Rong Li¹, Jinsu Huang¹, Meili Ma¹, Yuqing Lou¹, Yanwei Zhang¹, Lixia Wu², David w. Chang³, Picheng Zhao⁴, Qianggang Dong², Xifeng Wu³, Baohui Han¹

¹Department of Pulmonary Medicine,Shanghai Chest Hospital, Shanghai Jiao Tong University, ²Cancer stem cells researchgroup, Shanghai Jiaotong University Cancer Institute, ³Department ofEpidemiology, The University of Texas MD Anderson Cancer Center, ⁴Department of Pathologyand Genomic Medicine, Houston Methodist Hospital Research Institute

Objective:Stem-like cells in solid tumors arepurported to contribute to cancer development and poor treatment outcome. Theabilities to self-renew, differentiate, and resist anticancer therapies arehallmarks of these rare cells, and steering them into lineage commitment may beone strategy to curb cancer development or progression. Vitamin D is aprohormone that can alter cell growth and differentiation and may induce thedifferentiation cancer stem-like cells. Method: In this study, octamer-bindingtranscription factor 4 (OCT4) -positive/Nanog homeobox (Nanog)-positive lungadenocarcinoma stem-like cells (LACSCs) were enriched from spheroid culturedSPC-A1 cells and differentiated by a two-stage induction (TSI) method, whichinvolved knockdown of hypoxia-inducible factor 1-alpha (HIF1α) expression(first stage) followed by sequential induction with 1alpha, 25-dihydroxyvitaminD3 (1,25(OH)2D3, VD3) and suberoylanilide hydroxamic acid (SAHA) treatment(second stage). The tumor cell phenotypes were tested by vitro and vivo tests,including immunofluorescence analysis, colony formation assay, transwellmigration/invasion assay, microculture cytotoxicity assay and xenograft assay. Result:HIF1α-knockdowned cells displayed diminished cell invasion and clonogenicactivities. Moreover, the TSI cells highly expressed tumor suppressor proteinp63 (P63) and forkhead box J1 (FOXJ1) and lost stem cell characteristics, includingabsent expression of OCT4 and Nanog. These cells regained sensitivity tocisplatin in vitro while losing tumorigenic capacity and decreased tumor cellproliferation in vivo. Immunofluorescence analysis showed that HIF1α-KD cellsnegatively expressed stem cell markers and the other cell markers, includingOCT4, Nanog, SP-C, TTF-1, PLAGL2, Sox2, CCSP, FOXJ1 and P63. The HIF1α-KDLACSCs displayed decreased invasion activity compared to LACSCSs. The invasiverate was 22.5±0.3% for LACSCs compared to 3.31±0.1% for HIF1α-KD LACSCs (P<0.0001).Colony formation assay showed that the number of foci of HIF1α-KD LACSC weresignificantly less than that of LACSCs. Immunostaining of HIF1α-KD-VD3/SAHAcells showed P63 and FOXJ1 were positive, while OCT4, Nanog, SP-C, TTF-1,PLAGL2, and CCSP were all negative. Interestingly, Sox2 was reexpressedalthough weakly. LACSCs were highly resistant to cisplatin, while HIF1α-KD-VD3/SAHA induced cells were much more sensitive to cisplatin. The 50%inhibitory concentration (IC50) of cisplatin in HIF1α-KD -VD3/SAHA inducedcells was 0.51±0.03μg/ml, which is significantly lower than the peak plasmaconcentrations of cisplatin (about 4μg/ml) during standard chemotherapeutictreatment. The results for two independent experiments showed that thetumorigenicity rates were both 100% for LACSCs, while it was 0% and 50% forHIF1α-KD-VD3/SAHA induced cells. Therefore, the average tumorigenicity rate forHIF1α-KD-VD3/SAHA induced cells was 25%. Tumor volume of LACSCs and the inducedcells at the 56^th day were 905.2±13.5mm³ vs 0.5±0.04mm³,respectively, P<0.0001. This demonstratesthat the sequential differentiation of LACSCs could significantly reduce theirtumorigenic potential in vivo. Conclusion: Our results suggestthat induced transdifferentiation of LACSCs by vitamin D and SAHA may becomenovel therapeutic avenue to alter tumor cell phenotypes and improve patientoutcome.

Key Words: lung cancer  stem cell vitamin D