Bafilomycin A1 triggers proliferative potential of senescent cancer cellsin vitroand in NOD/SCID mice

Abstract

Anticancer therapies that induce DNA damage tend to trigger senescence in cancer cells, a process known as therapy-induced senescence (TIS). Such cells may undergo atypical divisions, thus contributing to tumor re-growth. Accumulation of senescent cancer cells reduces survival of patients after chemotherapy. As senescence interplays with autophagy, a dynamic recycling process, we sought to study whether inhibition of autophagy interferes with divisions of TIS cells. We exposed human colon cancer HCT116 cells to repeated cycles of a chemotherapeutic agentdoxorubicin (doxo) and demonstrated induction of hallmarks of TIS (e.g. growth arrest, hypertrophy, poliploidization and secretory phenotype) and certain properties of cancer stem cells (increased NANOG expression, percentages of CD24+ cells and side population). Colonies of small and highly proliferative progeny appeared shortly after drug removal. Treatment with bafilomycin A1 (BAF A1), an autophagy inhibitor, postponed short term in vitro cell re-population. It was associated with reduction in the number of diploid and increase in the number of poliploid cells. In a long term, a pulse of BAF A1 resulted in reactivation of autophagy in a subpopulation of HCT116 cells and increased proliferation. Accordingly, the senescent HCT116 cells treated with BAF A1 when injected into NOD/SCID mice formed tumors, in contrast to the controls.Our results suggest that senescent cancer cells that appear during therapy, can be considered as dormant cells that contribute to cancer re-growth, when chemotherapeutic treatment is stopped. These data unveil new mechanisms of TIS-related cancer maintenance and re-population, triggered by a single pulse of BAF A1 treatment.

Some chemotherapeutics-treated colon cancer cells display a specific phenotype being a combination of stem-like and senescent cell features

“…All image processing operations were implemented using CellProfiler 2.1. as previously described. 25…”

“…Data for 30 000 cells were analyzed using the BD LSRFortessa flow cytometer and BD FACSDiva 6.2 software as previously described. 25 Flow cytometry analysis of cancer stem cell markers Cells labeled with H33342 for SP were probed with specific fluorochrome-conjugated antibodies. Briefly, 1£ 10 5 cells were incubated with antibodies: APC-conjugated anti-CD133, FITCconjugated anti-CD24 and AlexaFluor 700-conjugated anti-CD44 antibodies for 30 minutes on ice.…”

“…Flow cytometry was performed using a BD LSRForessa instrument and BD FACSDiva 6.2 software as previously described. 25…”

“…In our previous studies we found that human colon cancer HCT116 cells treated with doxorubicin (DOXO) undergo senescence in cell cultures, but after withdrawal of the drug re-growth is observed. 25,33,34 However, DOXO is not used in therapy of colon cancer patients. Thus, here we tested the effects of the clinically relevant chemotherapeutics: 5-fluorouracil (5-FU), oxaliplatin (OXA) and irinotecan (IRINO).…”

“…20 Moreover, some studies demonstrated that senescent cells may display a stem cell features. [21][22][23][24][25][26][27] The cancer stem cell (CSC) model of cancer origin suggests that only a small subset of cancer cells is responsible for sustaining tumorigenesis. CSCs exhibit the stem cell properties of self-renewal and an ability to differentiate into various lineages.…”