Aldehyde dehydrogenase in regulatory T‐cell development, immunity and cancer

Summary

The role of aldehyde dehydrogenase (ALDH) in carcinogenesis and resistance to cancer therapies is well known. Mounting evidence also suggests a potentially important role for ALDH in the induction and function of regulatory T (Treg) cells. Treg cells are important cells of the immune system involved in promoting immune tolerance and preventing aberrant immune responses to beneficial or non‐harmful antigens. However, Treg cells also impair tumor immunity, leading to the progression of various carcinomas. ALDH expression and the subsequent production of retinoic acid by numerous cells, including dendritic cells, macrophages, eosinophils and epithelial cells, seems important in Treg induction and function in multiple organ systems. This is particularly evident in the gastrointestinal tract, pulmonary tract and skin, which are exposed to a myriad of environmental antigens and represent interfaces between the human body and the outside world. Expression of ALDH in Treg cells themselves may also be involved in the proliferation of these cells and resistance to certain cytotoxic therapies. Hence, inhibition of ALDH expression may be useful to treat cancer. Besides the direct effect of ALDH inhibition on carcinogenesis and resistance to cancer therapies, inhibition of ALDH could potentially augment the immune response to tumor antigens by inhibiting Treg induction, function and ability to promote immune tolerance to tumor cells in multiple cancer types.

Abbreviations

ALDH

aldehyde dehydrogenase

CSCs

cancer stem cells

DC

dendritic cell

GM‐CSF

granulocyte–macrophage colony‐stimulating factor

GVHD

graft‐versus‐host disease

IDO

indoleamine 2,3‐dioxygenase

LP

lamina propria

MLN

mesenteric lymph node

MLRs

mixed lymphocyte reactions

PPARγ

peroxisome proliferator‐activated receptor γ

pTreg

peripherally derived regulatory T cells

RA

retinoic acid

SCC

squamous cell carcinoma

TDO

tryptophan dioxygenase

TGF‐β

transforming growth factor‐β

Th

T helper

Treg

regulatory T

Introduction

The aldehyde dehydrogenases (ALDHs) are a superfamily of NADP⁺‐dependent enzymes that metabolize endogenous and exogenous aldehydes to corresponding carboxylic acids.1 This superfamily of proteins is comprised of 19 isozymes, with constitutive activity of at least one isozyme observed in a majority of mammalian tissues.2 The ALDHs play important roles, among other things, in cellular detoxification, the protection against ultraviolet radiation‐induced damage, and amino acid metabolism.3, 4, 5, 6 Retinoic acid (RA) biosynthesis and signaling (Fig. 1) is another crucial role of the ALDHs, with the conversion of retinal to RA performed by three isozymes in the ALDH1 family, specifically ALDH1A1 (RALDH1), ALDH1A2 (RALDH2) and ALDH1A3 (RALDH3).7, 8, 9 Research also implicates increased ALDH activity within tumors in the pathogenesis of various carcinomas and resistance to cancer therapies.

Figure 1.

Retinoic acid signaling pathway within a cell. Retinol is metabolized to retinal by ADH. ALDH1 (ALDH1A1, ALDH1A2, ALDH1A3) metabolizes retinal to retinoic acid. Retinoic acid binds to RAR (RAR α, RAR β, RAR γ) or RXR in the nucleus. The protein complex then binds to RAREs and modulates gene expression. Abbreviations: ADH, alcohol dehydrogenase; ALDH1, aldehyde dehydrogenase 1; RAR, retinoic acid receptor; RXR, retinoid X receptor; RARE, retinoic acid response element.

Overexpression of various ALDH isozymes in tumor tissue has been associated with a poor prognosis in multiple cancers, including gastric, esophageal, breast, lung, skin, pancreatic, prostate and head and neck squamous cell carcinomas (SCCs).10, 11, 12, 13, 14, 15, 16 Cancer stem cells (CSCs), which are a small subset of multipotent cells within tumors that can drive tumor proliferation, repopulation after various insults (e.g. radiation, chemotherapy, targeted therapy) and metastasis, have elevated ALDH expression.17, 18 CSCs are highly resistant to cancer therapies and can lead to relapse and progression to more aggressive disease.17 Examples include resistance to preoperative chemoradiation in esophageal carcinoma; cytarabine and bortezomib in acute myeloid leukemia; erlotinib, gefitinib and conventional chemotherapy in lung cancer; olaparib in breast cancer; doxorubicin and etoposide in Ewing sarcoma family tumors; cisplatin in mesothelioma and cyclophosphamide in a variety of tumors.16, 19, 20, 21, 22, 23, 24, 25 Hence, inhibition of ALDH has been an active field of research to suppress carcinogenesis and prevent resistance to cancer therapies.

