Aberrant signal transduction in Indian triple-negative breast cancer patients.

AIM OF STUDY: The aim of this study is to correlate the prominin-1 or CD133 association with functional pathway markers of cancer stemness in Indian triple-negative breast cancer (TNBC) patient samples. MATERIALS AND METHODS: TNBC samples were confirmed for the absence of hormone receptors (estrogen receptor-ER/progesterone receptor) and human epidermal growth factor receptor-2 or proto-oncogene neu or erbB2 or CD340 by immunohistochemical analysis. Formalin-fixed paraffin-embedded samples of patients were used to collect the total RNA. Then, one-step reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the cancer stemness-related transcript levels in the different samples. The RT-PCR products were analyzed semi-quantitatively on agarose gels. The band intensities of respective samples for different transcripts were analyzed by densitometry. RESULTS: TNBC-confirmed samples had shown increased levels of CD133 transcript than control tissues. Further, elevated CD133 transcripts are correlated with higher transcript levels of NOTCH1/FZD7/transforming growth factor-beta receptor Type III R/patched-1 pathway mediators. CONCLUSIONS: This work has clearly indicated that there is a correlation between CD133 and functional pathways that control cancer stem cells in TNBC. These observations may indicate the possible association between cancer stemness and TNBC malignancy.

Correction: Intermittent Hypoxia Effect on Osteoclastogenesis Stimulated by Neuroblastoma Cells.

[This corrects the article DOI: 10.1371/journal.pone.0105555.].

Implications of mitogen-activated protein kinase signaling in glioma.

Gliomas are the most common primary central nervous system tumors. Gliomas originate from astrocytes, oligodendrocytes, and neural stem cells or their precursors. According to WHO classification, gliomas are classified into four different malignant grades ranging from grade I to grade IV based on histopathological features and related molecular aberrations. The induction and maintenance of these tumors can be attributed largely to aberrant signaling networks. In this regard, the mitogen-activated protein kinase (MAPK) network has been widely studied and is reported to be severely altered in glial tumors. Mutations in MAPK pathways most frequently affect RAS and B-RAF in the ERK, c-Jun N-terminal kinase (JNK), and p38 pathways leading to malignant transformation. Also, it is linked to both inherited and sequential accumulations of mutations that control receptor tyrosine kinase (RTK)-activated signal transduction pathways, cell cycle growth arrest pathways, and nonresponsive cell death pathways. Genetic alterations that modulate RTK signaling can also alter several downstream pathways, including RAS-mediated MAP kinases along with JNK pathways, which ultimately regulate cell proliferation and cell death. The present review focuses on recent literature regarding important deregulations in the RTK-activated MAPK pathway during gliomagenesis and progression.

Intermittent hypoxia effect on osteoclastogenesis stimulated by neuroblastoma cells.

BACKGROUND: Neuroblastoma is the most common extracranial pediatric solid tumor. Intermittent hypoxia, which is characterized by cyclic periods of hypoxia and reoxygenation, has been shown to positively modulate tumor development and thereby induce tumor growth, angiogenic processes, and metastasis. Bone is one of the target organs of metastasis in advanced neuroblastoma Neuroblastoma cells produce osteoclast-activating factors that increase bone resorption by the osteoclasts. The present study focuses on how intermittent hypoxia preconditioned SH-SY5Y neuroblastoma cells modulate osteoclastogenesis in RAW 264.7 cells compared with neuroblastoma cells grown at normoxic conditions. METHODS: We inhibited HIF-1α and HIF-2α in neuroblastoma SH-SY5Y cells by siRNA/shRNA approaches. Protein expression of HIF-1α, HIF-2α and MAPKs were investigated by western blotting. Expression of osteoclastogenic factors were determined by real-time RT-PCR. The influence of intermittent hypoxia and HIF-1α siRNA on migration of neuroblastoma cells and in vitro differentiation of RAW 264.7 cells were assessed. Intratibial injection was performed with SH-SY5Y stable luciferase-expressing cells and in vivo bioluminescence imaging was used in the analysis of tumor growth in bone. RESULTS: Upregulation of mRNAs of osteoclastogenic factors VEGF and RANKL was observed in intermittent hypoxia-exposed neuroblastoma cells. Conditioned medium from the intermittent hypoxia-exposed neuroblastoma cells was found to enhance osteoclastogenesis, up-regulate the mRNAs of osteoclast marker genes including TRAP, CaSR and cathepsin K and induce the activation of ERK, JNK, and p38 in RAW 264.7 cells. Intermittent hypoxia-exposed neuroblastoma cells showed an increased migratory pattern compared with the parental cells. A significant increase of tumor volume was found in animals that received the intermittent hypoxia-exposed cells intratibially compared with parental cells. CONCLUSIONS: Intermittent hypoxic exposure enhanced capabilities of neuroblastoma cells in induction of osteoclast differentiation in RAW 264.7 cells. Increased migration and intratibial tumor growth was observed in intermittent hypoxia-exposed neuroblastoma cells compared with parental cells.

