Byong Hoon Yoo

Detachment-induced upregulation of XIAP and cIAP2 delays anoikis of intestinal epithelial cells

Detachment of normal epithelial cells from the extracellular matrix triggers apoptosis, a phenomenon called anoikis. Conversely, carcinoma cells tend to be relatively more anoikis-resistant than their normal counterparts, and this increased resistance represents a critical feature of the malignant phenotype. Mechanisms that control susceptibility and resistance to anoikis are not fully understood. It is now known that detachment of non-malignant epithelial cells triggers both pro- and antiapoptotic signals, and it is the balance between these signals and the duration of detachment that determine further fate of the cells. Detachment-induced antiapoptotic events delay anoikis and if cells reattach relatively soon after detachment they survive. Direct regulators of apoptosis responsible for this delay of anoikis are unknown. We found that detachment of non-malignant intestinal epithelial cells triggers upregulation of inhibitors of apoptosis protein (IAP) family, such as X-chromosome-linked inhibitor of apoptosis protein and cellular inhibitor of apoptosis-2 (cIAP2). We demonstrated that this upregulation requires detachment-dependent activation of the transcription factor nuclear factor-κB. We further observed that various IAP antagonists accelerate anoikis, indicating that upregulation of the IAPs delays detachment-triggered apoptosis. We conclude that the IAPs are important regulators of the balance between detachment-triggered life and death signals. Perhaps, not by coincidence, these proteins are often upregulated in carcinomas, tumors composed of cells that tend to be anoikis-resistant.

Oncogenic ras-induced Down-regulation of Autophagy Mediator Beclin-1 Is Required for Malignant Transformation of Intestinal Epithelial Cells

Detachment of non-malignant epithelial cells from the extracellular matrix causes their growth arrest and, ultimately, death. By contrast, cells composing carcinomas, cancers of epithelial origin, can survive and proliferate without being attached to the extracellular matrix. These properties of tumor cells represent hallmarks of malignant transformation and are critical for cancer progression. Previously we identified several mechanisms by which ras, a major oncogene, blocks detachment-induced apoptosis of intestinal epithelial cells, but mechanisms by which Ras promotes proliferation of those cells that remain viable following detachment are unknown. We show here that detachment of non-malignant intestinal epithelial cells promotes formation of autophagosomes, vacuole-like structures that mediate autophagy (a process of cellular self-cannibalization), and that oncogenic ras prevents this autophagosome formation. We also found that ras activates a GTPase RhoA, that RhoA promotes activation of a protease calpain, and that calpain triggers degradation of Beclin-1, a critical mediator of autophagy, in these cells. The reversal of the effect of ras on Beclin-1 (achieved by expression of exogenous Beclin-1) promoted autophagosome formation following cell detachment, significantly reduced the fraction of detached cells in the S phase of the cell cycle and their rate of proliferation without affecting their viability. Furthermore, RNA interference-induced Beclin-1 down-regulation in non-malignant intestinal epithelial cells prevented detachment-dependent reduction of the fraction of these cells in the S phase of the cell cycle. Thus, ras oncogene promotes proliferation of those malignant intestinal epithelial cells that remain viable following detachment via a distinct novel mechanism that involves Ras-induced down-regulation of Beclin-1.

Down-regulation of Death-associated Protein Kinase-2 Is Required for β-Catenin-induced Anoikis Resistance of Malignant Epithelial Cells

Resistance of solid tumor cells to anoikis, apoptosis induced by cell detachment from the extracellular matrix, is thought to be critical for the ability of these cells to grow anchorage independently within thee-dimensional tumor masses and from metastases. β-Catenin, a major oncoprotein, can inhibit anoikis of cancer cells via unknown mechanisms. In an effort to identify these mechanisms we found that β-catenin blocks anoikis of malignant kidney and intestinal epithelial cells and promotes their anchorage-independent growth by down-regulating death-associated protein kinase-2 (DAPk-2), a pro-apoptotic protein whose cellular functions have so far remained unexplored. We found that β-catenin-induced down-regulation of DAPk-2 requires the presence of the transcription factor Tcf-4, a known mediator of β-catenin signaling. We also observed that DAPk-2 contributes to the execution of anoikis of the non-malignant epithelial cells. Thus, β-catenin-induced down-regulation of DAPk-2 represents a novel signaling mechanism by which β-catenin promotes the survival of malignant epithelial cells following their detachment from the ECM and enables these cells to grow in an anchorage-independent manner.

