Epigenetic developmental mechanisms underlying sex differences in cancer.

Cancer risk is modulated by hereditary and somatic mutations, exposures, age, sex, and gender. The mechanisms by which sex and gender work alone and in combination with other cancer risk factors remain underexplored. In general, cancers that occur in both the male and female sexes occur more commonly in XY compared with XX individuals, regardless of genetic ancestry, geographic location, and age. Moreover, XY individuals are less frequently cured of their cancers, highlighting the need for a greater understanding of sex and gender effects in oncology. This will be necessary for optimal laboratory and clinical cancer investigations. To that end, we review the epigenetics of sexual differentiation and its effect on cancer hallmark pathways throughout life. Specifically, we will touch on how sex differences in metabolism, immunity, pluripotency, and tumor suppressor functions are patterned through the epigenetic effects of imprinting, sex chromosome complement, X inactivation, genes escaping X inactivation, sex hormones, and life history.

Gonadal sex patterns p21-induced cellular senescence in mouse and human glioblastoma.

Males exhibit higher incidence and worse prognosis for the majority of cancers, including glioblastoma (GBM). Disparate survival may be related to sex-biased responses to treatment, including radiation. Using a mouse model of GBM, we show that female cells are more sensitive to radiation, and that senescence represents a major component of the radiation therapeutic response in both sexes. Correlation analyses revealed that the CDK inhibitor p21 and irradiation induced senescence were differentially regulated between male and female cells. Indeed, female cellular senescence was more sensitive to changes in p21 levels, a finding that was observed in wildtype and transformed murine astrocytes, as well as patient-derived GBM cell lines. Using a novel Four Core Genotypes model of GBM, we further show that sex differences in p21-induced senescence are patterned during early development by gonadal sex. These data provide a rationale for the further study of sex differences in radiation response and how senescence might be enhanced for radiation sensitization. The determination that p21 and gonadal sex are required for sex differences in radiation response will serve as a foundation for these future mechanistic studies.

A Novel Therapeutic Mechanism of Imipridones ONC201/ONC206 in MYCN-Amplified Neuroblastoma Cells via Differential Expression of Tumorigenic Proteins.

Neuroblastoma is the most common extracranial nervous system tumor in children. It presents with a spectrum of clinical prognostic measures ranging from benign growths that regress spontaneously to highly malignant, treatment evasive tumors affiliated with increased mortality rates. MYCN amplification is commonly seen in high-risk neuroblastoma, rendering it highly malignant and recurrence prone. In our current study, we investigated the therapeutic potential of small molecule inducers of TRAIL, ONC201, and ONC206 in MYCN-amplified IMR-32 and non-MYCN-amplified SK-N-SH human neuroblastoma cell lines. Our results exhibit potent antitumor activity of ONC201 and ONC206 via a novel inhibition of EGF-induced L1CAM and PDGFRß phosphorylation in both cell lines. Drug treatment significantly reduced cellular proliferation, viability, migration, invasion, tumorsphere formation potential, and increased apoptosis in both cell lines. The protein expression of tumorigenic NMYC, Sox-2, Oct-4, FABP5, and HMGA1 significantly decreased 48 h post-drug treatment, whereas cleaved PARP1/caspase-3 and γH2AX increased 72 h post-drug treatment, compared with vehicle-treated cells in the MYCN-amplified IMR-32 cell line. We are the first to report this novel differential protein expression after ONC201 or ONC206 treatment in human neuroblastoma cells, demonstrating an important multitarget effect which may yield added therapeutic benefits in treating this devastating childhood cancer.

Mesenchymal Epithelial Transition Factor Signaling in Pediatric Nervous System Tumors: Implications for Malignancy and Cancer Stem Cell Enrichment.

