inhibit- ing GSE56 virus showed a much stronger response to cyclophosphamide treatment (B).

· For radiation, GSE56 virus- producing tumors showed significant size reduction, whereas Δ ψ-GSE56 tumors responded only with a slight decline in growth rate (C.D)· PFTa strongly sensitized Δ ψ-GSE56 tumors to radiation.

Conclusion

• Tumors with p53-deficient stroma were significantly more sensitive to experimental chemotherapy and

radiotherapy.

• Potentiation of the anticancer effect of chemotherapy and radiotherapy by p53 suppression in the tumor

stroma is likely to be due to the increased sensitivity of p53-deficient endothelium to genotoxic stress as

shown both in cell culture and in experimental tumors.

Burdelya LG ,et al. Cancer Res 2006, 66, 9356-61

Summary

• p53 pathway plays a significant role in chemotherapy.

• p53 Induce cell death and cell circle arrest. Their balance contribute to the response of chemotherapy.

• Effects of p53 in chemotherapy varied dramatically

depending on the drug.

• Tumors with p53-deficient stroma were significantly more sensitive to chemotherapy.

p53

Health & Medicine

p53 introduction

p53 pathway

p53 p53 mutation results

p53 activation

absence of p53

chemotherapy pros p53

p53dependent apoptosis

p53 ihc staining