Hypoxia-induced downregulation of autophagy mediator Beclin 1 reduces the susceptibility of malignant intestinal epithelial cells to hypoxia-dependent apoptosis

Disruption of tumor blood supply causes tumor hypoxia. Hypoxia can induce cell death, but cancer cells that remain viable in the absence of oxygen often possess an increased survival potential, and tumors formed by these cells tend to grow particularly aggressively. Thus, developing approaches aimed at increasing the susceptibility of malignant cells to hypoxia-induced death represents a potentially important avenue for cancer treatment. Molecular mechanisms that control the survival of cancer cells under hypoxia are not well understood. In an effort to understand them we found that hypoxia downregulates Beclin-1, a mediator of autophagy, in malignant intestinal epithelial cells. The reversal of this downregulation promoted autophagosome accumulation, enhanced the activation of a pro-apoptotic protease caspase-9 and subsequent caspase-9-dependent activation of two other pro-apoptotic proteases caspases 3 and 7 in these cells. Furthermore, the reversal of hypoxia-induced downregulation of Beclin-1 stimulated caspase-9-dependent apoptosis of the indicated cells under hypoxia. Interestingly, we found that Beclin-1-dependent caspase-9 activation in hypoxic cells was not associated with an increased release of cytochrome c from the mitochondria to the cytoplasm (such release represents a frequently occurring mechanism for caspase-9 activation). We also observed that Beclin-1-dependent apoptosis of hypoxic malignant cells was independent of FADD, a mediator of death receptor signaling. We conclude that hypoxia triggers a feedback mechanism that delays apoptosis of oxygen-deprived malignant intestinal epithelial cells and is driven by hypoxia-induced Beclin-1 downregulation. Thus, approaches aimed at the disruption of this mechanism can be expected to enhance the susceptibility of such cells to hypoxia-induced apoptosis.

Oncogenic ras-induced Down-regulation of Pro-apoptotic Protease Caspase-2 Is Required for Malignant Transformation of Intestinal Epithelial Cells

Background: Many epithelial tumors consist of cells that, unlike normal epithelial cells, survive outside of their original location. This viability is required for tumor growth. Results: ras oncogene promotes survival of cancer cells outside of their original location by down-regulating a cell death-promoting protein caspase-2. Conclusion: ras-induced caspase-2 down-regulation is required for ras-driven tumor growth. Significance: This is a novel mechanism of ras-dependent tumor progression. Resistance of carcinoma cells to anoikis, apoptosis that is normally induced by loss of cell-to-extracellular matrix adhesion, is thought to be essential for the ability of these cells to form primary tumors, invade adjacent tissues, and metastasize to distant organs. Current knowledge about the mechanisms by which cancer cells evade anoikis is far from complete. In an effort to understand these mechanisms, we found that ras, a major oncogene, down-regulates protease caspase-2 (which initiates certain steps of the cellular apoptotic program) in malignant human and rat intestinal epithelial cells. This down-regulation could be reversed by inhibition of a protein kinase Mek, a mediator of Ras signaling. We also found that enforced down-regulation of caspase-2 in nonmalignant intestinal epithelial cells by RNA interference protected them from anoikis. Furthermore, the reversal of the effect of Ras on caspase-2 achieved by the expression of exogenous caspase-2 in detached ras-transformed intestinal epithelial cells promoted well established apoptotic events, such as the release of the pro-apoptotic mitochondrial factors cytochrome c and HtrA2/Omi into the cytoplasm of these cells, significantly enhanced their anoikis susceptibility, and blocked their long term growth in the absence of adhesion to the extracellular matrix. Finally, the blockade of the effect of Ras on caspase-2 substantially suppressed growth of tumors formed by the ras-transformed cells in mice. We conclude that ras-induced down-regulation of caspase-2 represents a novel mechanism by which oncogenic Ras protects malignant intestinal epithelial cells from anoikis, promotes their anchorage-independent growth, and allows them to form tumors in vivo.

ras-Induced up-regulation of CTP:phosphocholine cytidylyltransferase α contributes to malignant transformation of intestinal epithelial cells.

