Lualhati Harkins

Specific localisation of human cytomegalovirus nucleic acids and proteins in human colorectal cancer

BACKGROUND Colorectal cancer is the second most frequent cause of death from cancer in the USA, and most tumours arise sporadically with no clear cause or genetic predisposition. Human cytomegalovirus is a beta-herpesvirus that is endemic in the human population and can cause life-threatening disease in immunosuppressed adults. In vitro, human cytomegalovirus can transform cells and dysregulate many cellular pathways relevant to colon adenocarcinoma pathogenesis, especially those affecting the cell cycle, mutagenesis, apoptosis, angiogenesis, and cyclo-oxygenase-2 (COX-2) expression. We aimed to assess whether gene products of human cytomegalovirus could be detected in colorectal cancers. METHODS We obtained formalin-fixed, paraffin-embedded pathological specimens of colorectal polyps, adenocarcinomas, and adjacent normal mucosa from 29 patients. To detect human cytomegalovirus proteins and nucleic acids, we used immunohistochemistry with two different monoclonal antibodies, in-situ hybridisation, and PCR with DNA sequencing. FINDINGS Human cytomegalovirus proteins IE1-72 and pp65 were detected in a tumour cell-specific pattern in 14 (82%) of 17 and seven (78%) of nine colorectal polyps, respectively, and 12 (80%) of 15 and 11 (92%) of 12 adenocarcinomas, respectively, but not in adjacent non-neoplastic colon biopsy samples from the same patients (none of seven and none of two, respectively). Human cytomegalovirus infection of colon-cancer cells (Caco-2) in vitro resulted in specific induction of Bcl-2 and cyclo-oxygenase-2 proteins, both of which are thought to contribute to progression of colon cancer. INTERPRETATION Human cytomegalovirus nucleic acids and proteins can be found that specifically localise to neoplastic cells in human colorectal polyps and adenocarcinomas, and virus infection can induce important oncogenic pathways in colon-cancer cells.

Detection of human cytomegalovirus in normal and neoplastic breast epithelium

IntroductionHuman cytomegalovirus (HCMV) establishes a persistent life-long infection, and can cause severe pathology in the fetus and the immunocompromised host[1]. Breast milk is the primary route of transmission in humans worldwide, and breast epithelium is thus a likely site of persistent infection and/or reactivation, though this phenomenon has not previously been demonstrated. Increasing evidence indicates HCMV infection can modulate signaling pathways associated with oncogenesis. We hypothesized that persistent HCMV infection occurs in normal adult breast epithelium and that persistent viral expression might be associated with normal and neoplastic ductal epithelium.MethodsSurgical biopsy specimens of normal breast (n = 38) breast carcinoma (n = 39) and paired normal breast from breast cancer patients (n = 21) were obtained. Specimens were evaluated by immunohistochemistry, in situ hybridization, PCR and DNA sequencing for evidence of HCMV antigens and nucleic acids.ResultsWe detected HCMV expression specifically in glandular epithelium in 17/27 (63%) of normal adult breast cases evaluated. In contrast, HCMV expression was evident in the neoplastic epithelium of 31/32 (97%) patients with ductal carcinoma in situ (DCIS) and infiltrating ductal carcinoma (IDC) cases evaluated (p = 0.0009).ConclusionsThese findings are the first to demonstrate that persistent HCMV infection occurs in breast epithelium in a significant percentage of normal adult females. HCMV expression was also evident in neoplastic breast epithelium in a high percentage of normal and neoplastic breast tissues obtained from breast cancer patients, raising the possibility that viral infection may be involved in the neoplastic process.

