Regulation of Tumor Progression by Programmed Necrosis.

Rapidly growing malignant tumors frequently encounter hypoxia and nutrient (e.g., glucose) deprivation, which occurs because of insufficient blood supply. This results in necrotic cell death in the core region of solid tumors. Necrotic cells release their cellular cytoplasmic contents into the extracellular space, such as high mobility group box 1 (HMGB1), which is a nonhistone nuclear protein, but acts as a proinflammatory and tumor-promoting cytokine when released by necrotic cells. These released molecules recruit immune and inflammatory cells, which exert tumor-promoting activity by inducing angiogenesis, proliferation, and invasion. Development of a necrotic core in cancer patients is also associated with poor prognosis. Conventionally, necrosis has been thought of as an unregulated process, unlike programmed cell death processes like apoptosis and autophagy. Recently, necrosis has been recognized as a programmed cell death, encompassing processes such as oncosis, necroptosis, and others. Metabolic stress-induced necrosis and its regulatory mechanisms have been poorly investigated until recently. Snail and Dlx-2, EMT-inducing transcription factors, are responsible for metabolic stress-induced necrosis in tumors. Snail and Dlx-2 contribute to tumor progression by promoting necrosis and inducing EMT and oncogenic metabolism. Oncogenic metabolism has been shown to play a role(s) in initiating necrosis. Here, we discuss the molecular mechanisms underlying metabolic stress-induced programmed necrosis that promote tumor progression and aggressiveness.

Induction of metastasis, cancer stem cell phenotype, and oncogenic metabolism in cancer cells by ionizing radiation.

Radiation therapy is one of the major tools of cancer treatment, and is widely used for a variety of malignant tumours. Radiotherapy causes DNA damage directly by ionization or indirectly via the generation of reactive oxygen species (ROS), thereby destroying cancer cells. However, ionizing radiation (IR) paradoxically promotes metastasis and invasion of cancer cells by inducing the epithelial-mesenchymal transition (EMT). Metastasis is a major obstacle to successful cancer therapy, and is closely linked to the rates of morbidity and mortality of many cancers. ROS have been shown to play important roles in mediating the biological effects of IR. ROS have been implicated in IR-induced EMT, via activation of several EMT transcription factors-including Snail, HIF-1, ZEB1, and STAT3-that are activated by signalling pathways, including those of TGF-ß, Wnt, Hedgehog, Notch, G-CSF, EGFR/PI3K/Akt, and MAPK. Cancer cells that undergo EMT have been shown to acquire stemness and undergo metabolic changes, although these points are debated. IR is known to induce cancer stem cell (CSC) properties, including dedifferentiation and self-renewal, and to promote oncogenic metabolism by activating these EMT-inducing pathways. Much accumulated evidence has shown that metabolic alterations in cancer cells are closely associated with the EMT and CSC phenotypes; specifically, the IR-induced oncogenic metabolism seems to be required for acquisition of the EMT and CSC phenotypes. IR can also elicit various changes in the tumour microenvironment (TME) that may affect invasion and metastasis. EMT, CSC, and oncogenic metabolism are involved in radioresistance; targeting them may improve the efficacy of radiotherapy, preventing tumour recurrence and metastasis. This study focuses on the molecular mechanisms of IR-induced EMT, CSCs, oncogenic metabolism, and alterations in the TME. We discuss how IR-induced EMT/CSC/oncogenic metabolism may promote resistance to radiotherapy; we also review efforts to develop therapeutic approaches to eliminate these IR-induced adverse effects.

Dlx-2 and glutaminase upregulate epithelial-mesenchymal transition and glycolytic switch.

Most cancer cells depend on enhanced glucose and glutamine (Gln) metabolism for growth and survival. Oncogenic metabolism provides biosynthetic precursors for nucleotides, lipids, and amino acids; however, its specific roles in tumor progression are largely unknown. We previously showed that distal-less homeobox-2 (Dlx-2), a homeodomain transcription factor involved in embryonic and tumor development, induces glycolytic switch and epithelial-mesenchymal transition (EMT) by inducing Snail expression. Here we show that Dlx-2 also induces the expression of the crucial Gln metabolism enzyme glutaminase (GLS1), which converts Gln to glutamate. TGF-ß and Wnt induced GLS1 expression in a Dlx-2-dependent manner. GLS1 shRNA (shGLS1) suppressed in vivo tumor metastasis and growth. Inhibition of Gln metabolism by shGLS1, Gln deprivation, and Gln metabolism inhibitors (DON, 968 and BPTES) prevented Dlx-2-, TGF-ß-, Wnt-, and Snail-induced EMT and glycolytic switch. Finally, shDlx-2 and Gln metabolism inhibition decreased Snail mRNA levels through p53-dependent upregulation of Snail-targeting microRNAs. These results demonstrate that the Dlx-2/GLS1/Gln metabolism axis is an important regulator of TGF-ß/Wnt-induced, Snail-dependent EMT, metastasis, and glycolytic switch.

