Paola Marcato

Aldehyde Dehydrogenase Activity of Breast Cancer Stem Cells Is Primarily Due To Isoform ALDH1A3 and Its Expression Is Predictive of Metastasis

Cancer stem cells (CSCs) are proposed to initiate cancer and propagate metastasis. Breast CSCs identified by aldehyde dehydrogenase (ALDH) activity are highly tumorigenic in xenograft models. However, in patient breast tumor immunohistological studies, where CSCs are identified by expression of ALDH isoform ALDH1A1, CSC prevalence is not correlative with metastasis, raising some doubt as to the role of CSCs in cancer. We characterized the expression of all 19 ALDH isoforms in patient breast tumor CSCs and breast cancer cell lines by total genome microarray expression analysis, immunofluorescence protein expression studies, and quantitative polymerase chain reaction. These studies revealed that ALDH activity of patient breast tumor CSCs and cell lines correlates best with expression of another isoform, ALDH1A3, not ALDH1A1. We performed shRNA knockdown experiments of the various ALDH isoforms and found that only ALDH1A3 knockdown uniformly reduced ALDH activity of breast cancer cells. Immunohistological studies with fixed patient breast tumor samples revealed that ALDH1A3 expression in patient breast tumors correlates significantly with tumor grade, metastasis, and cancer stage. Our results, therefore, identify ALDH1A3 as a novel CSC marker with potential clinical prognostic applicability, and demonstrate a clear correlation between CSC prevalence and the development of metastatic breast cancer. STEM CELLS 2011;29:32–45

Aldehyde dehydrogenase: Its role as a cancer stem cell marker comes down to the specific isoform

Recent evidence suggests that enhanced aldehyde dehydrogenase (ALDH) activity is a hallmark of cancer stem cells (CSC) measurable by the aldefluor assay. ALDH1A1, one of 19 ALDH isoforms expressed in humans, was generally believed to be responsible for the ALDH activity of CSCs. More recently, experiments with murine hematopoietic stem cells, murine progenitor pancreatic cells, and human breast CSCs indicate that other ALDH isoforms, particularly ALDH1A3, significantly contribute to aldefluor positivity, which may be tissue and cancer specific. Therefore, potential prognostic application involving the use of CSC prevalence in tumor tissue to predict patient outcome requires the identification and quantification of specific ALDH isoforms. Herein we review the suggested roles of ALDH in CSC biology and the immunohistological studies testing the potential application of ALDH isoforms as novel cancer prognostic indicators.

Oncolytic Reovirus Effectively Targets Breast Cancer Stem Cells

Recent evidence suggests that cancer stem cells (CSCs) play an important role in cancer, as these cells possess enhanced tumor-forming capabilities and are resistant to current anticancer therapies. Hence, novel cancer therapies will need to be tested for both tumor regression and CSC targeting. Herein we show that oncolytic reovirus that induces regression of human breast cancer primary tumor samples xenografted in immunocompromised mice also effectively targets and kills CSCs in these tumors. CSCs were identified based on CD24(-)CD44(+) cell surface expression and overexpression of aldehyde dehydrogenase. Upon reovirus treatment, the CSC population was reduced at the same rate as non-CSCs within the tumor. Immunofluorescence of breast tumor tissue samples from the reovirus- and mock-treated mice confirmed that both CSCs and non-CSCs were infectible by reovirus, and terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) assay showed that both populations died by apoptosis. Ras, which has been shown to mediate reovirus oncolysis, was found to be present at similar levels in all cell types, and this is consistent with their comparable sensitivity to reovirus. These experiments indicate that oncolytic reovirus has the potential to induce tumor regression in breast cancer patients. More important, the CSC population was equally reduced and was as susceptible to reovirus treatment as the non-CSC population.

Targeting Cancer-initiating Cells With Oncolytic Viruses

Recent studies in a variety of leukemias and solid tumors indicate that there is significant heterogeneity with respect to tumor-forming ability within a given population of tumor cells, suggesting that only a subpopulation of cells is responsible for tumorigenesis. These cells have been commonly referred to as cancer stem cells (CSCs) or cancer-initiating cells (CICs). CICs have been shown to be relatively resistant to conventional anticancer therapies and are thus thought to be responsible for disease relapse. As such, they represent a potentially critical therapeutic target. Oncolytic viruses are in clinical trials for cancer and kill cells through mechanisms different from conventional therapeutics. Because these viruses are not susceptible to the same pathways of drug or radiation resistance, it is important to learn whether CICs are susceptible to oncolytic virus infection. Here we review the available data regarding the ability of several different oncolytic virus types to target CICs for destruction.

