Mathematical Modeling of the Role of Survivin on Dedifferentiation and Radioresistance in Cancer.

We use a mathematical model to investigate cancer resistance to radiation, based on dedifferentiation of non-stem cancer cells into cancer stem cells. Experimental studies by Iwasa 2008, using human non-small cell lung cancer (NSCLC) cell lines in mice, have implicated the inhibitor of apoptosis protein survivin in cancer resistance to radiation. A marked increase in radio-sensitivity was observed, after inhibiting survivin expression with a specific survivin inhibitor YM155 (sepantronium bromide). It was suggested that these observations are due to survivin-dependent dedifferentiation of non-stem cancer cells into cancer stem cells. Here, we confirm this hypothesis with a mathematical model, which we fit to Iwasa's data on NSCLC in mice. We investigate the timing of combination therapies of YM155 administration and radiation. We find an interesting dichotomy. Sometimes it is best to hit a cancer with a large radiation dose right at the beginning of the YM155 treatment, while in other cases, it appears advantageous to wait a few days until most cancer cells are sensitized and then radiate. The optimal strategy depends on the nature of the cancer and the dose of radiation administered.

Low-dose γ-radiation inhibits IL-1ß-induced dedifferentiation and inflammation of articular chondrocytes via blockage of catenin signaling.

Although low-dose radiation (LDR) regulates a wide range of biological processes, limited information is available on the effects of LDR on the chondrocyte phenotype. Here, we found that LDR, at doses of 0.5-2 centiGray (cGy), inhibited interleukin (IL)-1ß-induced chondrocyte destruction without causing side effects, such as cell death and senescence. IL-1ß treatment induced an increase in the expression of α-, ß-, and γ-catenin proteins in chondrocytes via Akt signaling, thereby promoting dedifferentiation through catenin-dependent suppression of Sox-9 transcription factor expression and induction of inflammation through activation of the NF-κB pathway. Notably, LDR blocked cartilage disorders by inhibiting IL-1ß-induced catenin signaling and subsequent catenin-dependent suppression of the Sox-9 pathway and activation of the NF-κB pathway, without directly altering catenin expression. LDR also inhibited chondrocyte destruction through the catenin pathway induced by epidermal growth factor, phorbol 12-myristate 13-acetate, and retinoic acid. Collectively, these results identify the molecular mechanisms by which LDR suppresses pathophysiological processes and establish LDR as a potentially valuable therapeutic tool for patients with cytokine- or soluble factors-mediated cartilage disorders.

Technical brief: Constant intense light exposure to lesion and initiate regeneration in normally pigmented zebrafish.

Zebrafish are capable of robust and spontaneous regeneration of injured retina. Constant intense light exposure to adult albino zebrafish specifically causes apoptosis of rod and cone photoreceptor cells and is an excellent model to study the molecular mechanisms underlying photoreceptor regeneration. However, this paradigm has only been applied to lesion zebrafish of the nonpigmented albino genetic background, which precludes the use of numerous transgenic reporter lines that are widely used to study regeneration. Here, we explored the effectiveness of constant intense light exposure in causing photoreceptor apoptosis and stimulating regeneration in normally pigmented zebrafish retinas. We show that constant intense light exposure causes widespread photoreceptor damage in the dorsal-central retinas of pigmented zebrafish. Photoreceptor loss triggers dedifferentiation and proliferation of Müller glia as well as progenitor cell proliferation. We also demonstrate that the timeline of regeneration response is comparable between the albino and the pigmented retinas.

Radiation-induced reprogramming of breast cancer cells.

Breast cancers are thought to be organized hierarchically with a small number of breast cancer stem cells (BCSCs) able to regrow a tumor while their progeny lack this ability. Recently, several groups reported enrichment for BCSCs when breast cancers were subjected to classic anticancer treatment. However, the underlying mechanisms leading to this enrichment are incompletely understood. Using non-BCSCs sorted from patient samples, we found that ionizing radiation reprogrammed differentiated breast cancer cells into induced BCSCs (iBCSCs). iBCSCs showed increased mammosphere formation, increased tumorigenicity, and expressed the same stemness-related genes as BCSCs from nonirradiated samples. Reprogramming occurred in a polyploid subpopulation of cells, coincided with re-expression of the transcription factors Oct4, sex determining region Y-box 2, Nanog, and Klf4, and could be partially prevented by Notch inhibition. We conclude that radiation may induce a BCSC phenotype in differentiated breast cancer cells and that this mechanism contributes to increased BCSC numbers seen after classic anticancer treatment.

