Therapeutic and prophylactic gastrectomy in a family with hereditary diffuse gastric cancer secondary to a CDH1 mutation: a case series

Abstract Background Gastric cancer is the fifth most prevalent and the third most lethal cancer worldwide, causing approximately 720,000 deaths annually. Although most cases of gastric cancers are sporadic, one of its inherited forms, hereditary diffuse gastric cancer (HDGC), constitutes about 1–3% of cases. Interestingly, females in families with HDGC are also predisposed to developing lobular breast cancer (LBC). Recent analyses have identified loss-of-function germline mutations in cadherein-1 (CDH1) as a culprit in HDGC and LBC. This discovery fueled several sequencing analyses and case series reports analyzing the pattern of inheritance of CDH1 and its propensity to induce HDGC. In 2015, a multinational and multidisciplinary task force updated the guidelines and criteria for screening, diagnosing, and managing HDGC. Case presentation Here, we present a case series of three siblings with family history of HDGC who tested positive for the CDH1 mutation and describe their surgical treatment course, post-operative management, and follow-up as they pertain to the updated guidelines. Conclusions Despite recent updates in guidelines in the diagnosis and management of HDGC, the disease remains challenging to address with patients given the high level of uncertainty and the comorbidities associated with prophylactic intervention. We strongly recommend that an interdisciplinary team inclusive of clinical and surgical oncologists, along with geneticists, social work, and psychological support, should follow the patients in a longitudinal and comprehensive manner in order to achieve full recovery and return to normalcy, as with our patients.https://deepblue.lib.umich.edu/bitstream/2027.42/144774/1/12957_2018_Article_1415.pd

Evaluating the Response of Chemoradiation Combined with Immunotherapy in Treating Locally Advanced Cervical Cancer

https://openworks.mdanderson.org/sumexp23/1007/thumbnail.jp

Role of Nrf2 in KSHV Biology and Oncogenesis

Kaposi’s sarcoma-associated herpesvirus (KSHV), or human herpes virus 8 (HHV8), is a ?2 lymphotropic herpesvirus and is the etiological agent of Kaposi’s sarcoma (KS), primary effusion B-cell lymphoma (PEL), and the multicentric Castleman’s disease (MCD). KSHV malignancies are associated with immune suppression, such as in AIDS or organ transplantation patients, and with certain African and Mediterranean populations. In an effort to identify host factors involved in the establishment of KSHV infection, we focused on the nuclear factor E2-related factor 2 (Nrf2) a member of the Cap’n’Collar basic leucine zipper (bZIP) family of transcription factors. Nrf2 is induced by ROS, and has been shown to play an important role for infection by several viruses. Moreover, Nrf2 activation leads to the expression of a cohort of genes involved in apoptosis, angoigensis, metastasis, drug resistance, and the proliferative pentose pyrophosphate pathway (PPP) enzymes. Interestingly, several of these pathways are upregulated during KSHV infection through unknown mechanisms. Because KSHV infection, like other viruses that activate Nrf2, induces ROS, a powerful activator of Nrf2, we hypothesized that KSHV infection of target cells activates Nrf2 in order to induce Nrf2 target genes and create a microenvironment conducive to infection and tumorigenesis. To this end, we utilized cellular models to assess Nrf2 activity during de novo and latent KSHV infection, the mechanism of its activation, and its role in host and virus biology. In the first part of this study, we found that Kaposi's sarcoma (KS) skin tissue exhibited elevated Nrf2 levels compared to healthy skin tissue. De novo infection of endothelial (HMVEC-d) cells showed that ROS were essential for Nrf2 activation during the early stages of infection, but dispensable during latency, where the COX-2/PGE2/PKCζ axis played an essential role in the sustained activation. Interestingly, Nrf2 was essential for optimal COX-2 expression, a major pro-viral agent during KSHV infection, establishing a feed-forward loop between COX-2 and Nrf2 in KSHV biology. Nrf2 activation was also necessary for the KSHV-mediated induction of host Bcl-2, VEGF, and the PPP enzymes. Nrf2 colocalized with LANA-1 and the KSHV genome during latency, and played an important role in proper lytic (ORF50) and latent (ORF73) gene expression. This study demonstrated for the first time that KSHV induces Nrf2 during de novo infection of endothelial cells to aid with establishment of latency. In the second part of the study, we focused on long-term-infected telomerase-immortalized endothelial cells (TIVE-LTC), which provide a model of prolonged KSHV latency. We determined that ROS did not affect Nrf2 activity in these cells. More interestingly, we identified the existence of two simultaneous Nrf2 activating pathways. The first, the non-canonical pathway, involved the autophagic protein p62-mediated sequestration of the Nrf2 inhibitor Keap1, promoting intracellular Nrf2 protein accumulation. A second activating pathway involving the COX-2/PGE2/PKCζ axis further induced Nrf2 activation and phosphorylation, which was necessary for sustained expression of Nrf2 target genes, including GCS, NQO1, xCT, VEGF and IL6, all important agents in KSHV infection and oncogenesis. In the third part of the study, we shifted our attention to KSHV latency in PEL models. We determined that histopathological tissue obtained from PEL of the stomach exhibited significant Nrf2 activation. Investigation of PEL-derived cell lines revealed that the COX-2/PGE2/PKCζ axis required prostaglandin E receptor 4 (EP4) activation, which acted as the receptor used by PGE2 to activate Nrf2. Next-generation RNA sequencing (NGS) and qPCR experiments revealed that Nrf2 knockdown or inhibition with the chemical Brusatol resulted in elevated global lytic gene expression. Additionally, we identified a novel regulatory mechanism of the major lytic regulatory gene, ORF50, that involved Nrf2, viral LANA-1 and the host transcriptional repressor KAP1. We determined that Nrf2 played a crucial role in the ORF50-mediated lytic burst during early de novo infection, an effect that is repressed in latency by LANA-1 recruitment of KAP1 to the ORF50 promoter in an Nrf2-dependent manner. (Abstract shortened by UMI.

