Aldehyde dehydrogenase activity as a functional marker for lung cancer

Abstract

Aldehyde dehydrogenase (ALDH) activity has been implicated in multiple biological and biochemical pathways and has been used to identify potential cancer stem cells. Our main hypothesis is that ALDH activity may be a lung cancer stem cell marker. Using flow cytometry, we sorted cells with bright (ALDH^br) and dim (ALDH^lo) ALDH activity found in H522 lung cancer cell line. We used in vitro proliferation and colony assays as well as a xenograft animal model to test our hypothesis. Cytogenetic analysis demonstrated that the ALDH^br cells are indeed a different clone, but when left in normal culture conditions will give rise to ALDH^lo cells. Furthermore, the ALDH^br cells grow slower, have low clonal efficiency, and give rise to morphologically distinct colonies. The ability to form primary xenografts in NOD/SCID mice by ALDH^br and ALDH^lo cells was tested by injecting single cell suspension under the skin in each flank of same animal. Tumor size was calculated weekly. ALDH1A1 and ALDH3A1 immunohistochemistry (IHC) was performed on excised tumors. These tumors were also used to re-establish cell suspension, measure ALDH activity, and re-injection for secondary and tertiary transplants. The results indicate that both cell types can form tumors but the ones from ALDH^br cells grew much slower in primary recipient mice. Histologically, there was no significant difference in the expression of ALDH in primary tumors originating from ALDH^br or ALDH^lo cells. Secondary and tertiary xenografts originating from ALDH^br grew faster and bigger than those formed by ALDH^lo cells. In conclusion, ALDH^br cells may have some of the traditional features of stem cells in terms of being mostly dormant and slow to divide, but require support of other cells (ALDH^lo) to sustain tumor growth. These observations and the known role of ALDH in drug resistance may have significant therapeutic implications in the treatment of lung cancer.

Introduction

Aldehyde dehydrogenases (ALDH) are a group of NAD(P)⁺-dependent enzymes involved in oxidizing a wide variety of aldehydes into their corresponding carboxylic acids [1], [2]. The role of some of these ALDHs in endobiotic and xenobiotic metabolism has been reviewed extensively before [1], [2], [3], [4], [5], [6], [7], [8], [9], [10] and the specific metabolic pathways affected have been revealed in detail [1]. ALDHs have broad substrate specificity. Their role in alcohol metabolism, vitamin A metabolism, and resistance against oxazaphosphorines are some of the most studied aspects of their activities. Multiple tools have been used to study the role of the ALDH enzymes in cellular metabolism and organ development including antisense [11], siRNA [12], inhibitors such as diethylaminobenzaldehyde (DEAB) [13] and disulfiram [14] and knock out mice models [15], [16], [17], [18], [19].

Expression of ALDH isozymes has been shown to be increased in many cancer types including liver, pancreas, breast and colon cancers [20], [21], [22], [23], [24], [25]. Previously, we have profiled the ALDH expression levels of 12 different human lung cancer cell lines [26]. Specifically, ALDH1A1 and ALDH3A1 cytosolic forms of the enzymes were highly expressed in some non-small cell lung cancer cell lines as well as in patient lung cancer samples [27]. We also observed that expression of both enzymes gradually increases during the transition from normal to atypical pneumocyte, carcinoma in situ and then adenocarcinoma. Moreover, our results have shown that cigarette smoking alone seems sufficient to elevate ALDH expression in normal pneumocytes [27]. These observations are the basis for our hypothesis that the elevated expression of these isozymes may be related to the malignant transformation.

Major recent new hypotheses such as stem cell plasticity which means that somatic stem cells can regenerate and repair different types of tissues, and that cancer behaves like an organ with its own sustaining cancer stem cells (CSC), have intensified the search for a more practical way of defining stem cell. Stemness markers or genes are badly sought after. ALDH has been known to be highly expressed in hematopoietic stem cells (HSC) for years [28], [29] and it provides protection against alkylating agents in the oxazaphosphorines family, such as cyclophosphamide and its derivatives [9], [30], [31], [32], [33]. The use of ALDH activity as the basis of flow cytometry-based method to sort hematopoietic progenitors has opened the way to study high ALDH activity as a marker for stem cells in different tissues. This method (Aldefluor, Stem Cell Technologies, Inc., Vancouver, BC, Canada) [26] has allowed the isolation of viable progenitors that can now be studied for their functional characteristics in vitro and in vivo [34].

Recent publications and conference presentations have shown the existence of ALDH positive cells in several cancers including multiple myeloma, leukemia, head and neck, and breast [35], [36], [37], [38] which possess some stem cell characteristics and ability to initiate tumors in immunodeficient mice.

In this study we investigate the functional role of ALDH in proliferation and tumor formation using H522 human lung cancer cell line. We demonstrate that cells with high ALDH activity isolated from H522 cell line have distinct phenotypic and functional characteristics compatible with cancer initiating stem or early progenitor cells. On the other hand, low-level ALDH expressing cells endowed with characteristics similar to progenitor cells with limited proliferation and tumor formation. Most of the ALDH activity in this cell line is accounted for by ALDH1A1 and ALDH3A1 expression. These findings raise the question of which ALDH gene is the stemness marker and why. Future studies will need to address these important questions.