Numerous ALDH inhibitors have been developed in the fight against cancer. These include DEAB, DIMATE, disulfiram, CM026, CM037, NCT‐501, CVT‐10216, ALDH423 and CB29.26, 27, 28, 29, 30, 31, 32, 33 Among these, the broad‐spectrum ALDH inhibitors DEAB, DIMATE and disulfiram either remain in the early stages of development or are limited by their toxicity, while the isoform‐specific ALDH inhibitors CM026, CM037, NCT‐501, CVT‐10216, ALDH423 and CB29 have limited efficacy.26, 34, 35 Developing a broad‐spectrum, non‐toxic ALDH inhibitor would be a major advancement in the cancer field.

In addition to targeting ALDH activity with small molecule inhibitors, direct targeting of ALDH‐positive CSCs through immunological approaches has shown promise in the treatment of cancer.36 For instance, in vitro generation of ADLH1A1‐specific CD8⁺ T cells with transfer into immunodeficient mice with SCC xenograft led to inhibition of tumor growth and metastases as well as prolonged survival.37 Furthermore, ALDH^High CSC‐dendritic cell (DC) vaccines have been developed and show efficacy in cancer treatment by inducing targeted cellular and humoral responses to CSCs.38 Vaccination with ALDH^High CSC‐DCs following localized therapy (e.g. radiation, surgical excision) led to decreased tumor progression, metastasis and prolonged survival in murine models of SCC and melanoma using syngeneic immunocompetent hosts.38 However, these therapeutic approaches are early in development, with more preclinical work needed to establish efficacy and determine toxicity.

Besides the importance of ALDH in carcinogenesis and resistance to cancer therapy, some recent manuscripts suggest that ALDH may play an important role in the immune system. In this review, we present an overview of the current knowledge regarding the role of ALDH in immunity focusing on its effects on regulatory T (Treg) cells. The mounting evidence for the importance of ALDH in Treg induction, function and resistance to cytotoxic therapies is detailed. Finally, the effect of inhibition of ALDH on carcinogenesis is explored to predict possible future research directions that could affect clinical practice.

Role of Treg cells in immunity

Treg cells are immune cells essential for the maintenance of immunological self‐tolerance (e.g. the unresponsiveness of the immune system to self‐antigens).39 They do this through direct cytotoxic effects, the production of anti‐inflammatory cytokines, metabolic disruption and modulation of DC function.40 Deficiency in Treg cell number or function can lead to inflammatory and autoimmune disease. For instance, mutations in the gene encoding Foxp3, a Treg‐specific transcription factor important in Treg cell development, lead to the fatal multi‐organ autoimmune disease immune dysregulation, polyendocrinopathy, enteropathy and X‐linked (IPEX) syndrome.41 Stimulation of Treg cells on the other hand has been shown to help mitigate, and potentially be a treatment option for, numerous autoimmune diseases, such as inflammatory bowel disease.42

Unfortunately, immune tolerance induced by Treg cells against self‐antigens can also impair tumor immunity. Tumor antigens recognized by CD8⁺ T cells and CD4⁺ T cells, including Treg cells, are vast and separated into various categories.43 The major categories are: (i) unique antigens, which result from somatic mutations within tumors in ubiquitously expressed genes; (ii) shared antigens, which can be expressed, to various degrees depending on the antigen, in multiple tumor types and in normal tissue; and (iii) viral antigens, which are expressed in virus‐induced malignancies.43 Unlike the anti‐tumor effect of tumor‐infiltrating CD8⁺ T cells and some CD4⁺ T‐cell subsets, such as CD4⁺ T helper type 1 (Th1) cells, the presence of a large number of Treg cells in tumor tissue is generally associated with a pro‐tumor effect, disease progression and subsequently a poorer prognosis.39, 43, 44