Kaposi's sarcoma-associated herpesvirus induces Nrf2 during de novo infection of endothelial cells to create a microenvironment conducive to infection.

Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiological agent of Kaposi's sarcoma (KS) and primary effusion B-cell lymphoma. KSHV induces reactive oxygen species (ROS) early during infection of human dermal microvascular endothelial (HMVEC-d) cells that are critical for virus entry. One of the downstream targets of ROS is nuclear factor E2-related factor 2 (Nrf2), a transcription factor with important anti-oxidative functions. Here, we show that KS skin lesions have high Nrf2 activity compared to healthy skin tissue. Within 30 minutes of de novo KSHV infection of HMVEC-d cells, we observed Nrf2 activation through ROS-mediated dissociation from its inhibitor Keap1, Ser-40 phosphorylation, and subsequent nuclear translocation. KSHV binding and consequent signaling through Src, PI3-K and PKC-ζ were also important for Nrf2 stability, phosphorylation and transcriptional activity. Although Nrf2 was dispensable for ROS homeostasis, it was essential for the induction of COX-2, VEGF-A, VEGF-D, Bcl-2, NQO1, GCS, HO1, TKT, TALDO and G6PD gene expression in KSHV-infected HMVEC-d cells. The COX-2 product PGE2 induced Nrf2 activity through paracrine and autocrine signaling, creating a feed-forward loop between COX-2 and Nrf2. vFLIP, a product of KSHV latent gene ORF71, induced Nrf2 and its target genes NQO1 and HO1. Activated Nrf2 colocalized with the KSHV genome as well as with the latency protein LANA-1. Nrf2 knockdown enhanced ORF73 expression while reducing ORF50 and other lytic gene expression without affecting KSHV entry or genome nuclear delivery. Collectively, these studies for the first time demonstrate that during de novo infection, KSHV induces Nrf2 through intricate mechanisms involving multiple signal molecules, which is important for its ability to manipulate host and viral genes, creating a microenvironment conducive to KSHV infection. Thus, Nrf2 is a potential attractive target to intervene in KSHV infection and the associated maladies

Effect of Nrf2 modulation on KSHV biology.