Second messengers regulate RGS2 expression which is targeted to the nucleus

Regulators of G-protein Signaling (RGS) proteins attenuate signaling activities of G proteins, and modulation of expression appears to be a primary mechanism for regulating RGS proteins. In human astrocytoma 1321N1 cells RGS2 expression was increased by activation of muscarinic receptors coupled to phosphoinositide signaling with carbachol, or by increased cyclic AMP production, demonstrating that both signaling systems can increase the expression of a RGS family member in a single cell type. Carbachol-stimulated increases in endogenous RGS2 protein levels appeared by immunocytochemical analysis to be largely confined to the nucleus, and this localization was confirmed by Western blot analysis which showed increased nuclear, but not cytosolic, RGS2 after carbachol treatment. Additionally, transiently expressed green fluorescent protein (GFP)-tagged, 6xHis-tagged, or unmodified RGS2 resulted in a predominant nuclear localization, as well as a distinct accumulation of RGS2 along the plasma membrane. The intranuclear localization of GFP-RGS2 was confirmed with confocal microscopy. Thus, RGS2 expression is rapidly and transiently increased by phosphoinositide signaling and by cyclic AMP, and endogenous and transfected RGS2 is largely, although not entirely, localized in the nucleus.

Cytomegalovirus Immediate-Early Proteins Promote Stemness Properties in Glioblastoma

Glioblastoma (GBM) is the most common and aggressive human brain tumor. Human cytomegalovirus (HCMV) immediate-early (IE) proteins that are endogenously expressed in GBM cells are strong viral transactivators with oncogenic properties. Here, we show how HCMV IEs are preferentially expressed in glioma stem-like cells (GSC), where they colocalize with the other GBM stemness markers, CD133, Nestin, and Sox2. In patient-derived GSCs that are endogenously infected with HCMV, attenuating IE expression by an RNAi-based strategy was sufficient to inhibit tumorsphere formation, Sox2 expression, cell-cycle progression, and cell survival. Conversely, HCMV infection of HMCV-negative GSCs elicited robust self-renewal and proliferation of cells that could be partially reversed by IE attenuation. In HCMV-positive GSCs, IE attenuation induced a molecular program characterized by enhanced expression of mesenchymal markers and proinflammatory cytokines, resembling the therapeutically resistant GBM phenotype. Mechanistically, HCMV/IE regulation of Sox2 occurred via inhibition of miR-145, a negative regulator of Sox2 protein expression. In a spontaneous mouse model of glioma, ectopic expression of the IE1 gene (UL123) specifically increased Sox2 and Nestin levels in the IE1-positive tumors, upregulating stemness and proliferation markers in vivo. Similarly, human GSCs infected with the HCMV strain Towne but not the IE1-deficient strain CR208 showed enhanced growth as tumorspheres and intracranial tumor xenografts, compared with mock-infected human GSCs. Overall, our findings offer new mechanistic insights into how HCMV/IE control stemness properties in GBM cells.

CMV-Independent Lysis of Glioblastoma by Ex Vivo Expanded/Activated Vδ1+ γδ T Cells

Vδ2neg γδ T cells, of which Vδ1+ γδ T cells are by far the largest subset, are important effectors against CMV infection. Malignant gliomas often contain CMV genetic material and proteins, and evidence exists that CMV infection may be associated with initiation and/or progression of glioblastoma multiforme (GBM). We sought to determine if Vδ1+ γδ T cells were cytotoxic to GBM and the extent to which their cytotoxicity was CMV dependent. We examined the cytotoxic effect of ex vivo expanded/activated Vδ1+ γδ T cells from healthy CMV seropositive and CMV seronegative donors on unmanipulated and CMV-infected established GBM cell lines and cell lines developed from short- term culture of primary tumors. Expanded/activated Vδ1+ T cells killed CMV-negative U251, U87, and U373 GBM cell lines and two primary tumor explants regardless of the serologic status of the donor. Experimental CMV infection did not increase Vδ1+ T cell - mediated cytotoxicity and in some cases the cell lines were more resistant to lysis when infected with CMV. Flow cytometry analysis of CMV-infected cell lines revealed down-regulation of the NKG2D ligands ULBP-2, and ULBP-3 as well as MICA/B in CMV-infected cells. These studies show that ex vivo expanded/activated Vδ1+ γδ T cells readily recognize and kill established GBM cell lines and primary tumor-derived GBM cells regardless of whether CMV infection is present, however, CMV may enhance the resistance GBM cell lines to innate recognition possibly contributing to the poor immunogenicity of GBM.