Malignant nervous system cancers in children are the most devastating and worrisome diseases, specifically due to their aggressive nature and, in some cases, inoperable location in critical regions of the brain and spinal cord, and the impermeable blood-brain barrier that hinders delivery of pharmaco-therapeutic compounds into the tumor site. Moreover, the delicate developmental processes of the nervous system throughout the childhood years adds another limitation to the therapeutic modalities and doses used to treat these malignant cancers. Therefore, pediatric oncologists are charged with the daunting responsibility of attempting to deliver effective cures to these children, yet with limited doses of the currently available therapeutic options in order to mitigate the imminent neurotoxicity of radio- and chemotherapy on the developing nervous system. Various studies reported that c-Met/HGF signaling is affiliated with increased malignancy and stem cell enrichment in various cancers such as high-grade gliomas, high-risk medulloblastomas, and MYCN-amplified, high-risk neuroblastomas. Therapeutic interventions that are utilized to target c-Met signaling in these malignant nervous system cancers have shown benefits in basic translational studies and preclinical trials, but failed to yield significant clinical benefits in patients. While numerous pre-clinical data reported promising results with the use of combinatorial therapy that targets c-Met with other tumorigenic pathways, therapeutic resistance remains a problem, and long-term cures are rare. The possible mechanisms, including the overexpression and activation of compensatory tumorigenic mechanisms within the tumors or ineffective drug delivery methods that may contribute to therapeutic resistance observed in clinical trials are elaborated in this review.

Tideglusib attenuates growth of neuroblastoma cancer stem/progenitor cells in vitro and in vivo by specifically targeting GSK-3ß.

BACKGROUND: Neuroblastoma (NB) is the most frequently diagnosed extracranial solid tumor among the pediatric population. It is an embryonic tumor with high relapse rates pertaining to the presence of dormant slowly dividing cancer stem cells (CSC) within the tumor bulk that are responsible for therapy resistance. Therefore, there is a dire need to develop new therapeutic approaches that specifically target NB CSCs. Glycogen synthase kinase (GSK)-3ß is a serine/threonine kinase that represents a common signaling node at the intersection of many pathways implicated in NB CSCs. GSK-3ß sustains the survival and maintenance of CSCs and renders them insensitive to chemotherapeutic agents and radiation. METHODS: In our study, we aimed at evaluating the potential anti-tumor effect of Tideglusib (TDG), an irreversible GSK-3ß inhibitor drug, on three human NB cell lines, SK-N-SH, SH-SY5Y, and IMR-32. RESULTS: Our results showed that TDG significantly reduced cell proliferation, viability, and migration of the NB cells, in a dose- and time-dependent manner, and also significantly hindered the neurospheres formation eradicating the self-renewal ability of highly resistant CSCs. Besides, TDG potently reduced CD133 cancer stem cell marker expression in both SH-SY5Y cells and G1 spheres. Lastly, TDG inhibited NB tumor growth and progression in vivo. CONCLUSION: Collectively, we concluded that TDG could serve as an effective treatment capable of targeting the NB CSCs and hence overcoming therapy resistance. Yet, future studies are warranted to further investigate its potential role in NB and decipher the subcellular and molecular mechanisms underlying this role.

Drug repurposing towards targeting cancer stem cells in pediatric brain tumors.

In the pediatric population, brain tumors represent the most commonly diagnosed solid neoplasms and the leading cause of cancer-related deaths globally. They include low-grade gliomas (LGGs), medulloblastomas (MBs), and other embryonal, ependymal, and neuroectodermal tumors. The mainstay of treatment for most brain tumors includes surgical intervention, radiation therapy, and chemotherapy. However, resistance to conventional therapy is widespread, which contributes to the high mortality rates reported and lack of improvement in patient survival despite advancement in therapeutic research. This has been attributed to the presence of a subpopulation of cells, known as cancer stem cells (CSCs), which reside within the tumor bulk and maintain self-renewal and recurrence potential of the tumor. An emerging promising approach that enables identifying novel therapeutic strategies to target CSCs and overcome therapy resistance is drug repurposing or repositioning. This is based on using previously approved drugs with known pharmacokinetic and pharmacodynamic characteristics for indications other than their traditional ones, like cancer. In this review, we provide a synopsis of the drug repurposing methodologies that have been used in pediatric brain tumors, and we argue how this selective compilation of approaches, with a focus on CSC targeting, could elevate drug repurposing to the next level.

A Novel Mechanism of 17-AAG Therapeutic Efficacy on HSP90 Inhibition in MYCN-Amplified Neuroblastoma Cells.