<p><b>A</b>) KSHV entry assay was performed on cells transduced with shRL or shNrf2 and infected with KSHV (20 DNA copies/cell). DNA real-time PCR was performed with ORF73 gene-specific primers, and the absolute KSHV copy number was calculated from a standard curve obtained by real-time PCR of standards with known concentration of ORF73. <b>B</b>) Starved HMVEC-d cells in a 48-well plate previously transduced with lentivirus vectors expressing either shGFP or shNrf2 were labeled with the ROS-measuring dye, CM-H₂DCFDA, and then infected with KSHV (40 DNA copies/cell) for the indicated time points prior to fluorescence measurement. Values indicate the mean ± SD for 3 independent replicates. <b>C</b>) KSHV nuclear delivery assay was performed on cells that were transduced with shRL or shNrf2 prior to infection with KSHV for 2 hr. Real-time PCR was performed using ORF73 gene-specific primers on DNA extracted from the nuclei of infected cells to determine the levels of viral DNA. The absolute copy number was calculated from a standard curve obtained by real-time PCR of standards with known concentrations of ORF73. Bars indicate mean ± SD for 3 independent replicates. <b>D and E</b>) Starved HMVEC-d cells transduced with either shRL or shNrf2 were infected for various times with KSHV (50 DNA copies/cell) and analyzed by one-step real-time PCR reaction and by WB using ORF50-specific primers and antibody, respectively. <b>F</b>) ORF73 (LANA-1) gene-specific primers were used to determine the expression levels of ORF73 from RNA as in panel 12D. The absolute copy number was calculated from a standard curve obtained by real-time PCR of RNA standards of ORF73 or ORF50 with known concentrations. Bars indicate mean copy number ± SD of 3 independent replicates. * = p<0.05, ns = p>0.05.</p

Nrf2 induction during UV-KSHV infection and during latent KSHV gene vFLIP overexpression.

<p><b>A</b>) Starved HMVEC-d cells were left uninfected, infected with functional KSHV, or infected with UV-treated KSHV for 2 hr (lanes 1–3) and 24 hr (lanes 4–6), and immunoblotted for pNrf2 and tNrf2. β-actin was used as loading control. Fold inductions normalized to β-actin and relative to the uninfected (U.I.) condition (arbitrarily set to 1) are indicated. <b>B–D</b>) HMVEC-d cells were transduced for 72 hr using vectors containing the four latent KSHV genes (ORF71/vFLIP, ORF72/vCyclin, ORF73/LANA-1 and ORFK12/Kaposin) and the level of relevant genes were determined by real-time RT-PCR. Bars indicate fold induction relative to pSIN A (arbitrarily set to 1) ± SD for 3 independent replicates. <b>E</b>) HEK293T cells transfected with control vector or with vector containing ORF71/vFLIP for 24 hr were assessed for levels of tNrf2 and pNrf2.</p

Colocalization of pNrf2 with KSHV genome and LANA-1.

<p><b>A</b>) Proximity ligation assay (PLA) on uninfected (U.I.) and KSHV-infected cells (20 DNA copies/cell) for 24 hr. Cells were incubated for 1 hr with antibodies against pNrf2 (rabbit) and LANA-1 (mouse monoclonal), washed, incubated for 1 hr with species-specific PLA probes and 2 additional oligonucleotides to facilitate the hybridization only in close proximity (<16 nm). A ligase was then added to join the two hybridized oligonucleotides to form a closed circle and initiate a rolling-circle amplification using the ligated circle as a template after adding an amplification solution to generate a concatemeric product extending from the oligonucleotide arm of the PLA probe. Lastly, a detection solution consisting of fluorescently-labeled oligonucleotides was added, and the labeled oligonucleotides were hybridized to the concatemeric products. The signal was detected as distinct fluorescent dots. Negative controls consisted of samples treated as described but with only secondary antibodies. Confocal microscopy was used for imaging. Red dots represent LANA-1 and pNrf2; blue staining = DAPI; white arrow = PLA dot [LANA-1+pNrf2] interaction. <b>B</b>) Quantification of the number of dots in the nuclei of infected HMVEC-d cells were obtained from 3 independent, representative fields, containing ∼30 cells each. <b>C</b>) HMVEC-d cells were infected with EdU-labeled KSHV and PLA for pNrf2 and LANA-1 (green dots) was performed as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004460#ppat-1004460-g011" target="_blank">Figure 11A</a> prior to staining for EdU-KSHV (red). White arrows indicate the yellow colocalization spots between LANA-1+pNrf2 (PLA green spots) and EdU-KSHV genome (red). Blue arrows indicate the LANA-1+pNrf2 (PLA red spots) not colocalizing with EdU-KSHV genome.</p

