Abstract 3821: Fusobacterium Nucleatum and HPV16 cooperate to reprogram the human primary oral keratinocyte to enhanced stemness state, a newly identified pre-malignant cell state
Authors: Akeel Ibrahim , Pal Bidisha , Saikia Partha Jyoti , Pathak Lekhika , Sorra Sandhya , Das Bikul 
Published: September 2020

The mechanism of HPV16 induced malignant transformation of oral epithelial cells is not yet clear. We speculated that HPV16 may induce stem cell altruism that reprogram specific subfraction of the keratinocyte population to enhanced stemness state, a previously reported pre-malignant phenotype in human ES cells by our lab (1). Indeed, using HPV16 E6 transduction method, we demonstrated that CD271+ primary human keratinocytes undergo enhanced stemness switch i.e. low p53 activity, and high expression of HIF-2a, Nanog, sox2 and oct4. Importantly, these cells indicated resistance to calcium and serum induced differentiation, and clonogenic activities and failed to undergo malignant transformation (2). Considering that HPV16 induced carcinogenesis may involve additional steps to transformation, next, we treated CD271+ cells from HPV16 E6 transduced cells with 4-NQO oral carcinogen. We found that 4-NQO tx induced the MYC-HIF-2a stemness pathway (3), which we recently characterized in cancer stem cells (4). However, MYC activity was transient and therefore, the cells did not exhibit reprograming of enhanced stemness to tumor stemness state, the later being identified in cancer stem cells (5). Thus, it seems that additional oncogenes, in addition to MYC may be involved in the reprograming of enhanced stemness to tumor stemness state. Here we evaluate the ability of oral bacteria F. nucleatum (F.N.) to accelerate malignant process and facilitate the enhanced stemness to acquire tumor stemness state. Previously, we showed that F.N. can induce HIF-2a stemness pathway in oral cancer cell line (6), which prompted us to use this model for this present work. Considering that F.N. surface protein Fap2 can modulate E cadherins, we speculated that F.N. might induce E. cadherin-EGFR axis to promote ras oncogene activity in collaboration with HPV16.

Methods: The HPV16 E6 transduced (by using the retroviral LXSN 16E6 system) primary oral keratinocytes were grown for two weeks as previously described (2-3). In these cells. F.N. was added in 1:10 MOI for 12 hours under anerobic condition (5), and then washed and cultured in vitro for 2 weeks. The CD271+ cells were then immunomagnetically sorted and subjected to evaluation for MYC/HIF2 stemness pathway. Because, ABCG2+ cells are endowed with this stemness pathway (1,5), we next sorted the CD271+/ABCG2+ cells.

Results: We found that addition of F.N. led to upregulation of MYC/HIF-2a stemness pathway in the CD271+/ABCG2+ cells. These cells showed rapid proliferation for 2-3 weeks, and resistance to calcium and serum induced terminal differentiation and activated the EGFR/HRas axis. Interestingly, F.N. lysate is sufficient to induce EGFR expression and the activation of MYC/stemness pathway in these E6 transduced cells. Notably silencing of E-cadherin by siRNA led to loss of F.N mediated induction of MYC and Ras, and spontaneous apoptosis of the cells.

Conclusions: These data indicate that oral bacteria like F.N. can cause enhanced stemness switch, a pre-malignant state by activating the EGFR/Ras pathway that activate MYC/stemness pathway.

1. Das B,etal. Stem Cells. 2012

2. AACR Abstract nr 4297

3. AACR Abstract nr 4073

4. Das B et al. Cancer Res. 2019

5. AACR Abstract nr 3064.

Citation Format: Ibrahim Akeel, Bidisha Pal, Partha Jyoti Saikia, Lekhika Pathak, Sandhya Sorra, Bikul Das. Fusobacterium Nucleatum and HPV16 cooperate to reprogram the human primary oral keratinocyte to enhanced stemness state, a newly identified pre-malignant cell state [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3821.