Tumor levels of the mediators of ErbB2-driven anoikis resistance correlate with breast cancer relapse in patients receiving trastuzumab-based therapies.

PURPOSE: Patients with ErbB2/Her2 oncoprotein-positive breast cancers often receive neoadjuvant therapies (NATs) containing the anti-ErbB2 antibody trastuzumab. Tumors that are still present after NATs are resected, and patients continue receiving trastuzumab. These cancers are associated with high relapse risk. Whether relapse will occur cannot be presently reliably predicted. The ability to make such predictions could improve disease management. We found previously that ErbB2 blocks breast tumor cell anoikis, apoptosis induced by cell detachment from the extracellular matrix, by downregulating the pro-apoptotic protein Irf6 and upregulating the anti-apoptotic protein Epidermal Growth Factor Receptor (EGFR) in the cells and, thus, promotes their three-dimensional growth. We now tested whether tumor levels of these proteins before and after NATs correlate with patients' relapse-free survival (RFS) and overall survival (OS). METHODS: We selected archival breast tumor samples collected from 37 women with ErbB2-positive stages II and III breast cancer before and after NATs. We used immunohistochemistry to test whether levels of the indicated proteins in respective tumors correlate with RFS and OS. RESULTS: We observed that the presence of high Irf6 levels in the tumors following NATs correlated with reduced RFS and OS. Perhaps not by coincidence, we noticed that trastuzumab-sensitive ErbB2-positive breast cancer cells selected for the ability to overproduce exogenous Irf6 in culture acquired trastuzumab resistance. Finally, EGFR presence in patients' tumors before or after NATs was associated with decreased RFS and OS. CONCLUSIONS: This study could help identify patients with ErbB2-positive tumors that are at increased risk of disease relapse following NATs.

Trastuzumab-induced upregulation of a protein set in extracellular vesicles emitted by ErbB2-positive breast cancer cells correlates with their trastuzumab sensitivity.

BACKGROUND: ErbB2/HER2 oncoprotein often drives breast cancers (BCs) which are treated with the anti-ErbB2 antibody trastuzumab. The efficacy of trastuzumab-based metastatic BC therapies is routinely assessed by imaging studies. Trastuzumab typically becomes ineffective in the case of this disease and is then replaced by other drugs. Biomarkers of BC trastuzumab response could allow imaging studies and the switch to other drugs to occur earlier than is now possible. Moreover, bone-only BC metastases can be hard to measure, and biomarkers of their trastuzumab response could facilitate further treatment decisions. Such biomarkers are presently unavailable. In this study, we searched for proteins whose levels in BC cell-emitted extracellular vesicles (EVs) potentially correlate with BC trastuzumab sensitivity. METHODS: We isolated EVs from cultured trastuzumab-sensitive and trastuzumab-resistant human BC cells before and after trastuzumab treatment and characterized these EVs by nanoparticle tracking analysis and electron microscopy. We found previously that ErbB2 drives BC by downregulating a pro-apoptotic protein PERP. We now tested whether trastuzumab-induced PERP upregulation in EVs emitted by cultured human BC cells correlates with their trastuzumab sensitivity. We also used mass spectrometry to search for additional proteins whose levels in such EVs reflect BC cell trastuzumab sensitivity. Once we identified proteins whose EV levels correlate with this sensitivity in culture, we explored the feasibility of testing whether their levels in the blood EVs of trastuzumab-treated metastatic BC patients correlate with patients' response to trastuzumab-based treatments. RESULTS: We found that neither trastuzumab nor acquisition of trastuzumab resistance by BC cells affects the size or morphology of EVs emitted by cultured BC cells. We established that EV levels of proteins PERP, GNAS2, GNA13, ITB1, and RAB10 correlate with BC cell trastuzumab response. Moreover, these proteins were upregulated during trastuzumab-based therapies in the blood EVs of a pilot cohort of metastatic BC patients that benefited from these therapies but not in those derived from patients that failed such treatments. CONCLUSIONS: Upregulation of a protein set in EVs derived from cultured breast tumor cells correlates with tumor cell trastuzumab sensitivity. It is feasible to further evaluate these proteins as biomarkers of metastatic BC trastuzumab response.

