Accumulating evidence suggests that diverse solid tumors are hierarchically organized and sustained by a distinct subpopulation of tumor-initiating cells or cancer stem cells (CSCs), which share functional and phenotypic similarity with normal somatic stem cells (SSCs)1. The CSC hypothesis not only presents an attractive mechanism underlying the therapeutic refractoriness and dormant behavior exhibited by many human cancers, but also provides a rationale for novel therapeutic strategies beyond traditional anti-proliferative agents1.

With support from the Cancer Research Foundation, we investigated molecular mechanisms controlling stemness of CSCs. To this end, we used hair follicle stem cells as a model system. In mammalian skin, hair follicles continuously cycle through different stages of anagen (active growth), catagen (regression phase), and telogen (rest phase), and follicle stem cell lineage (bulge stem cell) is temporally and spatially chronicled2. Hair follicle also serves as an important model for mammalian carcinogenesis, as aberrant regulation of hair follicle stem cells leads to skin squamous cell carcinoma (SQCC), which is the most dangerous non-melanoma skin cancer, afflicting more than 700,000 patients every year in the United States alone3. Stem cells in SQCC have been identified4, and their self-renewal requires Wnt signaling4, which plays a fundamental role in maintenance and lineage -commitment of hair follicle stem cells as well5.

During the last funding period, we focused on examining the central hypothesis that stemness of skin CSCs is regulated by cytoskeletal dynamics and cytoskeletal coordination mediated by mammalian spectraplakin protein provides an intriguing target for treatment of skin malignancy. Skin cells develop an elaborate cytoskeletal system that is dynamically regulated in response to environmental stimuli. However, despite its potential importance, little is known about the function of cytoskeletal coordination in skin SSCs or CSCs. My previous studies identified MACF1 (microtubule actin crosslinking factor 1)/ACF7 (actin crosslinking factor 7) as an important cytoskeletal crosslinker with a novel ATPase activity in skin SSCs6,7. Specifically, as outlined below, we studied the role of ACF7 in skin tumorigenesis and examined the possibility that inhibition of ACF7 ATPase activity can restrain malignant self-renewal of CSCs.

1. We used Cre/LoxP technology to conditionally ablate expression of ACF7 in skin epidermis. With this genetic model, we examined skin carcinogenesis upon loss of ACF7 by two-step carcinogenesis protocol.

2. With fluorescence activated sorting, we isolated both skin SSC and CSCs from WT and ACF7-cKO animals. We systematically analyzed the consequence of ACF7 deletion on cell cycle progression, cell death, stem cell self-renewal, and differentiation. Our results revealed the potential molecular mechanisms underlying skin CSC stemness and skin carcinogenesis.

3. Taking advantage of skin epidermal system, we performed grafting experiments with genetically altered skin CSCs. We followed and studied secondary tumorigenesis with grafted cells. Our results provided conclusive evidence on the molecular role of ACF7 in skin carcinogenesis.

4. We tested the hypothesis that ACF7 can regulate Wnt signal transduction in skin stem cells. We further tested the potential mechanism whereby ACF7 control Wnt pathway through its role in cytoskeletal coordination and intracellular membrane trafficking.

5. ACF7 harbors a unique ATPase activity, making it an appealing drug target for skin disorders. By mutagenesis approaches, we tested the role of the ATPase activity in skin stem cell activities and skin tumorigenesis.

6. To develop potential chemical inhibitor of ACF7’s ATPase activity, we collaborated with Dr. Kozmin at Department of Chemistry, University of Chicago. We established a robust platform to examine the ATPase activity in vitro, and tested the possibility to carry out a large scale screen of chemical library.

Increasing evidence now suggests an important role of the cytoskeleton and motor proteins in transmitting the niche signaling to regulate stem cell functions8. However, the underlying molecular mechanism remains poorly understood. Our results uncover an essential role of ACF7, a cytoskeletal crosslinking protein with ATPase activity, in stem cell self-renewal and skin malignancy. With support from CRF, my research on ACF7 has greatly expanded our understanding of the elaborate and critical machinery underlying human SSC and CSC functions from a unique perspective. In addition, ACF7 serves as a promising drug target, and our studies utilizing cutting-edge approaches in chemical biology may lead to the development of novel therapeutics against different human cancers.

With all the promising results generated in the last funding period, we are now preparing a proposal for NIH R01 grant. Skin carcinogenesis shares significant similarity with cance rs in other tissues. Thus, the knowledge that we gained by studying ACF7 function in skin may apply to other systems. Indeed, with some of the results that we obtained last year, we have submitted a proposal entitled “Spectraplakin and coordinated cytoskeletal dynamics in colorectal cancer” to the V Foundation for Cancer Research. This proposal has recently been awarded.