The RAS/Raf1/MEK/ERK Signaling Pathway Facilitates VSV-mediated Oncolysis: Implication for the Defective Interferon Response in Cancer Cells

Vesicular stomatitis virus (VSV) can replicate in malignant cells more efficiently than in normal cells. Although the selective replication appears to be caused by defects in the interferon (IFN) system in malignant cells, the mechanisms which render these cells less responsive to IFN remain poorly understood. Here we present evidence that an activated RAS/Raf1/MEK/ERK pathway plays a critical role in the defects. NIH 3T3 or human primary cells stably expressing active RAS or Raf1 were rapidly killed by VSV. Although IFNα treatment no longer protected the RAS- or Raf1-overexpressing cells from VSV infection, responsiveness to IFNα was restored following treatment with the mitogen-activated protein kinase kinase (MEK) inhibitor U0126. Similarly, human cancer-derived cell lines became more responsive to IFNα in conjunction with U0126 treatment. Intriguingly, dual treatment with both IFNα and U0126 severely reduced the levels of viral RNAs in the infected cells. Moreover, cancer cells showed defects in inducing an IFNα-responsive factor, MxA, which is known to block VSV RNA synthesis, and U0126 restored the MxA expression. Our observations suggest that activation of the extracellular signal-regulated protein kinase (ERK) signaling leads to the defect in IFNα-mediated upregulation of MxA protein, which facilitates VSV oncolysis. In view of the fact that 30% of all cancers have constitutive activation of the RAS/Raf1/MEK/ERK pathway, VSV would be an ideal oncolytic virus for targeting such cancers.

Unshackling the links between reovirus oncolysis, Ras signaling, translational control and cancer

Reovirus has an inherent preference for replicating in cells with dysregulated growth factor signaling cascades that comprise Ras activation. Precisely how reovirus exploits the host cell Ras pathway is unclear, but there is evidence suggesting that activated Ras signaling is important for efficient viral protein synthesis. Defining the molecular mechanism of reovirus oncolysis will shed light on reovirus replication and important aspects of cellular transformation, Ras signaling cascades and regulation of protein translation.

Reovirus Virotherapy Overrides Tumor Antigen Presentation Evasion and Promotes Protective Antitumor Immunity

Tumor-associated immunosuppressive strategies, such as lack of tumor antigen recognition and failure of lymphocyte activation and homing, resist the development of tumor-specific immunity and hamper the immune response–mediated elimination of cancerous cells. In this report, we show that reovirus virotherapy overrides such a tumor immune evasion and establishes clinically meaningful antitumor immunity capable of protecting against subsequent tumor challenge. Reovirus-mediated destruction of tumor cells facilitates the recognition of tumor antigens by promoting the display of otherwise inaccessible tumor-specific immunogenic peptides on the surface of dendritic cells (DC). Furthermore, on exposure to reovirus, DCs produce IL-1α, IL-1β, IL-6, IL-12p40/70, IL-17, CD30L, eotaxin, GM-CSF, KC, MCP-1, MCP-5, M-CSF, MIG, MIP-1α, RANTES, TNF-α, VCAM-1, VSGF, CXCL-16, AXL, and MCP-2; undergo maturation; and migrate into the tumor microenvironment along with CD8 T cells. These reovirus-activated DCs also acquire the capacity to prime tumor antigen–specific transgenic T cells in vitro and intrinsic antitumor T-cell response in vivo. Further, reovirus virotherapy augments the efficacy of DC- or T cell–based anticancer immunotherapies and synergistically enhances the survival in tumor-bearing mice. Most importantly, antitumor cellular immune responses initiated during reovirus oncotherapy protect the host against subsequent tumor challenge in a reovirus-independent but antigen-dependent manner. These reovirus oncotherapy–initiated antitumor immune responses represent an anticancer therapeutic entity that can maintain a long-term cancer-free health even after discontinuation of therapy. Mol Cancer Ther; 9(11); 2924–33. ©2010 AACR.

