Loss of the putative tumor suppressor, Cav-1, is believed to be one of the causes for development of several types of cancers, but evidence also show that overexpression or re-expression of Cav-1 in advanced stages of the disease may contribute to tumor progression. Yet, how loss of Cav-1 facilitates tumorigenesis and how re-induction of Cav-1 promotes tumor progression remain an open question. It has been reported that expression of Cav-1 favors cancer cell proliferation by regulating survival pathways such as Rac, Erk and PtdIns 3-kinase 
and inhibits detachment-induced apoptosis (anoikis) either through suppressing p53 activation 
or up-regulating the transcription of the IGF-I receptor gene 
. We previously demonstrated that Cav-1- regulated calcium homeostasis plays a role in growth and survival of breast cancer cells 
. In the present study, we sought to determine the functional significance of Cav-1 up-regulation caused by treatments with DNA damaging agents, a phenomenon that was also observed by others 
. Our study reveals a new function of Cav-1 as a possible sensor and mediator in the DNA damage response/repair process. We show that expression of Cav-1 can be rapidly up-regulated by DNA damaging agents such as IR (), and that the up-regulation of Cav-1 protein plays a critical role in activating the DNA repair signaling cascade, since depletion of Cav-1 expression by siRNA impairs the cells' ability to repair DNA, as evidenced by increased accumulation of g-H2
AX () and ssDNA (), reduced phosphorylation of ATM at Ser1981
and CHK2 at Thr68
(), and decreased formation of BRCA1 foci (). Moreover, our study reveals for the first time that Cav-1 is able to regulate both HR and NHEJ pathways, two major mechanisms responsible for repair of DNA DSB. This conclusion is supported by use of two assays specific for detecting HR and NHEJ. In the current study, the repair of DSBs induced by the I-SceI endonuclease is monitored using artificial chromosome-integrated reporters, namely HT1080-1885 for HR pathway () and EJ5-GFP for NHEJ pathway (). Each individual reporter is designed such that repair of I-SceI-induced DSBs by a specific pathway restores a puromycin resistance or a GFP expression cassette. In each of the reporter-containing cell lines, the activation of the reporter is confirmed to be dependent upon expression of I-SceI. The restoration of puromycin resistance in HT1080-1885 can only be achieved by HR repair of I-SceI induced DSB using downstream homologue as template. For EJ5-GFP cells, a promoter is separated from a GFP coding cassette by a puro gene that is flanked by two I-SceI sites in the same orientation. Once the puro gene is excised by NHEJ repair of the two I-SceI-induced DSBs, the promoter is joined to the rest of the expression cassette, leading to restoration of the GFP+ gene.
We demonstrated that IR induced the expression of Cav-1 (), a phenomenon previously reported by others 
, (), but we also found that the increased expression of Cav-1 protein by IR does not appear to result from activation of Cav-
1 transcription, as the mRNA level of Cav-1 was not affected by the treatments (). The exact mechanism in which Cav-1 increased after DNA damage remains to be elucidated. Our observation on the roles of Cav-1 in activating DNA repair signaling (, , and ) may explain the pro-survival function of Cav-1 in IR-treated cells, as shown by us () and others 
. Notably, we found that Cav-1 could be up-regulated within 30 min following IR treatment (), earlier than the 24 h shown by Cordes et al 
, suggesting that Cav-1 may act as a sensor and early mediator in response to DNA damage.
In this study we have begun to elucidate the mechanisms by which Cav-1 regulates DNA repair. We demonstrated that Cav-1 participates in both HR and NHEJ repair pathways. The effect of Cav-1 on HR was demonstrated by the experiments showing that silencing of Cav-1 expression decreased HR frequency (). The role of Cav-1 in HR might be related to, at least in part, its effect on the accumulation of BRCA1 foci in nuclei after DNA damage (), which was verified by cell cycle analysis showing that knockdown of Cav-1 did not alter cell cycle distribution, a factor known to affect the foci formation of BRCA1 protein 
. Reciprocal regulation of the expression of Cav-1 and BRCA1 has been reported 
, but whether this is associated with the Cav-1-mediated BRCA1 nuclear accumulation remains to be clarified. The role of Cav-1 in NHEJ was supported by our observation that suppression of Cav-1 by siRNA dramatically inhibited the IR-activated phosphorylation (Ser2056) of DNA-PK (), one of the key executers in the NHEJ system, and by the GFP-based chromosomal reporter assay showing that the frequency of NHEJ was significantly higher in HEK293 cells transfected with a Cav-1 expression vector than in the cells transfected with a control vector (). The mechanism of these effects might involve the Cav-1-mediated nuclear translocation of EGFR, an activator of DNA-PK 
, as Cav-1-targeted siRNA also inhibited the co-translocation of Cav-1 and EGFR following IR treatment (). Therefore, it is likely that the signaling – modulating molecule, Cav-1, may facilitate DNA repair via multiple pathways. How precisely Cav-1 regulates HR and NHEJ and whether Cav-1 is involved in other DNA repair pathways remain to be studied.
Our results may provide a possible explanation for the differential expression of Cav-1 at various stages of tumor progression. As genome instability triggered by endogenous or exogenous DNA damaging agents is one of the main causes of cancer, loss or deficiency of Cav-1 at early stages of cancer development may cause a defect in DNA damage response leading to genomic alteration and oncogenic transformation. However, re-expression of Cav-1 at later stages of cancer may provide a protective mechanism for cancer cells to survive various harsh conditions such as DNA damage. In fact, the protective effects of Cav-1 against mechanical shearing damage, hypoxia, and nutrient depletion, the stresses that are considered the causes for death of tumor cells during their migration and metastasis, have been reported recently 
. Thus, re-expression of Cav-1 at advanced stages of cancer may provide a survival mechanism for tumor cells, and targeting Cav-1 may represent a new stratagem for cancer treatment.
Taken together, our study reveals a novel function for Cav-1 in repairing DNA, which involves both HR and NHEJ, and suggests that Cav-1 may play a critical role in orchestrating the response of cells to DNA damage and in mediating DNA repair.