Large numbers of tumor‐infiltrating Treg cells have been found in many cancers, including tumors of the ovary, head and neck, pancreas, gastrointestinal tract, liver, lung and breast.45, 46, 47, 48, 49, 50, 51, 52, 53 Increased ratios of tumor‐infiltrating FOXP3⁺ Treg cells to CD8⁺ T cells are associated with a poor prognosis, especially for patients with ovarian, gastric and breast carcinomas.50, 52, 53, 54 Similarly, survival for patients with melanoma, and tumors of the breast, kidney and cervix, is significantly reduced when a large number of FOXP3⁺ Treg cells are present in the tumor.55 Early studies in mice showed that depletion of Treg cells led to the effective eradication of a variety of inoculated syngeneic tumors, partly due to tumor‐specific CD8⁺ T cells.56 Re‐challenge with the same tumor cells in these mice led to more rapid eradication of the tumor cells, demonstrating the establishment of tumor‐specific immunity.56 Subsequently, numerous therapeutic strategies have been developed to inhibit Treg cells in the hopes of treating various cancers more effectively.

Cellular proteins that have been investigated and targeted in an attempt to deplete and/or functionally impair Treg cells include CD15, CD25, LAG3, OX‐40, PD‐1, CCR4, GITR and CTLA‐4.39 Besides the non‐specificity of these molecules to Treg cells, a major concern with these therapies, particularly anti‐CTLA‐4 and anti‐PD‐1 monoclonal antibodies, is the development of serious autoimmunity, such as enterocolitis, dermatitis or autoimmune thyroid disease, which may prompt discontinuation of the therapy.57 Targeted inhibition of indoleamine 2,3‐dioxygenase (IDO) and tryptophan dioxygenase (TDO), enzymes involved in the rate‐limiting step of tryptophan catabolism, has also shown promise in the treatment of cancer. These enzymes are often overexpressed in neoplastic cells, the tumor microenvironment and draining lymph nodes, leading to immunosuppression, partly through up‐regulation of Treg cells, as well as neovascularization and metastasis.58 Accordingly, inhibition of these enzymes leads to tumor immunity through increased CD8⁺ T effector cells, natural killer cells and reductions in Treg cells and myeloid‐derived suppressor cells.58 IDO/TDO inhibitors (e.g. epacadostat and navoximod) and the tryptophan mimetic, indoximod, have advanced to phase II/III clinical trials and are generally well tolerated.58 However, they have little activity on their own, but rather enhance ‘immunogenic’ chemotherapy or immune checkpoint drugs.58 Hence, novel approaches are still needed for effective targeting of Treg cells in cancer.

ALDH in Treg induction and function

Several recent manuscripts suggest a potentially important role for ALDH in immunity. This research has focused on the role of ALDH in Treg induction, function and resistance to cytotoxic therapy.59, 60, 61, 62 The gastrointestinal tract,59 pulmonary tract60 and skin61 are the main organ systems that have been identified in which Treg cell activity is enhanced through ALDH (Fig. 2). In short, an immunological stimulus leads to, among other things, up‐regulation of ALDH in multiple cells.59, 60, 61 RA produced from ALDH then enhances Treg induction and function from naive CD4⁺ T cells.59, 60, 61 These antigen‐specific Treg cells then function to promote immune tolerance (Fig. 2).59, 60, 61

Figure 2.

The currently accepted roles of ALDH in the induction and function of Treg cells in the gastrointestinal tract, pulmonary tract and skin. (a) An immunological stimulus leads to the up‐regulation of ALDH in various cells in the gastrointestinal tract. GM‐CSF and RA secreted by LP macrophages helps induce ALDH in intestinal DCs. RA produced by intestinal DCs, LP eosinophils and macrophages up‐regulates Treg cell number and function, leading to immune tolerance. (b) An immunological stimulus leads to the up‐regulation of ALDH in various cells in the pulmonary tract. GM‐CSF and RA secreted by airway epithelium may help induce ALDH in pulmonary DCs. RA produced by pulmonary DCs, alveolar and resident tissue macrophages up‐regulates Treg cell number, leading to immune tolerance. (c) An immunological stimulus leads to the up‐regulation of ALDH in various cells in the skin. GM‐CSF and RA secreted by fibroblasts, keratinocytes and hair follicles may help induce ALDH in skin DCs. RA produced by skin DCs up‐regulates Treg cell number, leading to immune tolerance. Abbreviations: RA, retinoic acid; LP, lamina propria; GM‐CSF, granulocyte–macrophage colony‐stimulating factor; Eo, eosinophil; DC, dendritic cell; Mo, macrophage; Fo, fibroblast; Treg, regulatory T.