Correction to: Trastuzumab-induced upregulation of a protein set in extracellular vesicles emitted by ErbB2-positive breast cancer cells correlates with their trastuzumab sensitivity.

An amendment to this paper has been published and can be accessed via the original article.

ErbB2-driven downregulation of the transcription factor Irf6 in breast epithelial cells is required for their 3D growth.

BACKGROUND: The ability of solid tumor cells to resist anoikis, apoptosis triggered by cell detachment from the extracellular matrix (ECM), is thought to be critical for 3D tumor growth. ErbB2/Her2 oncoprotein is often overproduced by breast tumor cells and blocks their anoikis by partially understood mechanisms. In our effort to understand them better, we observed that detachment of nonmalignant human breast epithelial cells from the ECM upregulates the transcription factor Irf6. Irf6 is thought to play an important role in mammary gland homeostasis and causes apoptosis by unknown mechanisms. We noticed that ErbB2, when overproduced by detached breast epithelial cells, downregulates Irf6. METHODS: To test whether ErbB2 downregulates Irf6 in human ErbB2-positive breast cancer cells, we examined the effect of ErbB2 inhibitors, such as the anti-ErbB2 antibody trastuzumab or the ErbB2/epidermal growth factor receptor small-molecule inhibitor lapatinib, on Irf6 in these cells. Moreover, we performed Irf6 IHC analysis of tumor samples derived from the locally advanced ErbB2-positive breast cancers before and after neoadjuvant trastuzumab-based therapies. To examine the role of Irf6 in anoikis of nonmalignant and ErbB2-overproducing breast epithelial cells, we studied anoikis after knocking down Irf6 in the former cells by RNA interference and after overproducing Irf6 in the latter cells. To examine the mechanisms by which cell detachment and ErbB2 control Irf6 expression in breast epithelial cells, we tested the effects of genetic and pharmacological inhibitors of the known ErbB2-dependent signaling pathways on Irf6 in these cells. RESULTS: We observed that trastuzumab and lapatinib upregulate Irf6 in ErbB2-positive human breast tumor cells and that neoadjuvant trastuzumab-based therapies tend to upregulate Irf6 in human breast tumors. We found that detachment-induced Irf6 upregulation in nonmalignant breast epithelial cells requires the presence of the transcription factor ∆Np63α and that Irf6 mediates their anoikis. We showed that ErbB2 blocks Irf6 upregulation in ErbB2-overproducing cells by activating the mitogen-activated protein kinases that inhibit ∆Np63α-dependent signals required for Irf6 upregulation. Finally, we demonstrated that ErbB2-driven Irf6 downregulation in ErbB2-overproducing breast epithelial cells blocks their anoikis and promotes their anchorage-independent growth. CONCLUSIONS: We have demonstrated that ErbB2 blocks anoikis of breast epithelial cells by downregulating Irf6.

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

Three-dimensional growth sensitizes breast cancer cells to treatment with ferroptosis-promoting drugs.