Astrocytes reverted to a neural progenitor-like state with transforming growth factor alpha are sensitized to cancerous transformation.

Gliomas, the most frequent primitive central nervous system tumors, have been suggested to originate from astrocytes or from neural progenitors/stem cells. However, the precise identity of the cells at the origin of gliomas remains a matter of debate because no pre-neoplastic state has been yet identified. Transforming growth factor (TGF)-alpha, an epidermal growth factor family member, is frequently overexpressed in the early stages of glioma progression. We previously demonstrated that prolonged exposure of astrocytes to TGF-alpha is sufficient to trigger their reversion to a neural progenitor-like state. To determine whether TGF-alpha dedifferentiating effects are associated with cancerous transforming effects, we grafted intracerebrally dedifferentiated astrocytes. We show that these cells had the same cytogenomic profile as astrocytes, survived in vivo, and did not give birth to tumors. When astrocytes dedifferentiated with TGF-alpha were submitted to oncogenic stress using gamma irradiation, they acquired cancerous properties: they were immortalized, showed cytogenomic abnormalities, and formed high-grade glioma-like tumors after brain grafting. In contrast, irradiation did not modify the lifespan of astrocytes cultivated in serum-free medium. Addition of TGF-alpha after irradiation did not promote their transformation but decreased their lifespan. These results demonstrate that reversion of mature astrocytes to an embryonic state without genomic manipulation is sufficient to sensitize them to oncogenic stress.

Mebendazole Potentiates Radiation Therapy in Triple-Negative Breast Cancer.

PURPOSE: The lack of a molecular target in triple-negative breast cancer (TNBC) makes it one of the most challenging breast cancers to treat. Radiation therapy (RT) is an important treatment modality for managing breast cancer; however, we previously showed that RT can also reprogram a fraction of the surviving breast cancer cells into breast cancer-initiating cells (BCICs), which are thought to contribute to disease recurrence. In this study, we characterize mebendazole (MBZ) as a drug with potential to prevent the occurrence of radiation-induced reprogramming and improve the effect of RT in patients with TNBC. METHODS AND MATERIALS: A high-throughput screen was used to identify drugs that prevented radiation-induced conversion of TNBC cells into cells with a cancer-initiating phenotype and exhibited significant toxicity toward TNBC cells. MBZ was one of the drug hits that fulfilled these criteria. In additional studies, we used BCIC markers and mammosphere-forming assays to investigate the effect of MBZ on the BCIC population. Staining with propidium iodide, annexin-V, and γ-H2AX was used to determine the effect of MBZ on cell cycle, apoptosis, and double-strand breaks. Finally, the potential for MBZ to enhance the effect of RT in TNBC was evaluated in vitro and in vivo. RESULTS: MBZ efficiently depletes the BCIC pool and prevents the ionizing radiation-induced conversion of breast cancer cells into therapy-resistant BCICs. In addition, MBZ arrests cells in the G2/M phase of the cell cycle and causes double-strand breaks and apoptosis. MBZ sensitizes TNBC cells to ionizing radiation in vitro and in vivo, resulting in improved tumor control in a human xenograft model of TNBC. CONCLUSIONS: The data presented in this study support the repurposing of MBZ as a combination treatment with RT in patients with TNBC.

Dedifferentiation process driven by radiotherapy-induced HMGB1/TLR2/YAP/HIF-1α signaling enhances pancreatic cancer stemness.