ALDH expression in gastrointestinal immunity

Much evidence for the role of ALDH in Treg induction and function comes from the study of the gastrointestinal tract.59 The intestinal immune system is exposed to a myriad of beneficial and non‐harmful antigens derived from ingested substances and organisms, such as foods and commensal bacteria.59 Oral tolerance is critical to prevent excess immune responses leading to food allergy and intestinal as well as systemic inflammatory disease.63 Antigen‐specific immune suppression through induction of Treg cells plays an important role in this regard.64, 65 In particular, peripherally derived Treg (pTreg) cells appear necessary for oral tolerance, with a majority of intestinal pTreg cells dependent upon food‐derived antigens.66 Lamina propria (LP) and mesenteric lymph node (MLN) DCs, LP macrophages, LP eosinophils and epithelial cells have the ability to induce Treg cell differentiation and promote immune tolerance, partly through increased expression of ALDH.59, 67, 68

LP DCs capture antigens in the intestinal lumen, migrate to MLNs and present the antigens to T cells, which results in the production of Treg cells specific for the antigen.59 The process of Treg cell induction is enhanced by several factors mediated by DCs including stimulation by transforming growth factor‐β (TGF‐β), IDO and RA.69, 70, 71 Both LP and MLN DCs highly express the gene Aldh1a2, which encodes RALDH2.59 RA produced by intestinal DCs also aids in Treg cell function by inducing the gut‐homing receptors CCR9 and integrin α ₄ β ₇ on T cells, which in turn induces gut‐homing Treg cells able to migrate to the intestine to suppress immune responses to intestinal antigens.72 ALDH produced by intestinal DCs also appears to be important in the intestinal immune tolerance induced by bacteria such as Lactobacillus planatarum, an intestinal bacterium known to have anti‐allergenic and anti‐inflammatory effects.73 In vitro and in vivo studies show induction of Treg cells by L. planatarum through RA‐ and TGF‐β‐mediated intestinal DC stimulation.73

LP macrophages and eosinophils, as well as intestinal epithelium, also aid in the process of Treg induction.68, 74, 75 Evidence suggests thata LP macrophages assist LP DCs by transferring luminal antigens to LP DCs.76 LP macrophages also produce RA through the expression of Aldh1a1 and Aldh1a2 and subsequently RALDH1 and RALDH2, respectively.61, 74 Accordingly, co‐incubation of murine LP macrophages with naive CD4⁺ T cells induces Treg cell production through interleukin‐10‐ (IL‐10), TGF‐β‐ and RA‐dependent mechanisms.74 Furthermore, LP macrophages produce granulocyte–macrophage colony‐stimulating factor (GM‐CSF), which is important for ALDH induction in intestinal DCs.75 As for eosinophils, co‐incubation of murine LP eosinophils with naive CD4⁺ T cells induces Treg cell differentiation, which may be important in intestinal immune tolerance, as eosinophils constitute a significant proportion of inflammatory cells within the LP of humans.77 The mechanism by which murine LP eosinophils induce Treg cell differentiation is through expression of ALDH and TGF‐β ₁.68 Accordingly, blocking either all‐trans‐RA or TGF‐β ₁ inhibits the capability of the eosinophils to induce Treg cells.68 Intestinal epithelial cells contribute to Treg activity, as they are able to produce RA, which is critical for the induction of ALDH in intestinal DCs.75

