**4. FA oxidant hypersensitivity**

44 Cancer Prevention – From Mechanisms to Translational Benefits

number of committed BM progenitors are normal in FA mice as compared to WT mice; however, when subjected to sublethal dose of DNA cross-linking agent mitomycin C (MMC), which does not affect WT mouse cells, to the mutant mice experienced progressive decrease of all peripheral blood parameters, as well as early and committed progenitors, and eventually died within 8 weeks (Chen *et al.,* 1996; Whitney *et al.,* 1996). These results suggest that loss of FA genes in mouse models results in compromised defects in response to environmental insults (Chen *et al.,* 1996; Whitney *et al.,* 1996; Pang *et al.,* 2000; Rathbun *et al.,*

Similar to FA-C patients, BM cells from *Fancc-/-* mice show compromised colony growth capacity following IFN-, TNF- and MIP-1 treatment (Haneline *et al.,* 1998). Literatures suggest that IFN- and TNF- suppress colony growth forming ability of FA mouse BM cells by upregulating other cellular receptors, such as the fas receptor (CD95) (Young *et al.,* 1997). Increase in CD95 expression has been found in CD34+ cells from children with FA as well as the CD34+ fraction of hematopoietic progenitors in *Fancc-/-* mice, which is associated with increased apoptosis (Cumming *et al.,* 1996; Otsuki *et al.,* 1999). The hypersensitivity of *Fancc- /-* hematopoietic cells to IFN- and TNF- is also mediated through activation of the RNAdependent protein kinase (PKR) pathway, which is reported to initiate apoptosis in some instances, as an elevated level of activated PKR was found in *Fancc-/-* mouse embryonic fibroblasts (Pang *et al.,* 2001, 2002; Zhang *et al.,* 2004). Several groups independently showed compromised hematopoietic engraftment and reconstitution after BM transplantation of FA HSCs (Haneline *et al.,* 2003; Zhang *et al.,* 2007). Deregulation of apoptotic responses in hematopoietic cells may account at least in part for the nearly universal development of BM

Inflammation is a biological process orchestrated mainly by myeloid cells and accompanied by infection or phagocytosis (Balwill *et al.,* 2001). Increased oxidative stress in FA patients may be the result of an increased burden of endogenously produced oxidants as well as increased amounts of ROS generated by various inflammatory cytokines. Many studies indicate a correlation between elevated circulating pro-inflammatory cytokines and anemia in patients with leukemia-related BM diseases (Hakim *et al.,* 1993), but direct evidence for the

There is evidence showing that patients with FA have abnormally high levels of TNF- (Fagerlie *et al.,* 2001; Fiers *et al.,* 1999; Freie *et al.,* 2003), which is a major mediator of inflammation and ROS production (Liu *et al.,* 2003; Lohrum *et al.,* 1999). Inappropriate induction or activation of TNF-signaling has been implicated in the pathogenesis of numerous common diseases such as arthritis, heart attacks, and cancer (Ekbom *et al.,* 1990; Jonsson *et al.,* 2005; Mantovani *et al.,* 2002; Marx *et al.,* 2004). It is conceivable that the presence of TNF- and increased oxidative stress in FA BM may account for profound physiologic

Similar to TNF-, IL-1 and IL-6 are also well-known pro-inflammatory cytokines with a wide range of biological activities in immune regulation, hematopoiesis, inflammation and oncogenesis (Ibanez *et al.,* 2009). It has been demonstrated that IL-1 is overexpressed in FA-A patients (de Cremoux *et al.,* 1996). The elevated levels of IL-1*β* were completely reverted

mechanistic link between inflammation and BMF or leukemia is lacking.

changes, including the development of BMF and progression to leukemia.

1997; Haneline *et al.,* 1998; Wong & Buchwald, 2002).

failure in children with inactivating FA mutations.

**3. Inflammation and FA** 

Even in steady state, hematopoietic cells are exposed to various ROS, which are routinely generated during metabolic or inflammatory process. ROS induce a variety of responses in hematopoietic cells, including cellular proliferation and growth inhibition (Howlett *et al.,* 2002; Ichijo *et al.,* 1997). Like cells from other tissues, hematopoietic cells have developed several mechanisms to prevent the damage induced by oxidative stress. First, antioxidant enzymes, including superoxide dismutases (SODs), catalase, glutathione peroxidases and peroxiredoxins, can directly eliminate ROS. Secondly, other cellular enzymes can function to repair DNA damage induced by ROS in hematopoietic tissues. While FA murine models do not recapitulate some of the major FA clinical manifestations such as BM failure and leukemia, hematopoietic cells from FA knockout mice exhibit extreme oxidant sensitivity. Extensive studies have demonstrated FA oxidant hypersensitivity by using primary and immortalized cell cultures as well as *ex vivo* materials from patients (Bogliolo *et al.,* 2002; Cohen–Haguenauer *et al.,* 2006; Cumming *et al.,* 1996; Futaki *et al.,* 2002; Hadjur *et al.,* 2001; Kruyt *et al.,* 1998; Pagano *et al.,* 2005; Park *et al.,* 2004; Saadatzadeh *et al.,* 2004). It has also been shown that reoxygenation-generated oxidative stress, which is associated with significant DNA damage and inhibition of colony formation capacity (Ames *et al.,* 1993; Hammond *et al.,* 2003; Chen *et al.,* 2000), induced senescence of bone marrow progenitor cells from *Fancc-/-* mice compared to their counterparts. While these studies suggest a correlation between oxidative stress and FA disease progression, the mechanism by which oxidative stress influences the function of FA HSC/P cells has not been systematically studied. A number of hypotheses regarding the effect of oxidative stress in FA have been suggested, including the proposal that ROS could damage DNA and inability of FA HSC/P cells to repair such damage would result in exacerbated genomic instability leading to apoptosis and malignant transformation.