Oncolytic Virus-initiated Protective Immunity Against Prostate Cancer

Recently reovirus-based oncotherapy has been successfully implemented for the treatment of prostate cancer. In this report, we show that apart from its primary direct cancer-killing activity, reovirus oncotherapy overrides tumor-associated immune evasion strategies and confers protective antiprostate cancer immunity. Prostate cancer represents an ideal target for immunotherapies. However, currently available immune interventions fail to induce clinically significant antiprostate cancer immune responses, owing to the immunosuppressive microenvironment associated with this disease. We show here that during the process of oncolysis, reovirus acts upon prostate cancer cells and initiates proinflammatory cytokines and major histocompatibility complex (MHC) class I molecule expression. In an immunocompetent transgenic adenocarcinoma of mouse prostate (TRAMP) model, reovirus oncotherapy induces the homing of CD8(+) T and NK cells in tumors and the display of tumor-associated antigens (TAAs) on antigen-presenting cells (APCs), and endows dendritic cells (DCs) with a capacity to successfully present TAAs to tumor-specific CD8(+) T cells. These newly generated immunological events lead to the development of strong antiprostate cancer T cell responses, which restrict the growth of subsequently, implanted syngeneic tumor in an antigen-specific, but reovirus-independent manner. Such reovirus-initiated antiprostate cancer immunity represents a clinically valuable entity that can promote long-term cancer-free health even after discontinuation of the primary oncotherapy.Recently reovirus-based oncotherapy has been successfully implemented for the treatment of prostate cancer. In this report, we show that apart from its primary direct cancer-killing activity, reovirus oncotherapy overrides tumor-associated immune evasion strategies and confers protective antiprostate cancer immunity. Prostate cancer represents an ideal target for immunotherapies. However, currently available immune interventions fail to induce clinically significant antiprostate cancer immune responses, owing to the immunosuppressive microenvironment associated with this disease. We show here that during the process of oncolysis, reovirus acts upon prostate cancer cells and initiates proinflammatory cytokines and major histocompatibility complex (MHC) class I molecule expression. In an immunocompetent transgenic adenocarcinoma of mouse prostate (TRAMP) model, reovirus oncotherapy induces the homing of CD8+ T and NK cells in tumors and the display of tumor-associated antigens (TAAs) on antigen-presenting cells (APCs), and endows dendritic cells (DCs) with a capacity to successfully present TAAs to tumor-specific CD8+ T cells. These newly generated immunological events lead to the development of strong antiprostate cancer T cell responses, which restrict the growth of subsequently, implanted syngeneic tumor in an antigen-specific, but reovirus-independent manner. Such reovirus-initiated antiprostate cancer immunity represents a clinically valuable entity that can promote long-term cancer-free health even after discontinuation of the primary oncotherapy.

Human Serum Amyloid P Component Protects against Escherichia coli O157:H7 Shiga Toxin 2 In Vivo: Therapeutic Implications for Hemolytic-Uremic Syndrome

Shiga toxin (Stx) 2 causes hemolytic-uremic syndrome (HUS), an intractable and often fatal complication of enterohemorrhagic Escherichia coli O157:H7 infection. Here, we show that serum amyloid P component (SAP), a normal human plasma protein, specifically protects mice against the lethal toxicity of Stx2, both when injected into wild-type mice and when expressed transgenically; in the presence of human SAP, there was greatly reduced in vivo localization of Stx2 to the kidneys, suggesting a possible mechanism of protection. In humans, circulating SAP concentrations did not differ between patients with suspected enterohemorrhagic E. coli infection with antibodies to E. coli O157:H7 lipopolysaccharide and those without antibodies or between patients with HUS and those without it. However, the potent protection conferred by human SAP in the mouse model suggests that infusion of supplemental SAP may be a useful novel therapeutic approach to the treatment of this devastating condition.

Recombinant Shiga Toxin B-Subunit-Keyhole Limpet Hemocyanin Conjugate Vaccine Protects Mice from Shigatoxemia

ABSTRACT Enterohemorrhagic Escherichia coli (EHEC) causes hemorrhagic colitis in humans and, in a subgroup of infected subjects, a more serious condition called hemolytic-uremic syndrome (HUS). These conditions arise because EHEC produces two antigenically distinct forms of Shiga toxin (Stx), called Stx1 and Stx2. Despite this, the production of Stx2 by virtually all EHEC serotypes and the documented role this toxin plays in HUS make it an attractive vaccine candidate. Previously, we assessed the potential of a purified recombinant Stx2 B-subunit preparation to prevent Shigatoxemia in rabbits. This study revealed that effective immunization could be achieved only if endotoxin was included with the vaccine antigen. Since the presence of endotoxin would be unacceptable in a human vaccine, the object of the studies described herein was to investigate ways to safely augment, in mice, the immunogenicity of the recombinant Stx2 B subunit containing <1 endotoxin unit per ml. The study revealed that sera from mice immunized with such a preparation, conjugated to keyhole limpet hemocyanin and administered with the Ribi adjuvant system, displayed the highest Shiga toxin 2 B-subunit-specific immunoglobulin G1 (IgG1) and IgG2a enzyme-linked immunosorbent assay titers and cytotoxicity-neutralizing activities in Ramos B cells. As well, 100% of the mice vaccinated with this preparation were subsequently protected from a lethal dose of Stx2 holotoxin. These results support further evaluation of a Stx2 B-subunit-based human EHEC vaccine.