Differentiated cancer cells reacquiring stem cell traits following radiotherapy may enrich cancer stem cells and accelerate tumor recurrence and metastasis. We are interested in the mechanistic role of dying cells-derived HMGB1 in CD133- pancreatic cancer cells dedifferentiation following radiotherapy. We firstly confirmed that X-ray irradiation induced differentiation of CD133- pancreatic cancer cells, from either sorted from patient samples or established cell lines, into cancer stem-like cells (iCSCs). Using an in vitro coculture model, X-ray irradiation induced dying cells to release HMGB1, which further promoted CD133- pancreatic cancer cells regaining stem cell traits, such as higher sphere forming ability and expressed higher level of stemness-related genes and proteins. Inhibiting the expression and activity of HMGB1 attenuated the dedifferentiation stimulating effect of irradiated, dying cells on C133- pancreatic cancer cells in vitro and in PDX models. Mechanistically, HMGB1 binding with TLR2 receptor functions in a paracrine manner to affect CD133- pancreatic cancer cells dedifferentiation via activating Hippo-YAP pathway and HIF-1α expression in oxygen independent manner in vitro and in vivo. We conclude that X-ray irradiation induces CD133- pancreatic cancer cell dedifferentiation into a CSC phenotype, and inhibiting HMGB1 may be a strategy to prevent CSC enrichment and further pancreatic carcinoma relapse.

Essential role of METTL3-mediated m6A modification in glioma stem-like cells maintenance and radioresistance.

Despite advances in biology and therapeutic modalities, existence of highly tumorigenic glioma stem-like cells (GSCs) makes glioblastomas (GBMs) invincible. N6-methyl adenosine (m6A), one of the abundant mRNA modifications catalyzed by methyltransferase-like 3 and 14 (METTL3/14), influences various events in RNA metabolism. Here, we report the crucial role of METTL3-mediated m6A modification in GSC (neurosphere) maintenance and dedifferentiation of glioma cells. METTL3 expression is elevated in GSC and attenuated during differentiation. RNA immunoprecipitation studies identified SOX2 as a bonafide m6A target of METTL3 and the m6A modification of SOX2 mRNA by METTL3 enhanced its stability. The exogenous overexpression of 3'UTR-less SOX2 significantly alleviated the inhibition of neurosphere formation observed in METTL3 silenced GSCs. METTL3 binding and m6A modification in vivo required intact three METTL3/m6A sites present in the SOX2-3'UTR. Further, we found that the recruitment of Human antigen R (HuR) to m6A-modified RNA is essential for SOX2 mRNA stabilization by METTL3. In addition, we found a preferential binding by HuR to the m6A-modified transcripts globally. METTL3 silenced GSCs showed enhanced sensitivity to γ-irradiation and reduced DNA repair as evidenced from the accumulation of γ-H2AX. Exogenous overexpression of 3'UTR-less SOX2 in METTL3 silenced GSCs showed efficient DNA repair and also resulted in the significant rescue of neurosphere formation from METTL3 silencing induced radiosensitivity. Silencing METTL3 inhibited RasV12 mediated transformation of mouse immortalized astrocytes. GBM tumors have elevated levels of METTL3 transcripts and silencing METTL3 in U87/TIC inhibited tumor growth in an intracranial orthotopic mouse model with prolonged mice survival. METTL3 transcript levels predicted poor survival in GBMs which are enriched for GSC-specific signature. Thus our study reports the importance of m6A modification in GSCs and uncovers METTL3 as a potential molecular target in GBM therapy.

Chordoma dedifferentiation after proton beam therapy: a case report and review of the literature.

BACKGROUND: Chordoma is a rare invasive bone tumor that may occur anywhere along the neuraxis. A total of three primary histological varieties have been identified: conventional, chondroid, and dedifferentiated. CASE PRESENTATION: We report a case of an 8-year-old white girl who presented with conventional chordoma, was treated with surgical resection and mixed proton and photon beam therapy, and had a recurrence in the resection cavity 2.5 years later with dedifferentiated morphology. The recurrent tumor did not express brachyury, a recently identified protein specific to tissue of notochordal origin. CONCLUSIONS: The short time period between radiation therapy and dedifferentiation, low dose of photons, and rarity of dedifferentiated skull base chordomas in pediatric patients should alert clinicians to the possibility of chordoma dedifferentiation after proton beam therapy.

