Abstract
It has been reported that the expression and activity of the interferon-inducible, dsRNA-dependent protein kinase, PKR, is increased in mammary carcinoma cell lines and primary tumor samples. To extend these findings and determine how PKR signaling may affect breast cancer cell sensitivity to chemotherapy, we measured PKR expression by immunohistochemical staining of 538 cases of primary breast cancer and normal tissues. Significantly, PKR expression was elevated in ductal, lobular and squamous cell carcinomas or lymph node metastases but not in either benign tumor specimens or cases of inflammation compared to normal tissues. Furthermore, PKR expression was increased in precancerous stages of mammary cell hyperplasia and dysplasia compared to normal tissues, indicating that PKR expression may be upregulated by the process of tumorigenesis. To test the function of PKR in breast cancer, we generated MCF7, T-47D and MDA-MB-231 breast cancer cell lines with significantly reduced PKR expression by siRNA knockdown. Importantly, while knockdown of PKR expression had no effect on cell proliferation under normal growth conditions, MCF7, T-47D or MDA-MB-231 cells with reduced PKR expression or treated with a small molecule PKR inhibitor were significantly less sensitive to doxorubicin or H2O2-induced toxicity compared to control cells. In addition, the rate of eIF2α phosphorylation following treatment with doxorubicin was delayed in breast cancer cell lines with decreased PKR expression. Significantly, treatment of breast cancer lines with reduced PKR expression with either interferon-α, which increases PKR expression, or salubrinal, which increases eIF2α phosphorylation, restored doxorubicin sensitivity to normal levels. Taken together these results indicate that increased PKR expression in primary breast cancer tissues may serve as a biomarker for response to doxorubicin-containing chemotherapy and that future therapeutic approaches to promote PKR expression/activation and eIF2α phosphorylation may be beneficial for the treatment of breast cancer.
Highlights
The interferon (IFN)-inducible, double-stranded RNA-activated protein kinase, PKR, is present in most mammalian cells in a latent or inactive state
PKR expression is increased in precancerous stages of mammary cell hyperplasia and dysplasia compared to normal tissues but not cases of breast tissue inflammation, indicating that PKR expression may be upregulated during tumorigenesis
We report that PKR expression is significantly upregulated in primary breast cancer compared to normal or benign breast epithelial tissue
Summary
The interferon (IFN)-inducible, double-stranded RNA-activated protein kinase, PKR, is present in most mammalian cells in a latent or inactive state. It has been well studied as an important component of the IFN-stimulated host antiviral defense mechanism. In this context, PKR is induced by IFN and activated by viral double-stranded RNA to catalyze phosphorylation of eIF2a resulting in global protein synthesis inhibition and initiation of apoptosis. [1,2,3] In addition to an inhibitor of translation, PKR has been reported to have an important role in signaling pathways such as NF-kB, p53 and STAT1 that regulate proliferation and apoptosis during cellular stress. Mutant forms of PKR and PKR’s downstream target, eIF2a, as well as inhibitors of PKR such as TRBP or p58 can induce transformation of cells. [19,20] the loss of PKR catalytic activity has been observed in B-cell chronic lymphocytic leukemia patient samples, and an inactivating point mutant in PKR’s dsRNA binding has been detected in a small set of patients with acute lymphoblastic leukemia of T-cell lineage. [21,22] The PKR gene is located on 2p21-22, a chromosomal region that has been associated with large cell lymphoma, myelodysplastic syndrome and acute myelogenous leukemia. [23,24,25,26,27] In addition, the PKR gene is transcriptionally regulated by IFNs a and c via IRF-1, and down regulation of PKR has been shown to occur in 5q- associated leukemias that delete the IRF1 gene. [28,29,30,31] Significantly, it has been recently reported that primary non-small cell lung cancer (NSCLC) samples have decreased PKR expression compared to normal bronchial epithelium. [32] loss of PKR expression correlates with a more aggressive behavior while high PKR expression predicts a subgroup of NSCLC patients with a favorable outcome. [32] Collectively, these findings suggest that PKR may play an important role in tumor suppression and that inhibition of PKR activity is associated with tumorigenesis
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