The Transcription Factor YY1 Is a Substrate for Polo-Like Kinase 1 at the G2/M Transition of the Cell Cycle

Yin-Yang 1 (YY1) is an essential multifunctional zinc-finger protein. It has been shown over the past two decades to be a critical regulator of a vast array of biological processes, including development, cell proliferation and differentiation, DNA repair, and apoptosis. YY1 exerts its functions primarily as a transcription factor that can activate or repress gene expression, dependent on its spatial and temporal context. YY1 regulates a large number of genes involved in cell cycle transitions, many of which are oncogenes and tumor-suppressor genes. YY1 itself has been classified as an oncogene and was found to be upregulated in many cancer types. Unfortunately, our knowledge of what regulates YY1 is very minimal. Although YY1 has been shown to be a phosphoprotein, no kinase has ever been identified for the phosphorylation of YY1. Polo-like kinase 1 (Plk1) has emerged in the past few years as a major cell cycle regulator, particularly for cell division. Plk1 has been shown to play important roles in the G/M transition into mitosis and for the proper execution of cytokinesis, processes that YY1 has been shown to regulate also. Here, we present evidence that Plk1 directly phosphorylates YY1 in vitro and in vivo at threonine 39 in the activation domain. We show that this phosphorylation is cell cycle regulated and peaks at G2/M. This is the first report identifying a kinase for which YY1 is a substrate

Expression of immune checkpoint regulators, cytotoxic T lymphocyte antigen 4 (CTLA-4), and CD137 in cervical carcinoma.

Immune checkpoint inhibitors have a significant role in oncology. One of these immune checkpoints is cytotoxic T-lymphocyte-associated protein 4 (CTLA-4). Inhibition of the CTLA-4 pathway has already led to the FDA approval of Ipilimumab (anti-CTLA-4), a targeted therapy for melanoma and other malignancies. CD137 is an inducible, costimulatory receptor of the tissue-necrosis-factor-receptor superfamily expressed on the activated immune cells. Clinical trials have also been set for anti-CD137 in several malignancies. We assessed CTLA-4 and CD137 expression on a tissue microarray (TMA) comprising of 99 core tissues which included normal, non-neoplastic, and neoplastic cervical lesions. When detected as strong granular cytoplasmic reaction in the epithelial cells, CTLA-4 expression was scored as positive. For CD137, the results were recorded based on the presence or absence of staining reaction on the cell membranes of the lymphoplasmacytic infiltrates. Overall, CTLA-4 was positive in 30% (30/100) of the cervical malignancies. Sub-categorically, 20% of invasive endocervical adenocarcinomas, 63% of adenosquamous carcinomas, and 31% of squamous cell carcinomas were positive for CTLA-4 with a tendency toward lower grade squamous cell carcinomas (SCCs). CD137 was positive in 100% lymphoplasmacytic infiltrates of endocervical adenocarcinomas, 90.5% of SCCs, and 87.5% of adenosquamous carcinomas. This study has found a significant expression of CTLA-4 in cervical cancer cells and CD137 positivity of lymphoplasmacytic infiltrates with potential for future targeted immunotherapy

Tonsillar control tissue for CTLA-4 (10x objective).

<p>Photomicrograph of a portion of the tonsillar tissue used as control in this study. The upper prat of the photograph depicts the epithelial lining of a crypt with light brownish staining for CTLA-4. The rest of the tissue is formed of lymphoid cells in which the reactive cells show strong (3+ intensity) cytoplasmic and cell membrane reactions for CTLA-4 (presumably a subset of activated T-cells) scattered throughout, mostly in the germinal center (right lower quadrant of the picture). A few of these reactive cells are viewed at a high magnification in the right inset (60x objective). The arrows point to the squamous epithelial lining of a crypt showing a light granular reaction in the cytoplasm. A high magnification of the epithelial cells is shown in the left inset (60x objective). In the control tissue, the characteristic positive granules (light staining and lower in number) of the epithelial cells can be established as 1+ intensity.</p

Group I, summary of the breast tissue cores with ductal carcinoma in situ showing the CTLA-4 staining reactions, scores, interpretations, and percentages of stained lymphocytes.