Three major FA core complex components, FANCA, FANCC, and FANCG (Bagby *et al.,* 2003; Kennedy *et al.,* 2000; Green *et al.,* 2009), were found to interact with a variety of cellular factors that primarily function in redox-related processes (Table 2), such as FANCC protein interacts with NADPH cytochrome P450 reductase and glutathione S-transferase P1-1 (Cumming *et al.,* 1996; Kruyt *et al.,* 1998), which are involved in either triggering or detoxifying reactive intermediates including ROS. It has also been demonstrated that *Fancc- /-* mice with deficiency in the anti-oxidative enzyme Cu/Zn superoxide dismutase

Inflammatory ROS in Fanconi Anemia Hematopoiesis and Leukemogenesis 47

2005; Park *et al.,* 2004; Saadatzadeh *et al.,* 2004; Schindler *et al.,* 1988; Zhang *et al.,* 2005) and certain FA proteins interact with cellular factors involved in redox metabolism (Aggarwal *et al.,* 2003; Ames *et al.,* 1995; Bagby *et al.,* 2003), the molecular pathways in which FA proteins function to modulate physiologic oxidative stress have not been defined. Our recent identification of the FANCD2-FOXO3a complex (Li *et al.,* 2010) and preliminary characterization of impaired anti-oxidant defense in primary BM cells from FA patients opened new research opportunities to extend the functional study on the roles of FA proteins in the context of oxidative stress. We envision a model (Fig 2) in which the FA proteins regulate oxidative stress response through mechanisms involving functional interplay with the major oxidative stress-responsive transcription factor FOXO3a and protection of anti-oxidant genes from oxidative damage. Loss of these FA protein functions leads to elevated levels of ROS. As a consequence, FA HSC/P cells accumulate excessive DNA damage and increased genomic instability. However, further studies remains to be

Fig. 2. A model for the role of FA proteins in oxidative stress signaling. In WT cells, the FA pathway helps keep cellular levels of ROS in check through functional interaction with the FOXO3a oxidative stress responsive pathway and safeguarding cellular anti-oxidant genes. In FA cells, both the FOXO3a pathway and the anti-oxidant defense are impaired due to loss of the FA protein functions. As a result, FA cells accumulate high levels of ROS, which

Certain chronic inflammatory conditions have long been known to link to cancer. There is compelling evidence that chronic inflammation increases the risk of human cancers such as hepatocellular carcinoma, colon and bladder cancers, B cell lymphomas, and visceral malignancies (Kuper *et al.,* 2000; Mackay *et al.,* 2001; Martin *et al.,* 2011; Suematsu *et al.,* 2003; Umeda *et al.,* 2002; Ziech *et al.,* 2010), probably through the unbalanced machinery between

**6. The FA syndrome links inflammatory ROS to leukemogenesis** 

damages DNA leading to genomic instability.

DNA damage and repair (Fig. 3.).

done in this context.

demonstrated a defective hematopoiesis (Hadjur *et al.,* 2001). *Fancc-/-* cells exhibit hyperactivation of ASK1, a serine-threonine kinase that plays an important role in redox apoptotic signaling (Saadatzadeh *et al.,* 2004). Another FA protein, FANCG, interacts with cytochrome P450 2E1, which is associated with the production of reactive oxygen intermediates, and mitochondrial anti-oxidant enzyme peroxiredoxin-3 (Futaki *et al.,* 2002, Mukhopadhyay *et al.,* 2006), which suggested a possible role of FANCG in protection against oxidative DNA damage. Furthermore, FANCA and FANCG interact upon oxidative stress (Park *et al.,* 2004). These findings indicate a crucial role of FA proteins in oxidative stress signaling. We recently found that FANCD2 associated with FOXO3a, a master regulator in response to oxidative stress (Huang *et al.,* 2007; Li *et al.,* 2010; Tsai *et al.,* 2008). While these observations point to the involvement of FA proteins in oxidative stress response, the molecular pathways in which FA proteins function to modulate physiologic oxidative stress have not been defined.


Table 2. Fanconi anemia proteins in redox signaling.
