Frequent mutation of receptor protein tyrosine phosphatases provides a mechanism for STAT3 hyperactivation in head and neck cancer

The underpinnings of STAT3 hyperphosphorylation resulting in enhanced signaling and cancer progression are incompletely understood. Loss-of-function mutations of enzymes that dephosphorylate STAT3, such as receptor protein tyrosine phosphatases, which are encoded by the PTPR gene family, represent a plausible mechanism of STAT3 hyperactivation. We analyzed whole exome sequencing (n = 374) and reverse-phase protein array data (n = 212) from head and neck squamous cell carcinomas (HNSCCs). PTPR mutations are most common and are associated with significantly increased phospho-STAT3 expression in HNSCC tumors. Expression of receptor-like protein tyrosine phosphatase T (PTPRT) mutant proteins induces STAT3 phosphorylation and cell survival, consistent with a “driver” phenotype. Computational modeling reveals functional consequences of PTPRT mutations on phospho-tyrosine–substrate interactions. A high mutation rate (30%) of PTPRs was found in HNSCC and 14 other solid tumors, suggesting that PTPR alterations, in particular PTPRT mutations, may define a subset of patients where STAT3 pathway inhibitors hold particular promise as effective therapeutic agents.Fil: Lui, Vivian Wai Yan. University of Pittsburgh; Estados UnidosFil: Peyser, Noah D.. University of Pittsburgh; Estados UnidosFil: Ng, Patrick Kwok-Shing. University Of Texas Md Anderson Cancer Center;Fil: Hritz, Jozef. University of Pittsburgh at Johnstown; Estados Unidos. University of Pittsburgh; Estados Unidos. Masaryk University; República ChecaFil: Zeng, Yan. University of Pittsburgh at Johnstown; Estados Unidos. University of Pittsburgh; Estados UnidosFil: Lu, Yiling. University Of Texas Md Anderson Cancer Center;Fil: Li, Hua. University of Pittsburgh; Estados Unidos. University of Pittsburgh at Johnstown; Estados UnidosFil: Wang, Lin. University of Pittsburgh; Estados Unidos. University of Pittsburgh at Johnstown; Estados UnidosFil: Gilbert, Breean R.. University of Pittsburgh; Estados Unidos. University of Pittsburgh at Johnstown; Estados UnidosFil: General, Ignacio. University of Pittsburgh; Estados Unidos. University of Pittsburgh at Johnstown; Estados UnidosFil: Bahar, Ivet. University of Pittsburgh at Johnstown; Estados Unidos. University of Pittsburgh; Estados UnidosFil: Ju, Zhenlin. University Of Texas Md Anderson Cancer Center;Fil: Wang, Zhenghe. Case Western Reserve University; Estados UnidosFil: Pendleton, Kelsey P.. University of Pittsburgh; Estados Unidos. University of Pittsburgh at Johnstown; Estados UnidosFil: Xiao, Xiao. University of Pittsburgh at Johnstown; Estados Unidos. University of Pittsburgh; Estados UnidosFil: Du, Yu. University of Pittsburgh at Johnstown; Estados Unidos. University of Pittsburgh; Estados UnidosFil: Vries, John K.. University of Pittsburgh; Estados Unidos. University of Pittsburgh at Johnstown; Estados UnidosFil: Hammerman, Peter S.. Harvard Medical School; Estados UnidosFil: Garraway, Levi A.. Harvard Medical School; Estados UnidosFil: Mills, Gordon B.. University Of Texas Md Anderson Cancer Center;Fil: Johnson, Daniel E.. University of Pittsburgh at Johnstown; Estados Unidos. University of Pittsburgh; Estados UnidosFil: Grandis, Jennifer R.. University of Pittsburgh; Estados Unidos. University of Pittsburgh at Johnstown; Estados Unido

eIF2α Kinases Regulate Development through the BzpR Transcription Factor in Dictyostelium discoideum

A major mechanism of translational regulation in response to a variety of stresses is mediated by phosphorylation of eIF2α to reduce delivery of initiator tRNAs to scanning ribosomes. For some mRNAs, often encoding a bZIP transcription factor, eIF2α phosphorylation leads to enhanced translation due to delayed reinitiation at upstream open reading frames. Dictyostelium cells possess at least three eIF2α kinases that regulate various portions of the starvation-induced developmental program. Cells possessing an eIF2α that cannot be phosphorylated (BS167) show abnormalities in growth and development. We sought to identify a bZIP protein in Dictyostelium whose production is controlled by the eIF2α regulatory system.Cells disrupted in the bzpR gene had similar developmental defects as BS167 cells, including small entities, stalk defects, and reduced spore viability. β-galactosidase production was used to examine translation from mRNA containing the bzpR 5' UTR. While protein production was readily apparent and regulated temporally and spatially in wild type cells, essentially no β-galactosidase was produced in developing BS167 cells even though the lacZ mRNA levels were the same as those in wild type cells. Also, no protein production was observed in strains lacking IfkA or IfkB eIF2α kinases. GFP fusions, with appropriate internal controls, were used to directly demonstrate that the bzpR 5' UTR, possessing 7 uORFs, suppressed translation by 12 fold. Suppression occurred even when all but one uORF was deleted, and translational suppression was removed when the ATG of the single uORF was mutated.The findings indicate that BzpR regulates aspects of the development program in Dictyostelium, serving as a downstream effector of eIF2α phosphorylation. Its production is temporally and spatially regulated by eIF2α phosphorylation by IfkA and IfkB and through the use of uORFs within the bzpR 5' UTR

Cisplatin-Based Chemotherapy Options for Recurrent and/or Metastatic Squamous Cell Cancer of the Head and Neck