BACKGROUND: Neuroblastoma is the most common pediatric extra-cranial nervous system tumor, originating from neural crest elements and giving rise to tumors in the adrenal medulla and sympathetic chain ganglia. Amplification of MYCN confers increased malignancy and poorer prognosis in high-risk neuroblastoma. Our SILAC proteomics analysis revealed over-expression of HSP90 in MYCN-amplified IMR-32 compared to the non-MYCN amplified SK-N-SH human neuroblastoma cells, rendering them highly resistant to therapeutic intervention. METHODS: We used cellular bio-functional (proliferation, migration/invasion, apoptosis, viability and stem-cell self-renewal) assays and Western blot analysis to elucidate the therapeutic efficacy of HSP90 inhibition with 17-AAG. RESULTS: 17-AAG treatment significantly inhibited cellular proliferation, viability and migration/invasion and increased apoptosis in both cell lines. Moreover, drug treatment significantly abrogated stem-cell self-renewal potential in the MYCN-amplified IMR-32 cells. Differential tumorigenic protein expression revealed a novel mechanism of therapeutic efficacy after 17-AAG treatment with a significant downregulation of HMGA1, FABP5, Oct4, MYCN, prohibitin and p-L1CAM in SK-N-SH cells. However, we observed a significant up-regulation of p-L1CAM, MYCN and prohibitin, and significant down-regulation of Oct4, FABP5, HMGA1, p-ERK, cleaved/total caspase-3 and PARP1 in IMR-32 cells. CONCLUSIONS: HSP90 inhibition revealed a novel therapeutic mechanism of antitumor activity in MYCN-amplified neuroblastoma cells that may enhance therapeutic sensitivity.

Cancer Stem Cells in Neuroblastoma: Expanding the Therapeutic Frontier.

Neuroblastoma (NB) is the most common extracranial solid tumor often diagnosed in childhood. Despite intense efforts to develop a successful treatment, current available therapies are still challenged by high rates of resistance, recurrence and progression, most notably in advanced cases and highly malignant tumors. Emerging evidence proposes that this might be due to a subpopulation of cancer stem cells (CSCs) or tumor-initiating cells (TICs) found in the bulk of the tumor. Therefore, the development of more targeted therapy is highly dependent on the identification of the molecular signatures and genetic aberrations characteristic to this subpopulation of cells. This review aims at providing an overview of the key molecular players involved in NB CSCs and focuses on the experimental evidence from NB cell lines, patient-derived xenografts and primary tumors. It also provides some novel approaches of targeting multiple drivers governing the stemness of CSCs to achieve better anti-tumor effects than the currently used therapeutic agents.

Tumorigenic proteins upregulated in the MYCN-amplified IMR-32 human neuroblastoma cells promote proliferation and migration.

Childhood neuroblastoma is one of the most common types of extra-cranial cancer affecting children with a clinical spectrum ranging from spontaneous regression to malignant and fatal progression. In order to improve the clinical outcomes of children with high-risk neuroblastoma, it is crucial to understand the tumorigenic mechanisms that govern its malignant behaviors. MYCN proto-oncogene, bHLH transcription factor (MYCN) amplification has been implicated in the malignant, treatment-evasive nature of aggressive, high-risk neuroblastoma. In this study, we used a SILAC approach to compare the proteomic signatures of MYCN-amplified IMR-32 and non-MYCN-amplified SK-N-SH human neuroblastoma cells. Tumorigenic proteins, including fatty-acid binding protein 5 (FABP5), L1-cell adhesion molecule (L1-CAM), baculoviral IAP repeat containing 5 [BIRC5 (survivin)] and high mobility group protein A1 (HMGA1) were found to be significantly upregulated in the IMR-32 compared to the SK-N-SH cells and mapped to highly tumorigenic pathways including, MYC, MYCN, microtubule associated protein Tau (MAPT), E2F transcription factor 1 (E2F1), sterol regulatory element binding transcription factor 1 or 2 (SREBF1/2), hypoxia-inducible factor 1α (HIF-1α), Sp1 transcription factor (SP1) and amyloid precursor protein (APP). The transcriptional knockdown (KD) of MYCN, HMGA1, FABP5 and L1-CAM significantly abrogated the proliferation of the IMR-32 cells at 48 h post transfection. The early apoptotic rates were significantly higher in the IMR-32 cells in which FABP5 and MYCN were knocked down, whereas cellular migration was significantly abrogated with FABP5 and HMGA1 KD compared to the controls. Of note, L1-CAM, HMGA1 and FABP5 KD concomitantly downregulated MYCN protein expression and MYCN KD concomitantly downregulated L1-CAM, HMGA1 and FABP5 protein expression, while survivin protein expression was significantly downregulated by MYCN, HMGA1 and FABP5 KD. In addition, combined L1-CAM and FABP5 KD led to the concomitant downregulation of HMGA1 protein expression. On the whole, our data indicate that this inter-play between MYCN and the highly tumorigenic proteins which are upregulated in the malignant IMR-32 cells may be fueling their aggressive behavior, thereby signifying the importance of combination, multi-modality targeted therapy to eradicate this deadly childhood cancer.

