Polyploid/Multinucleated Giant and Slow-Cycling Cancer Cell Enrichment in Response to X-ray Irradiation of Human Glioblastoma Multiforme Cells Differing in Radioresistance and TP53/PTEN Status.

Radioresistance compromises the efficacy of radiotherapy for glioblastoma multiforme (GBM), the most devastating and common brain tumor. The present study investigated the relationship between radiation tolerance and formation of polyploid/multinucleated giant (PGCC/MGCC) and quiescent/senescent slow-cycling cancer cells in human U-87, LN-229, and U-251 cell lines differing in TP53/PTEN status and radioresistance. We found significant enrichment in MGCC populations of U-87 and LN-229 cell lines, and generation of numerous small mononuclear (called Raju cells, or RJ cells) U-87-derived cells that eventually form cell colonies, in a process termed neosis, in response to X-ray irradiation (IR) at single acute therapeutic doses of 2-6 Gy. For the first time, single-cell high-content imaging and analysis of Ki-67- and EdU-coupled fluorescence demonstrated that the IR exposure dose-dependently augments two distinct GBM cell populations. Bifurcation of Ki-67 staining suggests fast-cycling and slow-cycling populations with a normal-sized nuclear area, and with an enlarged nuclear area, including one resembling the size of PGCC/MGCCs, that likely underlie the highest radioresistance and propensity for repopulation of U-87 cells. Proliferative activity and anchorage-independent survival of GBM cell lines seem to be related to neosis, low level of apoptosis, fraction of prematurely stress-induced senescent MGCCs, and the expression of p63 and p73, members of p53 family transcription factors, but not to the mutant p53. Collectively, our data support the importance of the TP53wt/PTENmut genotype for the maintenance of cycling radioresistant U-87 cells to produce a significant amount of senescent MGCCs as an IR stress-induced adaptation response to therapeutic irradiation doses.

Autophagy modulating therapeutics inhibit ovarian cancer colony generation by polyploid giant cancer cells (PGCCs).

BACKGROUND: Genomic instability and chemoresistance can arise in cancer due to a unique form of plasticity: that of polyploid giant cancer cells (PGCCs). These cells form under the stress of chemotherapy and have higher than diploid chromosome content. PGCCs are able to then repopulate tumors through an asymmetric daughter cell budding process. PGCCs have been observed in ovarian cancer histology, including the deadly and common form high-grade serous ovarian carcinoma (HGSC). We previously discovered that drugs which disrupt the cellular recycling process of autophagy are uniquely efficacious in pre-clinical HGSC models. While autophagy induction has been associated with PGCCs, it has never been previously investigated if autophagy modulation interacts with the PGCC life cycle and this form of tumor cell plasticity. METHODS: CAOV3 and OVCAR3 ovarian cancer cell lines were treated with carboplatin or docetaxel to induce PGCC formation. Microscopy was used to characterize and quantify PGCCs formed by chemotherapy. Two clinically available drugs that inhibit autophagy, hydroxychloroquine and nelfinavir, and a clinically available activator of autophagy, rapamycin, were employed to test the effect of these autophagy modulators on PGCC induction and subsequent colony formation from PGCCs. Crystal violet-stained colony formation assays were used to quantify the tumor-repopulating stage of the PGCC life cycle. RESULTS: Autophagy inhibitors did not prevent PGCC formation in OVCAR3 or CAOV3 cells. Rapamycin did not induce PGCC formation on its own nor did it exacerbate PGCC formation by chemotherapy. However, hydroxychloroquine prevented efficient colony formation in CAOV3 PGCCs induced by carboplatin (27% inhibition) or docetaxel (41% inhibition), as well as in OVCAR3 cells (95% and 77%, respectively). Nelfinavir similarly prevented colony formation in CAOV3 PGCCs induced by carboplatin (64% inhibition) or docetaxel (94% inhibition) as well as in OVCAR3 cells (89% and 80%, respectively). Rapamycin surprisingly also prevented PGCC colony outgrowth (52-84% inhibition). CONCLUSIONS: While the autophagy previously observed to correlate with PGCC formation is unlikely necessary for PGCCs to form, autophagy modulating drugs severely impair the ability of HGSC PGCCs to form colonies. Clinical trials which utilize hydroxychloroquine, nelfinavir, and/or rapamycin after chemotherapy may be of future interest.

Amitotic Cell Division, Malignancy, and Resistance to Anticancer Agents: A Tribute to Drs. Walen and Rajaraman.