Accumulating evidence suggest that diverse solid tumors are hierarchically organized and sustained by a distinct subpopulation of tumor-initiating cells or cancer stem cells (CSCs), which share functional and phenotypic similarity with normal somatic stem cells (SSCs)1 . The CSC hypothesis not only presents an attractive mechanism underlying the therapeutic refractoriness and dormant behavior exhibited by many human cancers, but also provides a rationale for novel therapeutic strategies beyond traditional anti-proliferative agents1 . With support from CRF (cancer research foundation), we investigated molecular mechanisms controlling stemness of CSCs. To this end, we used hair follicle stem cells as a model system. In mammalian skin, hair follicles continuously cycle through different stages of anagen (active growth), catagen (regression phase), and telogen (rest phase), and follicle stem cell lineage (bulge stem cell) is temporally and spatially chronicled2 . Hair follicle also serves as an important model for mammalian carcinogenesis, as aberrant regulation of hair follicle stem cells leads to skin squamous cell carcinoma (SQCC), which is the most dangerous non-melanoma skin cancer, afflicting more than 700,000 patients every year in the United States alone3 . Stem cells in SQCC have been identified4 , and their self-renewal requires Wnt signaling4 , which plays a fundamental role in maintenance and lineage -commitment of hair follicle stem cells as well5 . With CRF funding, we focused on examining the central hypothesis that stemness of skin CSCs is regulated by cytoskeletal dynamics and cytoskeletal coordination mediated by mammalian spectraplakin protein provides an intriguing target for treatment of skin malignancy. Skin cells develop an elaborate cytoskeletal system that is dynamically regulated in response to environmental stimuli. However, despite its potential importance, little is known about the function of cytoskeletal coordination in skin SSCs or CSCs. My previous studies identified MACF1 (microtubule actin crosslinking factor 1)/ACF7 (actin crosslinking factor 7) as an important cytoskeletal crosslinker with a novel ATPase activity in skin SSCs6,7 . Specifically, as outlined below, we studied the role of ACF7 in skin tumorigenesis and examined the possibility that inhibition of ACF7 ATPase activity can restrain malignant self-renewal of CSCs.

1. We used Cre/LoxP technology to conditionally ablate expression of ACF7 in skin epidermis. With this genetic model, we examined skin carcinogenesis upon loss of ACF7 by two-step carcinogenesis protocol.

2. With fluorescence activated sorting, we isolated both skin SSC and CSCs from WT and ACF7-cKO animals. We systematically analyzed the consequence of ACF7 deletion on cell cycle progression, cell death, stem cell self-renewal, and differentiation. Our results revealed the potential molecular mechanisms underlying skin CSC stemness and skin carcinogenesis.

3. Taking advantage of skin epidermal system, we performed grafting experiments with genetically altered skin CSCs. We followed and studied secondary tumorigenesis with grafted cells. Our results provided conclusive evidence on the molecular role of ACF7 in skin carcinogenesis.

4. We tested the hypothesis that ACF7 can regulate Wnt signal transduction in skin stem cells. We further tested the potential mechanism whereby ACF7 control Wnt pathway through its role in cytoskeletal coordination and intracellular membrane trafficking.

5. ACF7 harbors a unique ATPase activity, making it an appealing drug target for skin disorders. By mutagenesis approaches, we tested the role of the ATPase activity in skin stem cell activities and skin tumorigenesis.

6. To develop potential chemical inhibitor of ACF7’s ATPase activity, we collaborated with Dr. Kozmin at Department of Chemistry, University of Chicago. We established a robust platform to examine the ATPase activity in vitro, and tested the possibility to carry out a large scale screen of chemical library.

Increasing evidence now suggests an important role of the cytoskeleton and motor proteins in transmitting the niche signaling to regulate stem cell functions8 . However, the underlying molecular mechanism remains poorly understood. Our results uncover an essential role of ACF7, a cytoskeletal crosslinking protein with ATPase activity, in stem cell self-renewal and skin malignancy. With support from CRF, my research on ACF7 has greatly expanded our understanding of the elaborate and critical machinery underlying human SSC and CSC functions from a unique perspective. In addition, ACF7 serves as a promising drug target, and our studies utilizing cutting-edge approaches in chemical biology may lead to the development of novel therapeutics against different human cancers.With all the promising results, we have submitted multiple proposals to national funding agencies. Our proposal to NIH R01, ACS research scholar grant, and V foundation grant have been awarded.

References: 1. Zhou, B.B. et al. Tumour-initiating cells: challenges and opportunities for anticancer drug discovery. Nat Rev Drug Discov 8, 806-23 (2009). 2. Fuchs, E. The tortoise and the hair: slow-cycling cells in the stem cell race. Cell 137, 811-9 (2009). 3. Euvrard, S., Kanitakis, J. & Claudy, A. Skin cancers after organ transplantation. N Engl J Med 348, 1681-91 (2003). 4. Malanchi, I. et al. Cutaneous cancer stem cell maintenance is dependent on beta-catenin signalling. Nature 452, 650-3 (2008). 5. Moon, R.T., Kohn, A.D., De Ferrari, G.V. & Kaykas, A. WNT and beta-catenin signalling: diseases and therapies. Nat Rev Genet 5, 691-701 (2004). 6. Wu, X. et al. Skin stem cells orchestrate directional migration by regulating microtubule-ACF7 connections through GSK3beta. Cell 144, 341-52 (2011). 7. Wu, X., Kodama, A. & Fuchs, E. ACF7 regulates cytoskeletal-focal adhesion dynamics and migration and has ATPase activity. Cell 135, 137-48 (2008). 8. McBeath, R., Pirone, D.M., Nelson, C.M., Bhadriraju, K. & Chen, C.S. Cell shape, cytoskeletal tension, and RhoA regulate stem cell lineage commitment. Dev Cell 6, 483-95 (2004).