Recurrent and/or metastatic head and neck squamous cell carcinoma (R/M HNSCC) is a devastating malignancy with a poor prognosis. Treatment is limited to chemotherapeutic approaches. Cisplatin is an established and effective treatment for R/M HNSCC, and many studies have investigated cisplatin treatment in combination with other agents. Even when being treated with first line therapy (cisplatin + 5-fluorouracil + cetuximab), overall survival is only 10 months, indicating the need for novel chemotherapeutics and treatment regimens. Current research is focused on molecular targeting therapies inhibiting epidermal growth factor receptor, phosphoinositide-3-kinase/Akt/mammalian target of rapamycin, and vascular endothelial growth factor pathways. A variety of clinical trials have been completed and are currently underway with encouraging results. Finally, future directions of cisplatin-based R/M HNSCC treatment may include targeting specific pathways known to induce cisplatin resistance, such as nucleotide excision repair and inhibition of apoptosis, in hopes to enhance response to cisplatin therapy

Oligonucleotides used in this study.

<p>Upper case sequence occurs in genomic DNA.</p

RT-PCR determination of relative levels of β-galactosidase mRNA in Ax4 and BS167 transformed with pbzpR-9.

<p>Wild type (Ax4) and eIF2α S51A knockin (BS167) cells were grown in the presence of bacteria, harvested, and plated for development. RNA was isolated from growing cells (0) and at the tipped mound (tm), finger/slug (fs), early culminant (ec), and late culminant (lc) stages. Primers used were bzr-10 and lacZ-4. H7 primers were used in parallel reactions to confirm RNA concentrations. Conditions were optimized to reveal differences in RNA levels of two to ten-fold.</p

Fruiting bodies of Ax4 (wild type), BS168 (<i>bzpR</i> null), and BS167 (eIF2α S51A knockin).

<p>Cells were grown in the presence of bacteria, harvested, and plated for development. Photographs were taken at 48× after the filter was adhered to a gel slice in a vertical position.</p

RT-PCR localization of transcriptional start site of <i>bzpR</i> gene and determination of relative mRNA levels.

<p><b>A</b>. RNA and genomic DNA were isolated from growing Ax4 cells and used as templates in RT-PCR (r) or PCR (g) reactions. A 3′ primer (bzr-3) corresponding to sequences at the beginning of the coding region was used with three different 5′ primers corresponding to sequences progressively further upstream of the coding region (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032500#pone-0032500-g001" target="_blank">Fig. 1A</a>). Bzr-9 lies within the first uORF, bzr-10 lies just upstream of uORF 1, and bzr-5 is 41 residues further upstream of -10. <b>B</b>. RNA was isolated from Ax4 growing cells (0) and cells plated for development for the indicated times (in hours) and used in RT-PCR reactions with bzr-3 and -10. <i>H7</i> specific primers were used as an internal control as <i>H7</i> is expressed constitutively during growth and development. Conditions were optimized to reveal differences in RNA levels of two to ten-fold.</p

Translational repression by the <i>bzpR</i> 5′ UTR.

<p>Ax4 cells were transformed with the various <i>bzpR</i> 5′ UTR constructs: pbzpR-14 (no-UTR GFP, no-UTR RFP), pbzpR-17 (5′ UTR GFP, no-UTR RFP), pbzpR-53 (GFP: modified 5′ UTR with a single uORF, that being the fusion of uORF 1 and 7; no-UTR RFP), or pbzpR-54 (GFP: modified 5′ UTR mutant with the single fused uORF possessing an AAG start codon mutation; no-UTR RFP). Flow cytometry was used to compare levels of GFP and RFP in cells of each strain. <b>A</b>. Colored contour plots of GFP versus RFP fluorescence intensities for cells transformed with the indicated constructs. <b>B</b>. The GFP and RFP mean fluorescence values from the contour plots were used to determine the GFP to RFP ratios for each strain. Mean values were: pbzpR-14, 34,372 (GFP), 8735 (RFP); pbzpR-17, 1863 (GFP), 5675 (RFP); pbzR-53, 172 (GFP), 9570 (RFP); pbzpR-54, 19,193 (GFP), 9966 (RFP). <b>C</b>. RNA was isolated from cells transformed with each construct, and RT-PCR was carried out with RFP and GFP specific primers to compare relative levels of RFP and GFP mRNA. Two independently transformed populations are shown for pbzpR-17 (a and b). Several independently transformed populations with pbzpR-53 or pbzpR-54 gave identical results to those shown, i.e., a slight reduction in relative levels of GFP mRNA in -53.</p

The <i>bzpR</i> 5′ UTR sequence and plasmids used to test translational suppression.

<p><b>A</b>. A ‘to scale’ schematic of the seven uORFs within the <i>bzpR</i> 5′ UTR, color coded to correspond with panels B and C. <b>B</b>. The <i>bzpR</i> 5′ UTR sequence and the first nine codons (blue) of the coding region. The 5′ ends of the three primers, bzr-9, bzr-10, and bzr-5, used in RT-PCR to localize the transcriptional start site, are indicated. Transcription likely starts between the two underlined regions. The sequence shown was fused to the GFP gene in pbzpR-17. The italicized region, consisting only of the ribosome binding site and the first 9 codons, is the no-UTR control fused to the GFP and RFP genes in pbzpR-14 and to the RFP gene in pbzpR-17, -53, and -54. The larger font T and A in the first and last uORFs represent the fusion junction in the internal deletion constructs (pbzpR-53 and -54). <b>C</b>. Schematic diagram of the plasmids used to demonstrate suppression of translation by the <i>bzpR</i> 5′ UTR. A15 represents the actin 15 promoter driving transcription of each fusion. Thin blue lines represent the remaining portions of the plasmids, including transcriptional terminators and selectable drug resistance genes.</p