Radiation-Induced Dedifferentiation of Head and Neck Cancer Cells Into Cancer Stem Cells Depends on Human Papillomavirus Status.

PURPOSE: To test the hypothesis that the radiation response of cancer stem cells (CSCs) in human papillomavirus (HPV)-positive and HPV-negative head and neck squamous cell carcinoma (HNSCC) differs and is not reflected in the radiation response of the bulk tumor populations, that radiation therapy (RT) can dedifferentiate non-stem HNSCC cells into CSCs, and that radiation-induced dedifferentiation depends on the HPV status. METHODS AND MATERIALS: Records of a cohort of 162 HNSCC patients were reviewed, and their outcomes were correlated with their HPV status. Using a panel of HPV-positive and HPV-negative HNSCC cell lines expressing a reporter for CSCs, we characterized HPV-positive and HPV-negative lines via flow cytometry, sphere-forming capacity assays in vitro, and limiting dilution assays in vivo. Non-CSCs were treated with different doses of radiation, and the dedifferentiation of non-CSCs into CSCs was investigated via flow cytometry and quantitative reverse transcription-polymerase chain reaction for re-expression of reprogramming factors. RESULTS: Patients with HPV-positive tumors have superior overall survival and local-regional control. Human papillomavirus-positive HNSCC cell lines have lower numbers of CSCs, which inversely correlates with radiosensitivity. Human papillomavirus-negative HNSCC cell lines lack hierarchy owing to enhanced spontaneous dedifferentiation. Non-CSCs from HPV-negative lines show enhanced radiation-induced dedifferentiation compared with HPV-positive lines, and RT induced re-expression of Yamanaka reprogramming factors. CONCLUSIONS: Supporting the favorable prognosis of HPV-positive HNSCCs, we show that (1) HPV-positive HNSCCs have a lower frequency of CSCs; (2) RT can dedifferentiate HNSCC cells into CSCs; and (3) radiation-induced dedifferentiation depends on the HPV status of the tumor.

p53 antibody: is it an indicator of dedifferentiated thyroid cancer?

AIM: Radioiodine is the most effective treatment modality in differentiated thyroid carcinoma, either in metastatic or residual thyroid tissue. However, sometimes dedifferentiation can develop and the effectiveness of radioactive I-131 decreases. The p53 is a tumor suppressor gene which plays an important role in controlling normal cell proliferation regulation. In the serum of healthy individuals, the presence of p53 autoantibodies is extremely rare. Mutations in this gene cause an accumulation of non-functional proteins and may lead to development of anti-p53 antibodies. The aim of the present study was to devise a simple blood test that could lead to early identification of patients with dedifferentiation. In this respect, we investigate whether the serum level of anti-p53 antibody is of diagnostic value in the follow-up of patients with high levels of thyroglobulin (Tg) and negative I-131 scan. MATERIALS AND METHODS: Patients who were diagnosed with thyroid cancer, treated with total or near total thyroidectomy and referred for I-131 therapy or low dose I-131 whole body scan were included in our study. Blood samples were taken before the administration of I-131 orally in the group of patients. Besides, 28 healthy subjects were included. We quantified the presence of p53 autoantibodies from serums. RESULTS: In the present study were enrolled 171 patients with a mean age of 47.7±13.5 years (range 16­80 years) and 28 healthy subjects with an age range of 18­52 years (mean 36.0±9.8 years). One hundred and forty-eight patients had papillary (86.5%), 7 (4.1%) follicular, 10 (5.8%) thyroid tumors of uncertain malignant potential, 2 (1.2%) Hürthle cell carcinoma, 3 (1.8%) poor differentiated, and 1 (0.6%) undifferentiated thyroid carcinoma. The p53 antibodies were positive in 16 (9.4%) patients and negative in 155 (90.6%). The p53 antibodies were positive in 3 (10.7%) healthy subjects, and negative in 25 (89.3%) healthy subjects. In five patients with high Tg level and negative radioiodine scan, who were accepted as dedifferentiated, p53 antibodies were also negative. CONCLUSION: The results of the present study suggested that the level of serum p53 antibody seems to be of limited value in the demonstration of dedifferentiation in thyroid cancer patients.