Background: PC synthesis by the CDP-choline pathway is regulated by CCTα. Results: Increased expression of nuclear CCTα in ras-transformed intestinal epithelial contributes to PC synthesis that is required for anchorage-independent growth. Conclusion: An expanded pool of nuclear CCTα is involved in malignant transformation by ras. Significance: CCTα is a potential target to restore anchorage-dependent growth sensitivity of cancer cells. Cancer cells have enhanced lipogenic capacity characterized by increased synthesis of fatty acids and complex lipids, including phosphatidylcholine (PC). As the rate-limiting enzyme in the CDP-choline pathway for PC synthesis, CTP:phosphocholine cytidylyltransferase α (CCTα) is implicated in the provision of membranes and bioactive lipids necessary of cell proliferation. In this study, we assessed the role of CCTα in malignant intestinal epithelial cells transformed with activated H-ras (IEC-ras). Three IEC-ras clones had significant up-regulation CCTα expression, but PC synthesis and in vitro activity of CCTα were similar to control IEC. RNA interference of CCTα in adherent IEC-ras did not affect PC synthesis, confirming that the enzyme was relatively inactive. However, CCTα silencing in ras-transformed IEC reduced anchorage-independent growth, a criterion for malignant transformation, as well as tumorigenicity in mice. Relative to their adherent counterparts, detached IEC-ras had increased PC synthesis that was attenuated by inducible CCTα silencing. Detachment of IEC-ras was accompanied by increased CCTα phosphorylation and cytosolic enzyme activity. We conclude that the expanded pool of CCTα in IEC-ras is activated by detachment. This provides the increased PC biosynthetic capacity that contributes to malignant transformation of intestinal epithelial cells when detached from the extracellular matrix.

Upregulation of ATG3 contributes to autophagy induced by the detachment of intestinal epithelial cells from the extracellular matrix, but promotes autophagy-independent apoptosis of the attached cells

Detachment of nonmalignant intestinal epithelial cells from the extracellular matrix (ECM) triggers their growth arrest and, ultimately, apoptosis. In contrast, colorectal cancer cells can grow without attachment to the ECM. This ability is critical for their malignant potential. We found previously that detachment-induced growth arrest of nonmalignant intestinal epithelial cells is driven by their detachment-triggered autophagy, and that RAS, a major oncogene, promotes growth of detached cells by blocking such autophagy. In an effort to identify the mechanisms of detachment-induced autophagy and growth arrest of nonmalignant cells we found here that detachment of these cells causes upregulation of ATG3 and that ATG3 upregulation contributes to autophagy and growth arrest of detached cells. We also observed that when ATG3 expression is artificially increased in the attached cells, ATG3 promotes neither autophagy nor growth arrest but triggers their apoptosis. ATG3 upregulation likely promotes autophagy of the detached but not that of the attached cells because detachment-dependent autophagy requires other detachment-induced events, such as the upregulation of ATG7. We further observed that those few adherent cells that do not die by apoptosis induced by ATG3 become resistant to apoptosis caused by cell detachment, a property that is critical for the ability of normal epithelial cells to become malignant. We conclude that cell-ECM adhesion can switch ATG3 functions: when upregulated in detached cells in the context of other autophagy-promoting events, ATG3 contributes to autophagy. However, when overexpressed in the adherent cells, in the circumstances not favoring autophagy, ATG3 triggers apoptosis.

Tumor suppressor protein kinase Chk2 is a mediator of anoikis of intestinal epithelial cells

Resistance of carcinoma cells to anoikis, apoptosis that is normally induced by detachment of nonmalignant epithelial cells from the extracellular matrix, is thought to be critical for carcinoma progression. Molecular mechanisms that control anoikis of nonmalignant and cancer cells are understood poorly. In an effort to understand them we found that detachment of nonmalignant intestinal epithelial cells triggers upregulation of Chk2, a pro‐apoptotic protein kinase that has never been implicated in anoikis and has been thought to kill cells mainly under the conditions compromising genome integrity. We found that enforced downregulation of Chk2 protects intestinal epithelial cells from anoikis. Chk2 can kill cells by stabilizing p53 tumor suppressor protein or via p53‐independent mechanisms, and we established that Chk2‐mediated anoikis of intestinal epithelial cells is p53‐independent. We further found that, unlike nonmalignant intestinal epithelial cells whose anoikis is triggered by detachment‐induced Chk2 upregulation, intestinal epithelial cells carrying oncogenic ras, a known inhibitor of anoikis, remain anoikis‐resistant in response to enforced Chk2 upregulation. By contrast, drugs, such as topoisomerase I inhibitors, that can kill cells via Chk2‐indpendent mechanisms, efficiently triggered anoikis of ras‐transformed cells. Thus, oncogenic ras can prevent Chk2 from triggering anoikis even when levels of this protein kinase are elevated in cancer cells, and the use of therapeutic agents that kill cells in a Chk‐2‐independent, rather than Chk‐2‐dependent, manner could represent an efficient strategy for overcoming ras‐induced anoikis resistance of these cells. We conclude that Chk‐2 is an important novel component of anoikis‐promoting machinery of intestinal epithelial cells.

