In the present work we show that Rac1 is required for adhesion, migration and lung colonization of NSCLA cells. SP cells isolated from human adenocarcinoma cell lines have enriched lung colonization activity in immunodeficient mice. We further determined that this is associated with their elevated Rac1 activity and increased migration and invasion, as well as increased anchorage-independent growth ability. Importantly, the lung colonization, migration, and invasion activities of both SP and non-SP cells can be effectively blocked by either a Rac1 inhibitor or Rac1 knockdown using shRNA, and the effect may be applicable to the CD133+
cell populations. These beneficial effects on tumor cell suppression also appear to apply to primary patient derived NSCLA cells and are irrespective of the p53 or K-ras mutation status (Table S1
). We propose that Rac1 plays a crucial role in regulating CSC tumor initiating, metastatic activities and thus represents a novel and useful therapeutic target in NSCLA.
The discovery of a population of self renewing cancer stem cells in multiple types of cancer including lung cancer has led to the proposal that CSCs, not non-CSCs in a given tumor, are responsible for tumor initiation and possibly metastasis 
. Clinical observations of secondary metastasis post-surgery or -chemotherapy has added to the concept that a residual population of tumor cells may escape conventional therapy and give rise to heterogeneous tumors at metastatic sites. Thus, CSCs are likely the source cells present in the primary tumors which possess unique proliferative and metastatic advantages. To date, CSCs are mostly identified by using various markers. Breast cancer cell lines enriched for CD44+
markers express higher levels of pro-invasive genes and display higher invasive potential 
. In pancreatic cancer, CD133+
cells are shown to be responsible for metastasis 
. In human liver cancer, CD45−
cells form metastatic lesions in the lungs of immune compromised mice, and blockage of CD44 activity by an antagonizing antibody is shown to block tumor growth and metastasis 
. In line with these observations, SP lung cancer cells have been shown to possess enriched CSC activity by forming subcutaneous tumors in xenograft mice at a reduced cell number 
. Here, we found that SP cells isolated from NSCLA cells display increased migration, invasion, homing and lung colonization activities, in addition to enriched tumorigenic capability. These observations are consistent with the notion that CSCs are unique in their metastatic potential as well as tumor initiation ability.
Conventional chemotherapeutic agents mainly target malignant cells by either inducing DNA damage or blocking DNA replication. CSCs may be resistant to the effect of these agents through their elevated drug resistance or relative quiescence 
. To apply the CSC theory, several innovative therapeutic strategies aimed at eradicating CSCs have been developed. To tackle leukemia CSC, neutralizing antibodies to autocrine signaling mediators important for CSC growth such as CD123 
, antagonist for leukemia CSC localization in the bone marrow niche such as CXCR4-inhibitor AMD-3100 and CD44 antibodies 
, and inhibitors of signaling pathways specifically upregulated in CSCs that are important for the self renewal such as NFκ-β inhibitor parthenolide 
, have been shown to have efficacy in AML or CML. In solid cancers, IL4 has been shown to be useful in colorectal cancer stem cell suppression 
, BMP4 was found to induce glioblastoma CSCs to differentiate into non-CSCs 
, and salinomycin, a selective potassium ionophore, could target breast cancer stem cell proliferation and induce differentiation 
. Our current study adds to this list of potential CSC targeting approaches by presenting evidence that Rac1 inhibition could be efficacious for suppressing both tumor initiation and metastasis of NSCLA CSCs.
Effective targeting of CSCs for therapeutic benefit requires accurate identification of the CSC population. In lung cancer, CD133+
, and SP have been used as markers to track CSC activity, and in vitro
anchorage-independent growth, sphere formation assays and subcutaneous xenograft models have been employed as readouts for the relative CSC activity 
. Although each of these markers may help enrich the CSCs, it is clear that they do not accurately identify the tumor initiating cells in human NSCLA tumors, as this population might be quite complex and could be represented by an overlap of several different markers. Thus, using SP or CD133 as the sole marker for CSC identification may not include all CSC activity in a bulk culture. To this end, it will be interesting to determine whether SP cells overlap with CD133+
cells so that a more pure population of CSCs can be isolated.
The issue of CSC plasticity has been raised recently by several studies 
. It seems possible that both CSCs and non-CSCs could be dynamic populations – CSCs able to give rise to non-CSCs, and non-CSCs may convert to CSCs in given conditions. A recent review 
raised an interesting point that the reprogramming from differentiating cancer cells to CSCs, unlike that of reprogramming of fully differentiated cells into iPS cells, might occur readily in cancer cells. Recent evidence from Boiko et al 
and Roesch et al 
have shown that non-CSCs can indeed covert to CSCs under suitable conditions. We have found that highly purified non-SP cells still retain residue CSC activity in vitro
and in mice, raising the possibility that non-CSCs of NSCLA can produce CSCs or SP marker is insufficient in identifying CSCs. This consideration highlights the difficulty of utilizing the CSC theory to design new strategies against cancer, since it can be inferred that it will be necessary to effectively target both CSCs and non-CSCs or multiple marker populations in order to achieve true therapeutic benefits. Importantly, we show that targeting Rac1 can effectively block the lung metastatic and tumor initiating activities of both SP and non-SP NSCLA cells, and such benefits may apply to the CD133+
tumor initiating populations. Future stringent evaluation how Rac1 contributes to various aspects of CSC activity in multiple marker positive subpopulations will significantly add to the understanding of Rac1 targeting in lung tumorigenesis.