RE: “Lack of association of herpesviruses with brain tumors”

In the April 2006 issue of the Journal of NeuroVirology, Poltermann et al (2006) report a lack of association between human cytomegalovirus (HCMV) and glioma. This subject is highly controversial, and the debate over the role of HCMV in glioma pathogenesis is ongoing. Given the fatal nature of these tumors, and the fact that very little is known about their etiology, extreme technical proficiency and scientific scrutiny must be used when examining potential causes. Several technical issues described in the article should be addressed to allowmore accurate evaluation of the findings. With regards to the immunohistochemistry protocol: First, the authors report using brain tumor sections 8 μm thick. Such thickness is typically used for surface staining and is known to be too thick for antigen-antibody binding. For optimal staining, a thickness of 6 μm is suggested (Cobbs et al, 2002). This allows complete deparaffinization,which is critical for proper enzyme digestion and antigen retrieval. Second, the authors fail to describe any postfixation of tissue sections with formalin prior to digestion, as well as any pepsin digestion before antigen retrieval. Formalin treatment (which stabilizes the antigen) is critical for epitope conditioning, and pepsin digestion is critical for HCMV detection because pepsin cleaves specific peptide residues that interfere with antigen-antibody binding. Third, the authors describe antigen retrieval using Citra buffer. This approach results in false-negative staining that could be overcome by combining both pepsin digestion and antigen retrieval (Polak et al, 2003) at 45◦C to 50◦C for 2.5 h; false-negative staining may result

Methods for the Detection of Cytomegalovirus in Glioblastoma Cells and Tissues

An increased awareness of the potential oncomodulatory properties of human cytomegalovirus (HCMV) has evolved over the last decade. We first reported the presence of HCMV in human glioblastomas, and subsequently these findings have been corroborated by other groups. However, some controversy has been associated with the immunohistochemical and in situ hybridization techniques used, since standard immunohistochemical and in situ hybridization techniques have been insufficient to detect low level HCMV antigens and nucleic acids in some tumor tissues. Here, we present detailed methods that can be used for the sensitive detection of low level HCMV antigens and nucleic acids in human glioblastoma specimens. Using these techniques, HCMV is frequently detected in frozen and formalin fixed paraffin-embedded tissue specimens. Furthermore, we demonstrate how human primary glioblastoma cells can be cultured in vitro, and how these cells can be used for detection of HCMV by immunofluorescence, in situ hybridization, western blot, and RT-PCR.

The safety of allogeneic innate lymphocyte therapy for glioma patients with prior cranial irradiation.

The standard treatment of high-grade glioma presents a combination of radiotherapy, chemotherapy and surgery. Immunotherapy is proposed as a potential adjunct to standard cytotoxic regimens to target remaining microscopic disease following resection. We have shown ex vivo expanded/activated γδ T cells to be a promising innate lymphocyte therapy based on their recognition of stress antigens expressed on gliomas. However, successful integration of γδ T cell therapy protocols requires understanding the efficacy and safety of adoptively transferred immune cells in the post-treatment environment. The unique features of γδ T cell product and the environment (hypoxia, inflammation) can affect levels of expression of key cell receptors and secreted factors and either promote or hinder the feasibility of γδ T cell therapy. We investigated the potential for the γδ T cells to injure normal brain tissue that may have been stressed by treatment. We evaluated γδ T cell toxicity by assessing actual and correlative toxicity indicators in several available models including: (1) expression of stress markers on normal primary human astrocytes (as surrogate for brain parenchyma) after irradiation and temozolomide treatment, (2) cytotoxicity of γδ T cells on normal and irradiated primary astrocytes, (3) microglial activation and expression of stress-induced ligands in mouse brain after whole-brain irradiation and (4) expression of stress-induced markers on human brain tumors and on normal brain tissue. The lack of expression of stress-induced ligands in all tested models suggests that γδ T cell therapy is safe for brain tumor patients who undergo standard cytotoxic therapies.

