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p73 and p53 Pathway in Human Breast Cancers

Medical Oncology Unit, University Hospital, Parma, Italy

Eugenia M. Martella, Pellegrino Crafa, Costanza A. Lagrasta

Pathology Department, University Hospital, Parma, Italy

Roberta Camisa

Medical Oncology Unit, University Hospital, Parma, Italy

Antonio Bonati

Department of Clinical Sciences, University of Parma, Italy; Unit of Hematology, University Hospital, Parma, Italy

Paolo Lunghi

Department of Clinical Sciences, University of Parma, Italy

Andrea Ardizzoni

Medical Oncology Unit, University Hospital, Parma, Italy

To the Editor:

We read with interest the study by Domìnguez et al¹ aimed to evaluate the expression of TAp73 and TAp73 variants in patients with colon and breast cancer and their possible associations with E2F-1, p53, and K-ras status. The clinical relevance of alterations in these genes was also assessed. In their breast cancer series, TAp73 suppressor and the TAp73 oncogenic isoforms Ex2p73 and Np73 were significantly coupregulated (P = .031 and P = .019, respectively). Statistical associations were also observed between wild-type p53 status and overexpression of the Np73 variant (P = .04), overexpression of E2F-1 and some TP73 forms, and upregulation of TAp73 variants (Ex2p73, Ex2/3p73, and Np73) and some tumor pathologic markers (vascular invasion and hormone receptor status).

The expression levels of TAp73 and TAp73 were determined by quantitative real-time reverse transcriptase polymerase chain reaction. Furthermore, 14 of the 60 breast cancer cases were also analyzed for TAp73 and Np73 protein expression by immunohistochemistry. TAp73 and Np73 were considered positive in tissue samples exhibiting nuclear staining in more than 10% of the epithelial cells. TAp73 and Np73 immunoreactivity was observed in 21% and 30% of the 14 breast cancer cases, respectively. TAp73 and Np73 nuclear protein expression correlated with mRNA quantification in 12 (86%) and 11 (79%) of the 14 breast samples, respectively.

The significant association between expression of wild-type p53 and upregulation of Np73 observed by Domìnguez et al led the authors to postulate that redundant alterations affecting the same pathway may not confer extra growth advantages on tumor cells during carcinogenesis and thereby alleviate the selective pressure on those cells harboring both alterations.

Our experience refers to the immunocytochemical evaluation of TAp73, Np73, p53, and other biologic variables on 73 fine needle aspirates from primary breast cancer patients. Immunocytochemical analyses were performed on cytospin preparations using the same reagent used by Domìnguez et al: a monoclonal TAp73 antibody (clone 5B429; this antibody recognizes the TAp73 isoforms but does not detect the Np73 variant or p53), a monoclonal Np73 antibody (clone 38C674; this antibody does not cross react with any TAp73 isoform or p53), and a revelation system (LSA; DAKO, Glostrup, Denmark) using diaminobenzidine chromogen as substrate. A monoclonal antibody to p53 (D0-7; Dako Corp, Carpinteria, CA) was used. TAp73 and Np73 immunoreactivity was evaluated in a semiquantitative way. Cells were classified as positive for TAp73 and Np73 when nuclei or cytoplasm or both were scored as 2+ or 3+ in more than 10% of tumor cells. Cells were classified as p53 positive when 10% of the nuclei showed specific nuclear staining. Immunostaining of TAp73 protein was detected in 25 (47%) of 53 carcinomas examined. Staining was confined to the cell nucleus in 10 (40%) of 25 cases and to the cytoplasm in 14 (56%) of 25 cases. One case (4%) showed both cytoplasmic and nuclear TAp73 localization. Np73 immunostaining was detected in 30 (45%) of 67 samples. Np73 staining was cytoplasmic in 28 (93%) of 30 cases and nuclear in two cases (Fig 1). NB4 and K562 cell lines were tested by immunocytochemistry as positive controls for TAp73 and Np73 expression. TAp73 and Np73 expression was confirmed on tumor samples and cell lines by Western blot analysis.

View larger version (92K): [in this window] [in a new window] [PowerPoint Slide for Teaching]   Fig 1. Np73 expressing tumor cells show strong (3+) cytoplasmic staining.

In our series, no correlation was found between TP73 isoforms and estrogen and progesterone receptor status, Ki67 growth fraction, and HER-2/neu amplification whereas, like in the series by Domìnguez et al, a significant correlation between TAp73 and Np73 (P < .05) was observed (Table 1).

View this table: [in this window] [in a new window]   Table 1. Associations Between Np73 and Biological Characteristics in Breast Cancer Patients

Significantly, Np73 overexpression was observed in NB4 and K562 leukemia cell lines carrying an inactive p53⁸ and Np73 transcript and protein were detectable in different subtypes of primary acute myelogenous leukemia (AML) blasts.^9,10 In AML, TP53 mutations are quite rare (5% to 10%), but murine double minute 2, its principal negative regulator, has been found to be frequently overexpressed in AML and to inactivate p53^11-13 suggesting that both alterations, inactivation of wild-type p53 protein and Np73 expression, can cooperate in the leukemic process.

In addition, a trend but no significance between overexpression of dominant-negative forms of p73 and concomitant wild-type p53 status on a set of 30 tumors was observed.¹⁴

These findings together with our results suggest that more samples in various type of tumors need to be analyzed to fully clarify the relationship between p53 status, p53 activity, and Np73 protein levels. Finally, further functional studies are needed to elucidate the mechanisms that differentially control TA and Np73 localization, activity, protein stability, and p73-protein interactions, and the role that they play in breast carcinogenesis.

AUTHORS' DISCLOSURES OF POTENTIAL CONFLICTS OF INTEREST

The authors indicated no potential conflicts of interest.

REFERENCES

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10. Lunghi P, Costanzo A, Salvatore L, et al: MEK1 inhibition sensitizes primary acute myelogenous leukemia to arsenic trioxide-induced apoptosis. Blood 107:4549-4553, 2006[Abstract/Free Full Text]

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12. Bueso-Ramos CE, Yang Y, deLeon E, et al: The human MDM-2 oncogene is overexpressed in leukemias. Blood 82:2617-2623, 1993[Abstract/Free Full Text]

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