Dlx-2 is implicated in TGF-ß- and Wnt-induced epithelial-mesenchymal, glycolytic switch, and mitochondrial repression by Snail activation.

Epithelial-mesenchymal transition (EMT) and oncogenic metabolism (including glycolytic switch) are important for tumor development and progression. Here, we show that Dlx-2, one of distal-less (Dlx) homeobox genes, induces EMT and glycolytic switch by activation of Snail. In addition, it was induced by TGF-ß and Wnt and regulates TGF-ß- and Wnt-induced EMT and glycolytic switch by activating Snail. We also found that TGF-ß/Wnt suppressed cytochrome c oxidase (COX), the terminal enzyme of the mitochondrial respiratory chain, in a Dlx-2/Snail-dependent manner. TGF-ß/Wnt appeared to downregulate the expression of various COX subunits including COXVIc, COXVIIa and COXVIIc; among these COX subunits, COXVIc was a common target of TGF-ß, Wnt, Dlx-2 and Snail, indicating that COXVIc downregulation plays an important role(s) in TGF-ß/Wnt-induced COX inhibition. Taken together, our results showed that Dlx-2 is involved in TGF-ß- and Wnt-induced EMT, glycolytic switch, and mitochondrial repression by Snail activation.

Human CD103(+) dendritic cells promote the differentiation of Porphyromonas gingivalis heat shock protein peptide-specific regulatory T cells.

PURPOSE: Regulatory T cells (Tregs), expressing CD4 and CD25 as well as Foxp3, are known to play a pivotal role in immunoregulatory function in autoimmune diseases, cancers, and graft rejection. Dendritic cells (DCs) are considered the major antigen-presenting cells (APCs) for initiating these T-cell immune responses, of which CD103(+) DCs are derived from precursor human peripheral blood mononuclear cells (PBMCs). The aim of the present study was to evaluate the capacity of these PBMC-derived CD103(+) DCs to promote the differentiation of antigen-specific Tregs. METHODS: Monocyte-derived DCs were induced from CD14(+) monocytes from the PBMCs of 10 healthy subjects. Once the CD103(+) DCs were purified, the cell population was enriched by adding retinoic acid (RA). Peptide numbers 14 and 19 of Porphyromonas gingivalis heat shock protein 60 (HSP60) were synthesized to pulse CD103(+) DCs as a tool for presenting the peptide antigens to stimulate CD3(+) T cells that were isolated from human PBMC. Exogenous interleukin 2 was added as a coculture supplement. The antigen-specific T-cell lines established were phenotypically identified for their expression of CD4, CD25, or Foxp3. RESULTS: When PBMCs were used as APCs, they demonstrated only a marginal capacity to stimulate peptide-specific Tregs, whereas CD103(+) DCs showed a potent antigen presenting capability to promote the peptide-specific Tregs, especially for peptide 14. RA enhanced the conversion of CD103(+) DCs, which paralleled the antigen-specific Treg-stimulating effect, though the differences failed to reach statistical significance. CONCLUSIONS: We demonstrated that CD103(+) DCs can promote antigen-specific Tregs from naive T cells, when used as APCs for an epitope peptide from P. gingivalis HSP60. RA was an effective reagent that induces mature DCs with the typical phenotypic expression of CD103 that demonstrated the functional capability to promote antigen-specific Tregs.

Role of extracellular signal-regulated kinase (ERK)1/2 in multicellular resistance to docetaxel in MCF-7 cells.

Cancer cells frequently fail to respond to chemotherapy due to acquisition of chemoresistance. Tumour cells are prone to die by necrosis when they are metabolically stressed by hypoxic and glucose depletion (OGD) due to insufficient vascularization, a common feature of solid tumours. Tumour necrosis indicates poor prognosis and emergence of drug resistance in cancer patients; however, its molecular mechanism remains unclear. In this study, we used multicellular tumour spheroids (MTS) as an in vitro tumour model to investigate the molecular mechanisms underlying necrosis-linked drug resistance. MCF-7 cells formed tight and spherical shape of spheroids and started to form the necrotic core at 8 days of culture. We found that docetaxel (DOC)-induced apoptosis was gradually reduced during MCF-7 spheroid culture compared to that in monolayers and that more prominent resistance to DOC was observed when spheroids containing the necrotic core were treated. ERK1/2 and Akt appeared to be activated in MCF-7 spheroids with necrotic core, but not in 2D culture cells and in spheroids without necrotic core. DOC resistance in spheroids was reversed by inhibition of ERK1/2, but not of Akt, suggesting an important role for ERK1/2 in the DOC resistance in MCF-7 spheroids. These results provide new insight into the possible relation between necrosis-linked ERK1/2 activation and acquisition of multicellular resistance.

Role of reactive oxygen species-dependent protein aggregation in metabolic stress-induced necrosis.