Cell division is crucial for the survival of living organisms. Human cells undergo three types of cell division: mitosis, meiosis, and amitosis. The former two types occur in somatic cells and germ cells, respectively. Amitosis involves nuclear budding and occurs in cells that exhibit abnormal nuclear morphology (e.g., polyploidy) with increased cell size. In the early 2000s, Kirsten Walen and Rengaswami Rajaraman and his associates independently reported that polyploid human cells are capable of producing progeny via amitotic cell division, and that a subset of emerging daughter cells proliferate rapidly, exhibit stem cell-like properties, and can contribute to tumorigenesis. Polyploid cells that arise in solid tumors/tumor-derived cell lines are referred to as polyploid giant cancer cells (PGCCs) and are known to contribute to therapy resistance and disease recurrence following anticancer treatment. This commentary provides an update on some of these intriguing discoveries as a tribute to Drs. Walen and Rajaraman.

Cancer cell repopulation after therapy: which is the mechanism?

Cancer cell repopulation after therapy is a phenomenon that leads to therapeutic failure with the consequent relapse of the disease. The process is understudied and mechanisms need to be uncovered. Here we discuss the issue of cancer cell repopulation after chemo- and radio-therapies. We compile evidence alleging that the repopulation of cancer cells can be originated from either cancer stem cells resistant to therapy, cancer cells that in response to therapy become polyploid and thereafter germinate into near-diploid rapid proliferating cells, and/or cells that respond to treatment undergoing senescence as a transient mechanism to survive, followed by the reinitiation of the cell cycle. Approaches targeted to prevent this post-therapy cancer cell repopulation should be uncovered to prevent tumor relapse and thus increase overall survival from this devastating disease.

Stress-Induced Polyploid Giant Cancer Cells: Unique Way of Formation and Non-Negligible Characteristics.

Polyploidy is a conserved mechanism in cell development and stress responses. Multiple stresses of treatment, including radiation and chemotherapy drugs, can induce the polyploidization of tumor cells. Through endoreplication or cell fusion, diploid tumor cells convert into giant tumor cells with single large nuclei or multiple small nucleuses. Some of the stress-induced colossal cells, which were previously thought to be senescent and have no ability to proliferate, can escape the fate of death by a special way. They can remain alive at least before producing progeny cells through asymmetric cell division, a depolyploidization way named neosis. Those large and danger cells are recognized as polyploid giant cancer cells (PGCCs). Such cells are under suspicion of being highly related to tumor recurrence and metastasis after treatment and can bring new targets for cancer therapy. However, differences in formation mechanisms between PGCCs and well-accepted polyploid cancer cells are largely unknown. In this review, the methods used in different studies to induce polyploid cells are summarized, and several mechanisms of polyploidization are demonstrated. Besides, we discuss some characteristics related to the poor prognosis caused by PGCCs in order to provide readers with a more comprehensive understanding of these huge cells.

Irradiation-induced polyploid giant cancer cells are involved in tumor cell repopulation via neosis.

Tumor repopulation occurs when residual tumor cells surviving therapies tenaciously proliferate and re-establish the tumor. The cellular and molecular mechanisms underlying this process remain poorly understood. In this study, we propose that polyploid giant cancer cells (PGCCs) are involved in tumor repopulation via neosis following radiotherapy. We found that although the majority of PGCCs induced by irradiation underwent cell death, some PGCCs exhibited proliferative capacity. Utilizing time-lapse microscopy and single-cell cloning assays, we observed that proliferating PGCCs underwent neosis, thereby contributing to tumor cell repopulation after irradiation. Notably, HMGB1 released from dying tumor cells rather than intracellular HMGB1 could promote neosis-based tumor repopulation, and the latter could be suppressed by the use of HMGB1 inhibitors. Taken together, our results indicate that PGCC can initiate tumor repopulation via neosis following radiation therapy.

Giants and monsters: Unexpected characters in the story of cancer recurrence.

Polyploid giant cancer cells (PGCC) constitute a dangerous subpopulation of cancer cells and are a driving force in cancer recurrence. These unique cells arise from diploid tumor cells in response to stress encountered in the tumor microenvironment or during cancer therapy. PGCC are greatly dedifferentiated, acquire pluripotency, and are able to replicate through a form of asymmetric division called neosis, which results in new populations that are themselves able to differentiate into new cell types or to re-establish tumors. Progeny tend to be more genetically unstable than the founding population due to the dysregulation required to transition through a PGCC state. Therefore, cancers that escape stressors through this mechanism tend to re-emerge with a more aggressive phenotype that is therapy resistant. This review focuses on the clinical significance of PGCC, the need for standardized nomenclature and molecular markers, as well as possible avenues to develop therapies aimed at PGCC and the process of neosis. The biology underlying the development of PGCC including cell cycle checkpoint dysregulation, stress responses, dedifferentiation, stemness and epithelial-mesenchymal transition is discussed.