Glutamate Secretion and Metabotropic Glutamate Receptor 1 Expression during Kaposi's Sarcoma-Associated Herpesvirus Infection Promotes Cell Proliferation

<div><p>Kaposi's sarcoma associated herpesvirus (KSHV) is etiologically associated with endothelial Kaposi's sarcoma (KS) and B-cell proliferative primary effusion lymphoma (PEL), common malignancies seen in immunocompromised HIV-1 infected patients. The progression of these cancers occurs by the proliferation of cells latently infected with KSHV, which is highly dependent on autocrine and paracrine factors secreted from the infected cells. Glutamate and glutamate receptors have emerged as key regulators of intracellular signaling pathways and cell proliferation. However, whether they play any role in the pathological changes associated with virus induced oncogenesis is not known. Here, we report the first systematic study of the role of glutamate and its metabotropic glutamate receptor 1 (mGluR1) in KSHV infected cell proliferation. Our studies show increased glutamate secretion and glutaminase expression during <i>de novo</i> KSHV infection of endothelial cells as well as in KSHV latently infected endothelial and B-cells. Increased mGluR1 expression was detected in KSHV infected KS and PEL tissue sections. Increased c-Myc and glutaminase expression in the infected cells was mediated by KSHV latency associated nuclear antigen 1 (LANA-1). In addition, mGluR1 expression regulating host RE-1 silencing transcription factor/neuron restrictive silencer factor (REST/NRSF) was retained in the cytoplasm of infected cells. KSHV latent protein Kaposin A was also involved in the over expression of mGluR1 by interacting with REST in the cytoplasm of infected cells and by regulating the phosphorylation of REST and interaction with β-TRCP for ubiquitination. Colocalization of Kaposin A with REST was also observed in KS and PEL tissue samples. KSHV infected cell proliferation was significantly inhibited by glutamate release inhibitor and mGluR1 antagonists. These studies demonstrated that elevated glutamate secretion and mGluR1 expression play a role in KSHV induced cell proliferation and suggest that targeting glutamate and mGluR1 is an attractive therapeutic strategy to effectively control the KSHV associated malignancies.</p></div

LANA-1 puncta during infection of Nrf2-deficient cells.

<p>shRL- and shNrf2-transduced HMVEC-d cells were infected with KSHV (40 DNA copies/cell) for 24 hr prior to IFA analysis using a rabbit LANA-1 specific antibody (red). The vector containing shRL expresses the green fluorescent protein (GFP), which explains the green color of shRL cells that have been successfully transduced with the vector. <b>B</b>) Quantification of the number of KSHV+ (LANA-1+) cells in the shRL-transduced cells that expressed GFP (successfully transduced) and cells that did not (unsuccessfully transduced). Bars represent mean ± SD for three individual fields containing at least 10 cells each (panel A, row 3, columns 1–2). <b>C</b>) Quantification of the number of KSHV+ (LANA-1+) cells in shRL vs. shNrf2 conditions. Bars represent mean ± SD for three individual fields containing at least 10 cells each (panel A, row 4). <b>D</b>) Quantification of the number of LANA-1 dots/nucleus in shRL vs. shNrf2 conditions. Bars represent mean ± SD for three individual fields containing at least 10 cells each (panel A, row 4).</p