Drugs causing ferroptosis, iron-mediated cell death, represent promising tools for cancer treatment. While exploring the effect of these drugs on breast cancer (BC), we found that a ferroptosis-inducing drug erastin dramatically inhibits tumorigenicity of human BC cells in mice but when used at a concentration known to effectively kill other cell types only modestly reduces such growth in 2D monolayer culture. BCs grow in vivo as 3D masses, and we found that ferroptosis inducers erastin and sulfasalazine inhibit growth of multiple human BC cell lines in 3D culture significantly stronger than in 2D culture. To understand the mechanism of this differential effect, we found that ferroptosis inducers upregulate mRNAs encoding multiple direct and indirect autophagy stimulators, such as ATG16L2, ATG9A, ATG4D, GABARAP, SQSTM/p62, SEC23A and BAX, in tumor cells growing in 2D but not in 3D culture. Furthermore, these drugs promoted autophagy of tumor cells growing in a 2D but not in a 3D manner. We observed that pharmacological inhibition of autophagy-stimulating protein kinase ULK1 or RNA interference-mediated knockdown of autophagy mediator ATG12 significantly sensitized tumor cells to erastin treatment in 2D culture. We also found that ferroptosis-promoting treatments upregulate heme oxygenase-1 (HO-1) in BC cells. HO-1 increases cellular free iron pool and can potentially promote ferroptosis. Indeed, we observed that HO-1 knockdown by RNA interference reversed the effect of ferroptosis inducers on BC cell 3D growth. Hence, the effect of these drugs on such growth is mediated by HO-1. In summary, autophagy triggered by ferroptosis-promoting drugs reduces their ability to kill BC growing in a 2D manner. This protection mechanism is inhibited in BC cells growing as a 3D mass, and ferroptosis-promoting drugs kill such cells more effectively. Moreover, this death is mediated by HO-1. Thus, ferroptosis induction represents a promising strategy for blocking 3D BC growth.

Oncogenic ras-induced down-regulation of pro-apoptotic protease caspase-2 is required for malignant transformation of intestinal epithelial cells.

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.

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/Her2-dependent downregulation of a cell death-promoting protein BLNK in breast cancer cells is required for 3D breast tumor growth.

A significant proportion of breast cancers are driven by ErbB2/Her2 oncoprotein that they overexpress. These malignancies are typically treated with various ErbB2-targeted drugs, but many such cancers develop resistance to these agents and become incurable. Conceivably, treatment of ErbB2-positive cancers could be facilitated by use of agents blocking oncogenic signaling mechanisms downstream of ErbB2. However, current understanding of these mechanisms is limited. The ability of solid tumor cells to resist anoikis, cell death triggered by cell detachment from the extracellular matrix (ECM), is thought to be critical for 3D tumor growth. In an effort to understand the mechanisms of ErbB2-driven breast cancer cell anoikis resistance we found that detachment of non-malignant breast epithelial cells from the ECM upregulates a cell death-promoting tumor suppressor adapter protein BLNK and that ErbB2 blocks this upregulation by reducing tumor cell levels of transcription factor IRF6. We further observed that trastuzumab, a therapeutic anti-ErbB2 antibody, upregulates BLNK in human trastuzumab-sensitive but not trastuzumab-resistant ErbB2-positive breast cancer cells. Moreover, we established that BLNK promotes anoikis by activating p38 MAP kinase and that ErbB2-dependent BLNK downregulation blocks breast cancer cell anoikis. In search for pharmacological approaches allowing to upregulate BLNK in tumor cells we found that clinically approved proteasome inhibitor bortezomib upregulates IRF6 and BLNK in human breast cancer cells and inhibits their 3D growth in a BLNK-dependent manner. In addition, we found that BLNK upregulation in human ErbB2-positive breast cancer cells blocks their ability to form tumors in mice. Furthermore, we used publicly available data on mRNA levels in multiple breast cancers to demonstrate that increased BLNK mRNA levels correlate with increased relapse-free survival in a cohort of approximately 400 patients with ErbB2-positive breast cancer. In summary, we discovered a novel mechanism of ErbB2-driven 3D breast tumor growth mediated by ErbB2-dependent BLNK downregulation.

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.

Oncogenic RAS-induced downregulation of ATG12 is required for survival of malignant intestinal epithelial cells.

