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.

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 Disclosure of potential conflicts of interest is found at the end of this article.

Aldehyde dehydrogenase (ALDH) 1A enzymes produce retinoic acid (RA), a transcription induction molecule. To investigate if ALDH1A1 or ALDH1A3-mediated RA signaling has an active role in breast cancer tumorigenesis, we performed gene expression and tumor xenograft studies. Analysis of breast patient tumors revealed that high levels of ALDH1A3 correlated with expression of RA-inducible genes with retinoic acid response elements (RAREs), poorer patient survival and triple-negative breast cancers. This suggests a potential link between ALDH1A3 expression and RA signaling especially in aggressive and/or triple-negative breast cancers. In MDA-MB-231, MDA-MB-468 and MDA-MB-435 cells, ALDH1A3 and RA increased expression of RA-inducible genes. Interestingly, ALDH1A3 had opposing effects in tumor xenografts, increasing tumor growth and metastasis of MDA-MB-231 and MDA-MB-435 cells, but decreasing tumor growth of MDA-MB-468 cells. Exogenous RA replaced ALDH1A3 in inducing the same opposing tumor growth and metastasis effects, suggesting that ALDH1A3 mediates these effects by promoting RA signaling. Genome expression analysis revealed that ALDH1A3 induced largely divergent gene expression in MDA-MB-231 and MDA-MB-468 cells which likely resulted in the opposing tumor growth effects. Treatment with DNA methylation inhibitor 5-aza-2'deoxycytidine restored uniform RA-inducibility of RARE-containing HOXA1 and MUC4 in MDA-MB-231 and MDA-MB-468 cells, suggesting that differences in epigenetic modifications contribute to differential ALDH1A3/RA-induced gene expression in breast cancer. In summary, ALDH1A3 induces differential RA signaling in breast cancer cells which affects the rate of breast cancer progression.

Reovirus, a potential cancer therapy, replicates more efficiently in Ras-transformed cells than in non-transformed cells. It was presumed that increased translation was the mechanistic basis of reovirus oncolysis. Analyses of each step of the reovirus life cycle now show that cellular processes deregulated by Ras transformation promote not one but three viral replication steps. First, in Ras-transformed cells, proteolytic disassembly (uncoating) of the incoming virions, required for onset of infection, occurs more efficiently. Consequently, threefold more Ras-transformed cells become productively infected with reovirus than non-transformed cells, which accounts for the observed increase of reovirus proteins in Ras-transformed cells. Second, Ras transformation increases the infectious-to-noninfectious virus particle ratio, as virions purified from Ras-transformed cells are fourfold more infectious than those purified from non-transformed cells. Progeny assembled in non- and Ras-transformed cells appear similar by electron microscopy and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis, suggesting that Ras transformation introduces a subtle change necessary for virus infectivity. Finally, reovirus release, mediated by caspase-induced apoptosis, is ninefold more efficient in Ras-transformed cells. The combined effects of enhanced virus uncoating, infectivity, and release result in >100-fold differences in virus titers within one round of replication. Our analysis reveals previously unrecognized mechanisms by which Ras transformation mediates selective viral oncolysis.

The therapeutic potential of 2 soluble multivalent receptor-based inhibitors of Shiga toxin (Stx) 1 and Stx2 was determined in mice. One of these, Starfish, protected mice when it was injected subcutaneously in admixture with a lethal dose of Stx1 but not Stx2. Starfish also reduced the distribution of (125)I-Stx1 but not (125)I-Stx2 to the murine kidney and brain. A modified version of Starfish, called "Daisy," in which the Stx alpha Gal(1,4)beta Gal(1,4)beta Glc receptors were installed on the core glucose structure via a modified tethering strategy, protected mice against both Stx1 and Stx2. Daisy also protected streptomycin-treated mice from Escherichia coli O91:H21 and did not interfere with the ability of the murine immune system to produce Stx-specific protective antibodies. These results extend the possibility of using soluble carbohydrate-based receptor inhibitors to prevent Stx-mediated complications arising from infections with enterohemorrhagic E. coli serotypes.

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 tumorspecific 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.

Here we studied the relevance and modulation of aldehyde dehydrogenase (ALDH) expression in malignant pleural mesothelioma (MPM) chemoresistant cell subpopulations (ALDHbright cells), which survive pemetrexed + cisplatin treatment in vitro and in vivo. Expression of the ALDH1A3 isoform was invariably enriched in purified ALDHbright cells from multiple MPM cell lines and accounted for the enzymatic activity of those cells. RNAi mediated downregulation of ALDH1A3 reduced the survival of the ALDHbright cells at steady state and, much more, after pemetrexed + cisplatin treatment. We demonstrated, for the first time, that a pSTAT3(tyr705)-NFkB(p65) complex is required for the repression of DDIT3 mRNA and this ensures high levels of CEBPβ-dependent ALDH1A3 promoter activity. Inhibition of STAT3-NFkB activity allowed high levels of DDIT3 expression with increased formation of a DDIT3-CEBPβ complex. This reduced the occupancy of the ALDH1A3 promoter by CEBPβ, thus largely reducing the ALDH1A3 expression. Consequently, survival of ALDHbright cells in pemetrexed + cisplatin-treated cultures was impaired, following increased apoptosis. We show that such a mechanism is relevant in vivo and underlies the action of butein, a dual STAT3-NFkB inhibitor capable of abating the chemoresistance of mesothelioma cells in vivo. The possible broad translational relevance of the described mechanism is discussed.

INTRODUCTION: Incisional biopsies, including the diagnostic core needle biopsy (CNB), routinely performed before surgical excision of breast cancer tumors are hypothesized to increase the risk of metastatic disease. In this study, we experimentally determined whether CNB of breast cancer tumors results in increased distant metastases and examine important resultant changes in the primary tumor and tumor microenvironment associated with this outcome. METHOD: To evaluate the effect of CNB on metastasis development, we implanted murine mammary 4T1 tumor cells in BALB/c mice and performed CNB on palpable tumors in half the mice. Subsequently, emulating the human scenario, all mice underwent complete tumor excision and were allowed to recover, with attendant metastasis development. Tumor growth, lung metastasis, circulating tumor cell (CTC) levels, variation in gene expression, composition of the tumor microenvironment, and changes in immunologic markers were compared in biopsied and non-biopsied mice. RESULTS: Mice with biopsied tumors developed significantly more lung metastases compared to non-biopsied mice. Tumors from biopsied mice contained a higher frequency of myeloid-derived suppressor cells (MDSCs) accompanied by reduced CD4 + T cells, CD8 + T cells, and macrophages, suggesting biopsy-mediated development of an increasingly immunosuppressive tumor microenvironment. We also observed a CNB-dependent up-regulation in the expression of SOX4, Ezh2, and other key epithelial-mesenchymal transition (EMT) genes, as well as increased CTC levels among the biopsy group. CONCLUSION: CNB creates an immunosuppressive tumor microenvironment, increases EMT, and facilitates release of CTCs, all of which likely contribute to the observed increase in development of distant metastases.