ALDH expression in pulmonary immunity

Induction of Treg cells by DCs, partly through ALDH, also appears to be involved in immune tolerance in the respiratory tract, which like the gastrointestinal tract is constantly exposed to environmental antigens.60 Lack of pulmonary immune tolerance can lead to unwarranted inflammatory immune responses to inhaled antigens in susceptible individuals, such as asthma.78 Recent studies have shown the importance of pulmonary DCs for inducing Treg cells and subsequently promoting airway tolerance.60, 79, 80, 81 Induction of Treg cells in this setting is largely through TGF‐β production and stimulation of peroxisome proliferator‐activated receptor γ (PPARγ) in DCs.60, 79 PPARγ is a member of the nuclear receptor superfamily with known anti‐inflammatory effects, and is expressed by multiple cell types in the lung, including DCs.82, 83 PPARγ mediates immune tolerance through various mechanisms. The receptor appears to be crucial for expression of Aldh1a2 leading to the production of RA through RALDH2, which along with TGF‐β is necessary for Treg cell induction.60, 84 PPARγ also actively dampens expression of pro‐inflammatory cytokines, such as IL‐6 and IL‐23, which preserves Treg cell function.60 Alveolar and lung‐resident tissue macrophages also express ALDH and are able to induce Treg cells from naive CD4⁺ T cells via RA and TGF‐β.61, 85, 86 Furthermore, airway epithelium may have the capability to induce ALDH expression in pulmonary DCs as they express Aldh1a1 and potentially could be a source of GM‐CSF.61

ALDH expression in skin immunity

DCs with the capability of Treg induction through ALDH expression and RA production are also present in the skin and draining cutaneous lymph nodes.61 These DCs have increased Aldh1a2 gene expression and the ability to induce skin‐specific Treg cells through RA and TGF‐β.61 Accordingly, inhibition of RA receptors with LE540 led to impaired generation of Treg cells.61 Keratinocytes and fibroblasts may induce ALDH expression in skin DCs as they produce GM‐CSF.61 Furthermore, hair follicles may stimulate ALDH expression in skin DCs by expressing RALDH2 and RALDH3.61 Hence, ALDH expression and the production of RA by skin DCs appear to be important in the cutaneous immune tolerance mediated by Treg cells. Treg cells constitute 5–10% of the T cells in normal skin.40 Deficiency in Treg number or function has been implicated in numerous inflammatory skin diseases, including psoriasis and atopic dermatitis.40

ALDH expression in Treg cells

There is minimal evidence for the role of ALDH action within Treg cells or its effect on Treg activity and function. Most of this evidence comes from work by Kanakry et al. in the study of the immunosuppressive effect of cyclophosphamide following allogeneic blood or bone marrow transplantation (alloBMT).62, 87 Treg cells with elevated ALDH expression may partially explain the success of cyclophosphamide in preventing acute and chronic graft‐versus‐host disease in these patients.62, 87 The utility of cyclophosphamide in preventing GVHD is well established,88 with early evidence suggesting that selective killing of proliferating, alloreactive T cells is the main mechanism by which cyclophosphamide exerts its effect.89 Other studies showed that generation of suppressor T cells following therapy might also contribute to the prevention of GVHD.90 More recent evidence suggests Treg cells play a crucial role in immune tolerance and the prevention of GVHD following post‐transplantation cyclophosphamide.91 Higher pTreg cell numbers are associated with a lower incidence of GVHD in these patients.92 The administration of Treg cells also modulates GVHD and may even be effective in GVHD prophylaxis.93

The mechanism by which Treg cells retain suppressive capability when exposed to cyclophosphamide is hypothesized to be in part through increased expression of ALDH.17, 62, 87 ALDH1 activity is a major pathway through which cyclophosphamide is detoxified in vivo, by oxidation of aldophosphamide to the inactive metabolite, carboxyphosphamide.94 Kanakry et al.62 found that allogeneic stimulation of mixed lymphocyte reactions followed by the administration of mafosfamide, an active cyclophosphamide analogue, led to increased numbers of Treg cells with up‐regulation of ALDH1A1, but not ALDH3A1. Furthermore, treatment of the mixed lymphocyte reactions with DEAB, a competitive ALDH inhibitor, increased sensitivity of Treg cells to mafosfamide.62 Similarly, peripheral blood taken from patients following allogeneic blood or bone marrow transplantation had increased numbers of Treg cells expressing ALDH. The Treg cells were also resistant to treatment with cyclophosphamide.62 Taken together, these results suggest that increased expression of ALDH by Treg cells following post‐transplantation cyclophosphamide may contribute to the immunosuppressive effects of cyclophosphamide in some patients (Fig. 3).