Molecular and functional analysis of anchorage independent, treatment-evasive neuroblastoma tumorspheres with enhanced malignant properties: A possible explanation for radio-therapy resistance.

Despite significant advances in cancer treatment and management, more than 60% of patients with neuroblastoma present with very poor prognosis in the form of metastatic and aggressive disease. Solid tumors including neuroblastoma are thought to be heterogeneous with a sub-population of stem-like cells that are treatment-evasive with highly malignant characteristics. We previously identified a phenomenon of reversible adaptive plasticity (RAP) between anchorage dependent (AD) cells and anchorage independent (AI) tumorspheres in neuroblastoma cell cultures. To expand our molecular characterization of the AI tumorspheres, we sought to define the comprehensive proteomic profile of murine AD and AI neuroblastoma cells. The proteomic profiles of the two phenotypic cell populations were compared to each other to determine the differential protein expression and molecular pathways of interest. We report exclusive or significant up-regulation of tumorigenic pathways expressed by the AI tumorspheres compared to the AD cancer cells. These pathways govern metastatic potential, enhanced malignancy and epithelial to mesenchymal transition. Furthermore, radio-therapy induced significant up-regulation of specific tumorigenic and proliferative proteins, namely survivin, CDC2 and the enzyme Poly [ADP-ribose] polymerase 1. Bio-functional characteristics of the AI tumorspheres were resistant to sutent inhibition of receptor tyrosine kinases (RTKs) as well as to 2.5 Gy radio-therapy as assessed by cell survival, proliferation, apoptosis and migration. Interestingly, PDGF-BB stimulation of the PDGFRß led to transactivation of EGFR and VEGFR in AI tumorspheres more potently than in AD cells. Sutent inhibition of PDGFRß abrogated this transactivation in both cell types. In addition, 48 h sutent treatment significantly down-regulated the protein expression of PDGFRß, MYCN, SOX2 and Survivin in the AI tumorspheres and inhibited tumorsphere self-renewal. Radio-sensitivity in AI tumorspheres was enhanced when sutent treatment was combined with survivin knock-down. We conclude that AI tumorspheres have a differential protein expression compared to AD cancer cells that contribute to their malignant phenotype and radio-resistance. Specific targeting of both cellular phenotypes is needed to improve outcomes in neuroblastoma patients.

Brain Cancer Stem Cells in Adults and Children: Cell Biology and Therapeutic Implications.

Brain tumors represent some of the most malignant cancers in both children and adults. Current treatment options target the majority of tumor cells but do not adequately target self-renewing cancer stem cells (CSCs). CSCs have been reported to resist the most aggressive radiation and chemotherapies, and give rise to recurrent, treatment-resistant secondary malignancies. With advancing technologies, we now have a better understanding of the genetic, epigenetic and molecular signatures and microenvironmental influences which are useful in distinguishing between distinctly different tumor subtypes. As a result, efforts are now underway to identify and target CSCs within various tumor subtypes based on this foundation. This review discusses progress in CSC biology as it relates to targeted therapies which may be uniquely different between pediatric and adult brain tumors. Studies to date suggest that pediatric brain tumors may benefit more from genetic and epigenetic targeted therapies, while combination treatments aimed specifically at multiple molecular pathways may be more effective in treating adult brain tumors which seem to have a greater propensity towards microenvironmental interactions. Ultimately, CSC targeting approaches in combination with current clinical therapies have the potential to be more effective owing to their ability to compromise CSCs maintenance and the mechanisms which underlie their highly aggressive and deadly nature.