ErbB2-dependent downregulation of a pro-apoptotic protein Perp is required for oncogenic transformation of breast epithelial cells

The ability of breast cancer cells to resist anoikis, apoptosis caused by detachment of the non-malignant epithelial cells from the extracellular matrix (ECM), is thought to be critical for breast tumor growth, invasion and metastasis. ErbB2, an oncoprotein that is often overproduced in breast tumors, can block breast cancer cell anoikis via mechanisms that are understood only in part. In an effort to understand them better we found that detachment of the non-malignant human breast epithelial cells from the ECM upregulates a protein Perp in these cells. Perp is a component of the desmosomes, multiprotein complexes involved in cell-to-cell adhesion. Perp can cause apoptosis via unknown mechanisms. We demonstrated that Perp upregulation by cell detachment is driven by detachment-induced loss of epidermal growth factor receptor (EGFR). We also found that Perp knockdown by RNA interference (RNAi) rescues detached cells from death which indicates that Perp contributes to their anoikis. We observed that ErbB2, when overexpressed in detached breast epithelial cells, causes Perp downregulation. Furthermore, ErbB2-directed RNAi or treatment with lapatinib, an ErbB2/EGFR small-molecule inhibitor used for breast cancer therapy, upregulated Perp in ErbB2-positive human breast and ovarian carcinoma cells. We established that ErbB2 downregulates Perp by activating an ErbB2 effector protein kinase Mek that blocks detachment-induced EGFR loss in a manner that requires the presence of a signaling protein Sprouty-2. Finally, we observed that restoration of the wild-type Perp levels in ErbB2-overproducing breast epithelial cells increases their anoikis susceptibility and blocks their clonogenicity in the absence of adhesion to the ECM. In summary, we have identified a novel mechanism of ErbB2-mediated mechanism of anoikis resistance of ErbB2-overproducing breast epithelial cells. This mechanism allows such cells to grow without adhesion to the ECM and is driven by ErbB2-induced activation of Mek, subsequent EGFR upregulation and further EGFR-dependent Perp loss.

Anoikis of colon carcinoma cells triggered by β-catenin loss can be enhanced by tumor necrosis factor receptor 1 antagonists.

Detachment of non-malignant epithelial cells from the extracellular matrix causes their apoptosis, a phenomenon called anoikis. By contrast, carcinoma cells are anoikis-resistant, and this resistance is thought to be critical for tumor progression. Many oncogenes trigger not only anti- but also pr-apoptotic signals. The proapoptotic events represent an aspect of a phenomenon called oncogenic stress, which acts as a safeguard mechanism blocking tumor initiation. In cells that become malignant, oncogene-induced antiapoptotic signals outbalance the proapoptotic ones. It is now thought that treatments blocking the antiapoptotic events but preserving the proapoptotic signals can be particularly effective in killing tumor cells. Whether or not oncogenes induce any proanoikis signals that can be used for enhancing the efficiency of approaches aimed at triggering anoikis of cancer cells has never been explored. β-Catenin is a major oncoprotein that is often activated in colorectal cancer and promotes tumor progression via mechanisms that are understood only in part. We found here that β-catenin triggers both anti- and proanoikis signals in colon cancer cells. We observed that the antianoikis signals prevail and the cells become anoikis-resistant. We further established that one proanoikis signal in these cells is triggered by β-catenin-induced downregulation of an apoptosis inhibitor tumor necrosis factor receptor 1 (TNFR1) and subsequent reduction of the activity of a transcription factor NF-κB (nuclear factor-κB), a mediator of TNFR1 signaling. We also found that the effect of β-catenin on TNFR1 requires the presence of transcription factor TCF1, a β-catenin effector. We demonstrated that ablation of β-catenin in colon cancer cells triggers their anoikis and that this anoikis is enhanced even further if low TNFR1 or NF-κB activity is artificially preserved in the β-catenin-deprived cells. Thus, inhibition of TNFR1 or NF-κB activity can be expected to enhance the efficiency of approaches aimed at blocking β-catenin-driven anoikis resistance of colon carcinoma cells.