TGFβ‐Responsive HMOX1 Expression Is Associated with Stemness and Invasion in Glioblastoma Multiforme

Glioblastoma multiforme (GBM) is the most common and lethal adult brain tumor. Resistance to standard radiation and chemotherapy is thought to involve survival of GBM cancer stem cells (CSCs). To date, no single marker for identifying GBM CSCs has been able to capture the diversity of CSC populations, justifying the needs for additional CSC markers for better characterization. Employing targeted mass spectrometry, here we present five cell‐surface markers HMOX1, SLC16A1, CADM1, SCAMP3, and CLCC1 which were found to be elevated in CSCs relative to healthy neural stem cells (NSCs). Transcriptomic analyses of REMBRANDT and TCGA compendiums also indicated elevated expression of these markers in GBM relative to controls and non‐GBM diseases. Two markers SLC16A1 and HMOX1 were found to be expressed among pseudopalisading cells that reside in the hypoxic region of GBM, substantiating the histopathological hallmarks of GBM. In a prospective study (N = 8) we confirmed the surface expression of HMOX1 on freshly isolated primary GBM cells (P0). Employing functional assays that are known to evaluate stemness, we demonstrate that elevated HMOX1 expression is associated with stemness in GBM and can be modulated through TGFβ. siRNA‐mediated silencing of HMOX1 impaired GBM invasion—a phenomenon related to poor prognosis. In addition, surgical resection of GBM tumors caused declines (18% ± 5.1SEM) in the level of plasma HMOX1 as measured by ELISA, in 8/10 GBM patients. These findings indicate that HMOX1 is a robust predictor of GBM CSC stemness and pathogenesis. Further understanding of the role of HMOX1 in GBM may uncover novel therapeutic approaches. Stem Cells 2016;34:2276–2289

In vivo expansion and activation of γδ T cells as immunotherapy for refractory neuroblastoma: A phase 1 study.

Introduction:CD3+ &ggr;&dgr;+ T cells comprise 2% to 5% of circulating T cells with V&ggr;9V&dgr;2+ cells the dominant circulating subtype. V&ggr;9V&dgr;2+ cells recognize non-peptide phosphoantigens and stress-associated NKG2D ligands expressed on malignant cells. Strategies that incorporate the tumoricidal properties of &ggr;&dgr; T cells represent a promising immunotherapeutic strategy for treatment of solid malignancies including neuroblastoma (NB). In this prospective, non-randomized Phase I trial, we assessed whether circulating V&ggr;9V&dgr;2+ cells could be safely expanded using intravenous ZOL (Zoledronate [Zometa®]) and subcutaneous Interleukin-2 (IL-2) in patients with refractory NB. Methods:Patients 2 to 21 years of age with refractory neuroblastoma with no known curative therapeutic options received ZOL on day 1, and IL-2 on days 1 to 5 and 15 to 19 of each 28-day cycle (n = 4). Lymphocyte immunophenotyping was assessed weekly. Immunophenotyping studies from the treatment group were compared with healthy pediatric controls (n = 16; range, 5y–15y) and of untreated NB disease controls (n = 9; range, 4m–18y). Results:Treatment was well tolerated with no unexpected grade 3 and 4 toxicities. Lymphocyte subset counts did not differ significantly between volunteers and disease controls with the exception of &ggr;&dgr;+ T cell counts that were significantly higher in healthy volunteers (212 + 93 vs. 89 + 42, P = 0.05). Study patients showed increases in circulating &ggr;&dgr;+ T cell count (3–10 fold) after the first week, increasing into the range seen in healthy volunteers (125 + 37, P = 0.1940). Interestingly, all ZOL + IL-2 treated patients showed significant increases in CD3+CD4+CD27hiCD127dim T cells that rose weekly in 2 patients throughout the 4 weeks of observation (maximum 41% and 24% of total CD3+CD4+ T cells, respectively). Conclusions:In summary, combined ZOL and IL-2 is well tolerated and restored &ggr;&dgr;+ T cell counts to the normal range with a moderate expansion of Natural Killer cells. Progressive increases in circulating CD4+ T cells with a regulatory phenotype cells may offset beneficial effects of this therapy.