Intermittent hypoxia regulates stem-like characteristics and differentiation of neuroblastoma cells.

BACKGROUND: Neuroblastomas are the most common extracranial solid tumors in children. Neuroblastomas are derived from immature cells of the sympathetic nervous system and are characterized by clinical and biological heterogeneity. Hypoxia has been linked to tumor progression and increased malignancy. Intermittent hypoxia or repeated episodes of hypoxia followed by re-oxygenation is a common phenomenon in solid tumors including neuroblastoma and it has a significant influence on the outcome of therapies. The present study focuses on how intermittent hypoxia modulates the stem-like properties and differentiation in neuroblastoma cells. METHODS AND FINDINGS: Cell survival was assessed by clonogenic assay and cell differentiation was determined by morphological characterization. Hypoxia-inducible genes were analyzed by real-time PCR and Western blotting. Immunofluorescence, real-time PCR and Western blotting were utilized to study stem cell markers. Analysis of neural crest/sympathetic nervous system (SNS) markers and neuronal differentiation markers were done by real-time PCR and Western blotting, respectively. Intermittent hypoxia stimulated the levels of HIF-1α and HIF-2 α proteins and enhanced stem-like properties of neuroblastoma cells. In intermittent hypoxia-conditioned cells, downregulation of SNS marker genes and upregulation of genes expressed in the neural crest were observed. Intermittent hypoxia suppressed the retinoic acid-induced differentiation of neuroblastoma cells. CONCLUSIONS: Our results suggest that intermittent hypoxia enhances stem-like characteristics and suppresses differentiation propensities in neuroblastoma cells.

Differential PARP cleavage: an indication for existence of multiple forms of cell death in human gliomas.

BACKGROUND: Gliomas represent a diverse range of clinical presentation, histological differentiation, and response to therapy. Altered cell proliferation and cell death signals in gliomas are of great interest to elucidate the key molecules involved and to find effective treatment modalities. By considering the role of different proteases in correlation with differential poly (ADP-ribose) polymerase (PARP) fragmentation we have studied the pattern of cell death in human glioma tissues. MATERIALS AND METHODS: In our study, five different human glioma biopsies were collected and analyzed for the PARP cleavage pattern by using western immunoblotting. Samples were also analyzed for pro-caspase 3, calpain I (micro) and II (m), granzyme-B and apoptosis-inducing factor (AIF). Parallel sections of histologically confirmed astrocytoma and glioblastoma multiforme (GBM) were used for immunohistochemical analysis of cleaved caspase-3, granzyme B, AIF and cyclo-oxygenase -2 (cox-2). RESULTS: We found PARP fragmentation, along with usual approximately 89 kDa and approximately 24 kDa fragments, into other fragments of different molecular weights. Caspase mediated cell death may lead to appearance of larger approximately 89 kDa fragment and smaller approximately 24 kDa fragment indicating existence of apoptosis in the tumors. However, other fragments corresponding to approximately 64 kDa, approximately 54 kDa, and approximately 40 kDa were observed concomitantly in all glial tumor tissues. CONCLUSIONS: These results may indicate, not only apoptosis and necrosis, but there occurs the co-existence of intermediate cell death pathways in human glial tumors.