Ionizing radiations sustain glioblastoma cell dedifferentiation to a stem-like phenotype through survivin: possible involvement in radioresistance.

Glioblastomas (GBM) are some bad prognosis brain tumors despite a conventional treatment associating surgical resection and subsequent radio-chemotherapy. Among these heterogeneous tumors, a subpopulation of chemo- and radioresistant GBM stem-like cells appears to be involved in the systematic GBM recurrence. Moreover, recent studies showed that differentiated tumor cells may have the ability to dedifferentiate and acquire a stem-like phenotype, a phenomenon also called plasticity, in response to microenvironment stresses such as hypoxia. We hypothesized that GBM cells could be subjected to a similar dedifferentiation process after ionizing radiations (IRs), then supporting the GBM rapid recurrence after radiotherapy. In the present study we demonstrated that subtoxic IR exposure of differentiated GBM cells isolated from patient resections potentiated the long-term reacquisition of stem-associated properties such as the ability to generate primary and secondary neurospheres, the expression of stemness markers and an increased tumorigenicity. We also identified during this process an upregulation of the anti-apoptotic protein survivin and we showed that its specific downregulation led to the blockade of the IR-induced plasticity. Altogether, these results demonstrated that irradiation could regulate GBM cell dedifferentiation via a survivin-dependent pathway. Targeting the mechanisms associated with IR-induced plasticity will likely contribute to the development of some innovating pharmacological strategies for an improved radiosensitization of these aggressive brain cancers.

Dedifferentiation of differentiated thyroid carcinoma cell line FTC-133 is enhanced by 131I pretreatment.

INTRODUCTION: Differentiated thyroid carcinoma (DTC) usually has a high iodine uptake. However, dedifferentiation of DTC with decreased or no radioiodine ((131)I) uptake is observed in clinical practice, with poor prognosis. The aim of this study was to investigate the effects of (131)I radiation on radioiodine uptake (RAIU) and the expression of thyroid-specific molecules. METHODS: FTC-133 cells were treated with (131)I, the dosage dictated by methylthiazol tetrazolium test results and preliminary experiments. The experimental cell group was incubated with (131)I for 48 h and then cultured for 3 months in (131)I-free medium. The control group was set without (131)I. Primary cells were defined as the blank group. Following treatment, RAIU was measured with a gamma counter as the counts/cell number. Na(+)/I(-) symporter (NIS), thyroid-stimulating hormone receptor (TSHR), thyroid peroxidase (TPO) and thyroglobulin (Tg) levels were detected by Western blotting and radioimmunoassay, and their mRNAs were detected by real-time polymerase chain reaction. RESULTS: RAIU of FTC-133 cells decreased gradually after coincubation with (131)I and did not recover even if (131)I was removed. The relative RAIU of the control and experimental groups was 0.567 and 0.182, respectively, a statistically significant difference (P<.01). Expression of NIS, TSHR, TPO and Tg decreased in the experimental group to a statistically significant degree compared to that of controls (P<.05). CONCLUSION: Changes in the mRNA levels were in accordance with the expression of thyroid-specific proteins. Thus, FTC-133 cells undergo dedifferentiation during long-term culture in vitro, and (131)I may promote this progress.

Iodine-refractory thyroid carcinoma.

The management of thyroid carcinomas commonly involves thyroidectomy, radioactive iodine to ablate any thyroid remnant in patients at high risk of recurrence, and suppressive thyroid hormone replacement. However many tumors will de-differentiate and become refractory to radioactive iodine. After progressive dedifferentiation the prognosis for these patients is poor, with no curative options available as these tumors respond poorly to currently available agents. In this review, we examine the unique biology of thyroid cancer and the various intracellular pathways which are currently the subject of intensive focus. Trials evaluating the efficacy of cytotoxic chemotherapeutic agents in iodine-refractory thyroid carcinoma and current novel therapeutic approaches will be emphasized.