Cancer cells in the inner region of avascularized solid tumours experience metabolical stress by hypoxic and glucose depletion (OGD) and are prone to die by necrosis to form a necrotic core, a common feature of solid tumours. Unlike in apoptosis, where the cellular contents remain packed in the apoptotic bodies that are removed by macrophages, necrosis is characterized by cell membrane rupture, and the release of many cellular proteins including tumour promoting cytokine high mobility group box 1 (HMGB1) into the extra-cellular space. Although ROS produced by metabolic stress are known to cause membrane damage leading to the plasma membrane rupture, its molecular mechanism remains unclear. In this study, we show that some cellular proteins including pro-apoptotic molecules p53, caspase-3, and caspase-9 and a pro-autophagic molecule beclin 1 are not released into the extracellular space but rather aggregated in the cytosol during GD-induced necrosis and that the protein aggregation occurs in a ROS-dependent manner. We also found that Snail, the transcription factor that is induced by GD, was not translocated to the nucleus and aggregated in the cytosol. In addition, Snail interference appeared to block metabolic stress-induced protein aggregation, indicating a critical role(s) of Snail in the protein aggregation. These results demonstrate that in metabolically stressed cancer cells, ROS induce a specific set of cellular proteins to form insoluble aggregates that are highly toxic to cells and trigger the necrosis-associated membrane rupture and HMGB1 release to promote tumour progression.

CuZnSOD and MnSOD inhibit metabolic stress-induced necrosis and multicellular tumour spheroid growth.

CuZnSOD and MnSOD have been shown to exert tumour suppressive activities; however, their exact molecular mechanism is still unclear. We investigated the molecular mechanism underlying the tumour suppressive activities of CuZnSOD and MnSOD using multicellular tumour spheroid (MTS), an in vitro tumour model. Overexpression of CuZnSOD and MnSOD significantly suppressed the growth of A549 and MCF-7 MTS, supporting a critical role(s) of reactive oxygen species (ROS) in tumour growth. In solid tumours, ROS is produced by metabolic stress due to insufficient oxygen and glucose supply and induces necrosis that is known to promote tumour progression by releasing the proinflammatory cytokine HMGB1. We observed that CuZnSOD and MnSOD overexpression prevents metabolic stress-induced necrosis and HMGB1 release by inhibiting mitochondrial ROS and intracellular O2- production in response to glucose depletion in two dimensional cell culture. CuZnSOD and MnSOD overexpression also significantly repressed the occurrence of necrosis that was observed during MTS culture. In human tumour tissues including lung pulmonary adenocarcinoma, CuZnSOD and MnSOD expression was detected in the para-necrotic region that was identified by the expression of a hypoxic marker carbonic anhydrase (CA) IX. These results suggest that CuZnSOD and MnSOD may suppress tumour growth through inhibiting metabolic stress-induced necrosis and HMGB1 release via inhibiting metabolic stress-induced mitochondrial ROS production.

Implication of necrosis-linked p53 aggregation in acquired apoptotic resistance to 5-FU in MCF-7 multicellular tumour spheroids.

Three-dimensional (3D) multicellular tumour spheroids (MTS) have been used as an in vitro model of solid tumours for drug resistance studies because they mimic the growth characteristics of in vivo tumours more closely than in vitro two-dimensional (2D) culture of cancer cell lines. As observed in solid tumours, MTS exhibits a proliferation gradient with outer regions consisting of proliferating cells that surround inner quiescent cells. The innermost cells in core regions undergo cell death mostly by necrosis to form necrotic core due to insufficient supply of oxygen and nutrient such as glucose with increasing size of spheroids. Tumour necrosis is thought to indicate a poor prognosis and to contribute to acquisition of chemoresistance in solid tumours; however, the mechanism underlying necrosis-mediated chemoresistance remains unclear. In this study, we examined the chemoresistance to 5-Fluorouracil (5-FU) using MCF-7 breast cancer MTS. 5-FU (400 microM) induced apoptosis in MCF-7 cell monolayer as determined by HO/PI staining, PARP cleavage, p53 induction, Bax induction, and Bcl-2 down-regulation. When MCF-7 breast tumour spheroids were cultured on agarose for 8 days, they reached approximately 700 microm in diameter, with a necrotic core. We found that 5-FU-induced apoptosis is markedly reduced in spheroids that were cultured for 9 days and had necrotic core, compared with MCF-7 monolayer cells and spheroids that were cultured for 6 days and had no necrotic core, indicating that the formation of necrotic core may be linked to acquisition of chemoresistance to 5-FU. We also found that a specific set of cellular proteins including p53 was aggregated into a RIPA-insoluble form during MTS culture. Furthermore, most of p53 induced by 5-FU was aggregated in MTS with necrotic core. Our results suggest that necrosis-linked p53 aggregation may contribute to acquired apoptotic resistance to 5-FU in MTS model system.