Figure 3.

Role of Treg cell ALDH expression in preventing GVHD in patients receiving post‐transplantation cyclophosphamide. Cyclophosphamide is metabolized by hepatic cytochrome p450 system to 4‐hydroxycyclophosphamide, which equilibrates with aldophosphamide. Both molecules readily diffuse into cells but are not cytotoxic. ALDH1 in Treg cells converts aldophosphamide to the inactive metabolite, carboxyphosphamide. Resistant Treg cells multiply and inhibit GVHD. Abbreviations: ALDH1, aldehyde dehydrogenase 1; Treg, regulatory T; GVHD, graft‐versus‐host disease.

Conclusion

ALDH signaling appears to be increasingly important for Treg cell induction and function through the production of RA by multiple cell types (e.g. DCs, macrophages, eosinophils, epithelial cells) as discussed above. Evidence also suggests that RA is important in Treg cell stability under inflammatory conditions and in inhibiting the differentiation of naive CD4⁺ T cells into pro‐inflammatory Th17 cells.95, 96 Furthermore, RA may indirectly induce Treg cells through inhibition of memory T cells, which block Treg induction through IL‐4, IL‐21 and interferon‐γ.95, 96 Taken together, ALDH expression and RA‐signaling appear critical for up‐regulating Treg cells and suppressing pathogenic T cells involved in autoimmunity. Also, ALDH expression by Treg cells seems to be critical for resistance of Treg cells to cytotoxic therapies, such as cyclophosphamide.62, 87 However, continued research is needed to further elucidate the contributions of ALDH isozymes and RA signaling, as well as other factors, in the activity and function of Treg cells specifically and immune cells in general.

Treg cells are important immune cells that promote immune tolerance but impair tumor immunity. The T‐cell receptor repertoire of Treg cells is broad, so Treg cells are able to recognize a wide variety of self and tumor antigens.39, 43, 44, 65, 97, 98 Due to the immunosuppressive role of Treg cells, these immune cells have been identified as a target in the treatment of various allergic and autoimmune diseases through Treg stimulation.42 Similarly, treatments aimed at Treg depletion and/or functional impairment are being investigated in various cancers.39

Evidence links ALDH expression by CSCs to the carcinogenesis of various tumors and resistance to cancer therapies. Hence, it is not inconceivable to envision that inhibiting ALDH could have a multipronged effect on cancer by modulating the immune system. Inhibition of ALDH could have a direct anti‐proliferative effect on cancers and increase sensitivity to anti‐cancer drugs or ionizing radiation.19, 26 ALDH inhibition could also increase the ratio of effector T cells to Treg cells within tumor tissue leading to increased tumor immunity.99 For instance, inhibition of RA receptors has been shown to increase the efficacy of anti‐tumor DC vaccines in a murine melanoma model through the suppression of tumor‐infiltrating Treg cells and up‐regulation of tumor‐infiltrating, interferon‐γ‐secreting CD4⁺ and CD8⁺ T cells.99 Measurement of the ratios of tumor‐infiltrating lymphocytes within tumor tissue following ALDH inhibition would be needed to confirm a similar mechanism of action.

Research into ALDH inhibitors has been hindered by the fact that broad‐spectrum ALDH inhibitors are generally toxic or remain in early stages of development, and isoform‐specific ALDH inhibitors are generally not effective.26, 34, 35 Hence, the development of a broad‐spectrum, non‐toxic ALDH inhibitor could prove to be a key advancement in this field of cancer research. With the successful development of such a drug, it could potentially be combined with other anti‐cancer drugs and/or radiation therapy to more effectively treat cancer. The sequence in which such an inhibitor is used in combinatorial therapy would need investigation. Anti‐cancer drugs and radiation cause release of tumor antigens and pro‐inflammatory signals, potentially priming or enhancing a Treg cell response.100 Further, Treg cells are more resistant to radiation compared with other T cells, with relative increases seen after radiation.101, 102 Hence, early administration of such an inhibitor in the context of combinatorial therapy could maximize inhibition of the pro‐tumor effect of Treg cells and enhancement of T‐cell‐mediated tumor rejection.

Disclosures

None.

Funding

None.