L1-CAM knock-down radiosensitizes neuroblastoma IMR-32 cells by simultaneously decreasing MycN, but increasing PTEN protein expression.

Childhood neuroblastoma is one of the most malignant types of cancers leading to a high mortality rate. These cancerous cells can be highly metastatic and malignant giving rise to disease recurrence and poor prognosis. The proto-oncogene myelocytomatosis neuroblastoma (MycN) is known to be amplified in this type of cancer, thus, promoting high malignancy and resistance. The L1 cell adhesion molecule (L1-CAM) cleavage has been found upregulated in many types of malignant cancers. In the present study, we explored the interplay between L1-CAM, MycN and PTEN as well as the role played by PDGFR and VEGFR on tumorigenicity in neuroblastoma cells. We investigated the effect of L1-CAM knock-down (KD) and PDGFR/VEGFR inhibition with sunitinib malate (Sutent®) treatment on subsequent tumorsphere formation and cellular proliferation and migration in the MycN-amplified IMR-32 neuroblastoma cells. We further examined the effect of combined L1-CAM KD with Sutent treatment or radiotherapy on these cellular functions in our cells. Tumorsphere formation is one of the indicators of aggressiveness in malignant cancers, which was significantly inhibited in IMR-32 cells after L1-CAM KD or Sutent treatment, however, no synergistic effect was observed with dual treatments, rather L1-CAM KD alone showed a greater inhibition on tumorsphere formation compared to Sutent treatment alone. In addition, cellular proliferation and migration were significantly inhibited after L1-CAM KD in the IMR-32 cells with no synergistic effect observed on the rate of cell proliferation when combined with Sutent treatment. Again, L1-CAM KD alone exhibited greater inhibitory effect than Sutent treatment on cell proliferation. L1-CAM KD led to the simultaneous downregulation of MycN, but the upregulation of PTEN protein expression. Notably, radiotherapy (2 Gy) of the IMR-32 cells led to significant upregulation of both L1-CAM and MycN, which was abrogated with L1-CAM KD in our cells. In addition, L1-CAM KD radiosensitized the cells as exhibited by the synergistic effect on the reduction in cell proliferation compared to radiotherapy alone. Taken together, our data show the importance of L1-CAM interplay with MycN and PTEN on the MycN amplified neuroblastoma cell radioresistance, proliferation and motility.

Effects of Radiotherapy on the Risk of Developing Secondary Malignant Neoplasms in Hodgkin's Lymphoma Survivors.

Extended follow-up of Hodgkin lymphoma (HL) survivors indicates that these patients are at high risk of secondary malignant neoplasms (SMNs) contributing to increased morbidity and mortality. This study examined the characteristics of HL survivors who developed SMNs with the aim to report any correlation with radiotherapy (RT) dose. In this retrospective multi-center cohort study of HL patients treated between 1990 and 2011 at three major teaching hospitals in Lebanon, classification was into two groups including those treated with combined modality (RT and chemotherapy-CHT) and those treated with CHT alone. Approval from the University Institutional Review Board (IRB) was obtained. Of the 112 patients evaluated, 52.7% (59) received the combined modality while 47.3% (53) received CHT alone. There were 6 cases of SMNs in the combined modality cohort and 5 cases in the CHT cohort. The mean RT dose in the combined modality cohort was 34.5 Gray (Gy) (SD ± 5.3). A statistically significant increase (1.5 fold) in the risk of developing SMNs was observed among patients who received a dose higher than 41 Gy compared to a dose between 20 to 30 Gy (OR= 1.5; 95% confidence interval= 0.674 to 3.339, p=0.012). The risk of SMNs was not significantly higher among patients who received extended field compared to involved field RT (p=0.964). This study showed that the risk of developing SMNs is higher among patients treated with RT dose greater than 31 Gy, independent of the RT type used.