<p>Table is primarily arranged based on the tumor grades and intensities.</p

Expression of immune checkpoint regulators, cytotoxic T lymphocyte antigen 4 (CTLA-4) and programmed death-ligand 1 (PD-L1), in female breast carcinomas

<div><p>Background</p><p>Immune checkpoint regulators, <i>cytotoxic T lymphocyte antigen 4</i> (CTLA-4) and the <i>programmed cell death protein-1/programmed death-ligand 1</i> (PD-1/PD-L1) have emerged as promising new targets for cancer therapeutics. While tumor expression of PD-L1 has been shown to have objective responses to anti-PD-L1 immunotherapies, the clinical implications of CTLA-4 expression in tumor cells or immune cells in the tumor microenvironment is still controversial. We investigated the expression of CTLA-4 and PD-L1 in human breast tumors and provided a scoring system for the systematic evaluation of CTLA-4 staining.</p><p>Methods</p><p>Immunohistochemical staining for PD-L1 and CTLA-4 expression was performed on a tissue microarray of 102 cores, which included normal and neoplastic breast tissues. Neoplastic cores were divided into four groups: <i>Ductal carcinoma in situ</i> (DCIS), <i>invasive ductal carcinoma</i> (IDC), <i>invasive lobular carcinoma</i> (ILC) and <i>invasive tubular carcinoma</i> (ITC). PD-L1 and CTLA-4 expressions were scored based on a system which accounted for the percentage and intensity of positivity and results provided in conjunction with available clinical and demographic data.</p><p>Results</p><p>Overall, CTLA-4 was over-expressed in 49 of 93 (52.7%) breast tumors. Subcategorically, CTLA-4 was positive in 3 of 8 (37.5%) ductal carcinoma in situ, 40 of 73 (55%) of invasive ductal carcinomas, 4 of 10 (40%) of invasive lobular carcinomas and 2 of 2 (100%) of invasive tubular carcinomas. All 6 normal breast tissues were interpreted as negative for CTLA-4 staining. Only 4.1% of the invasive ductal carcinomas were positive for PD-L1 reactivity and the remaining carcinomas stained negative.</p><p>Conclusions</p><p>This study shows a significant overexpression of CTLA-4 in >50% of breast carcinomas with no such overexpression of CTLA-4 in benign breast tissues. PDL-1 staining is seen in only a small number of invasive ductal carcinomas (4.1%). These findings suggest the need for further investigation of anti-CTLA-4 and anti-PD-L1 immunotherapies and their efficacy in the treatment of breast carcinomas with overexpression of these immune modulators. In addition, the proposed scoring system will facilitate a more systematic correlation between tumor reactivity and clinical outcome which can be applied to all intracytoplasmic tumor markers.</p></div

Scores 1b & 2b in IDC (20x objective).

<p>Examples of invasive ductal carcinomas are photomicrographed showing the neoplastic cells invading the interstitial tissues. <b>B</b> CTLA-4 IHC stain with 2+ intensity showing cytoplasmic reaction in about 40% of the neoplastic cells scored as <b>1b</b> (from #2; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0195958#pone.0195958.t003" target="_blank">Table 3</a>). <b>D</b> shows 3+ intensity of CTLA-4 cytoplasmic granular reaction in 100% of the ductal carcinoma cells scored as <b>2b</b> (from #56; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0195958#pone.0195958.t003" target="_blank">Table 3</a>). The photomicrographs of the counterpart hematoxylin & eosin stains are displayed to the left of each respective CTLA-4 IHC stain (panels <b>A</b> & <b>C</b>).</p

PD-L1 in IDC (40x objective).

<p>Photomicrographs of an invasive ductal carcinoma are displayed. <b>B</b> shows PD-L1 reaction involving cell membranes of the tumor cells with large open nuclear chromatin. The reaction was observed in 20% of the tumor cells in this core. Small mononuclear cells are also present in between the tumor cells. A hematoxylin & eosin stain photomicrograph of the same tumor is shown in panel <b>A</b> (#37; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0195958#pone.0195958.t003" target="_blank">Table 3</a>).</p