Activating mutations of RAS GTPase contribute to the progression of many cancers, including colorectal carcinoma. So far, attempts to develop treatments of mutant RAS-carrying cancers have been unsuccessful due to insufficient understanding of the salient mechanisms of RAS signaling. We found that RAS downregulates the protein ATG12 in colon cancer cells. ATG12 is a mediator of autophagy, a process of degradation and reutilization of cellular components. In addition, ATG12 can kill cells via autophagy-independent mechanisms. We established that RAS reduces ATG12 levels in cancer cells by accelerating its proteasomal degradation. We further observed that RAS-dependent ATG12 loss in these cells is mediated by protein kinases MAP2K/MEK and MAPK1/ERK2-MAPK3/ERK1, known effectors of RAS. We also demonstrated that the reversal of the effect of RAS on ATG12 achieved by the expression of exogenous ATG12 in cancer cells triggers both apoptotic and nonapoptotic signals and efficiently kills the cells. ATG12 is known to promote autophagy by forming covalent complexes with other autophagy mediators, such as ATG5. We found that the ability of ATG12 to kill oncogenic RAS-carrying malignant cells does not require covalent binding of ATG12 to other proteins. In summary, we have identified a novel mechanism by which oncogenic RAS promotes survival of malignant intestinal epithelial cells. This mechanism is driven by RAS-dependent loss of ATG12 in these cells.

Mek activity is required for ErbB2 expression in breast cancer cells detached from the extracellular matrix.

Detachment of non-malignant epithelial cells from the extracellullar matrix (ECM) triggers their growth arrest and apoptosis. Conversely, carcinoma cells can grow without adhesion to the ECM. This capacity for anchorage-independent growth is thought to be critical for tumor progression. ErbB2/Her2 oncoprotein is overproduced by a significant fraction of breast cancers and promotes anchorage-independent tumor cell growth by poorly understood mechanisms. In an effort to understand them we found that in order to produce ErbB2, detached breast cancer cells require the activity of an ErbB2 effector protein kinase Mek and that Mek-driven ErbB2 expression is neccesary for anchorage-independent growth of such cells. We observed that Mek inhibition does not alter ErbB2 mRNA levels in detached cancer cells and that ErbB2 protein loss induced by this inhibition can be blocked by a lysosomal inhibitor. We also noticed that an increase of the density of cancer cells detached from the ECM downregulates a Mek effector protein kinase Erk and causes ErbB2 loss. Those cells that survive after ErbB2 loss display resistance to trastuzumab, an anti-ErbB2 antibody used for ErbB2-positive breast cancer treatment. Thus, Mek-induced ErbB2 stabilization in detached breast cancer cells is critical for their ability to grow anchorage-independently and their trastuzumab sensitivity.

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.

Down-regulation of death-associated protein kinase-2 is required for beta-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 three-dimensional tumor masses and from metastases. Beta-catenin, a major oncoprotein, can inhibit anoikis of cancer cells via unknown mechanisms. In an effort to identify these mechanisms we found that beta-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 beta-catenin-induced down-regulation of DAPk-2 requires the presence of the transcription factor Tcf-4, a known mediator of beta-catenin signaling. We also observed that DAPk-2 contributes to the execution of anoikis of the non-malignant epithelial cells. Thus, beta-catenin-induced down-regulation of DAPk-2 represents a novel signaling mechanism by which beta-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.

Acquisition of anoikis resistance promotes the emergence of oncogenic K-ras mutations in colorectal cancer cells and stimulates their tumorigenicity in vivo.

Detachment from the extracellular matrix causes apoptosis of normal epithelial cells--a phenomenon called anoikis. K-ras oncogene, an established anoikis inhibitor, often occurs in colorectal carcinoma (CRC). In addition to blocking anoikis-inducing mechanisms, oncogenic K-ras can cause anoikis-unrelated changes in CRC cells, such as induction of events promoting their deregulated mitogenesis, ability to trigger angiogenesis, and so on. Thus, whether ras-induced anoikis resistance of CRC cells is essential for their ability to form tumors in vivo or represents a mere epiphenomenon is unclear. We found that when poorly tumorigenic, oncogenic, K-ras-negative, anoikis-susceptible human CRC cells were cultured under anoikis-inducing conditions in vitro, they spontaneously gave rise to an anoikis-resistant cell population harboring de novo oncogenic K-ras mutations and manifesting dramatically increased tumorigenicity. We further observed that a variant of the same oncogenic K-ras-negative anoikis-susceptible cells selected for increased tumorigenicity acquired de novo oncogenic K-ras mutations and manifested increased anoikis resistance. Unlike the case with anoikis, oncogenic K-ras did not rescue CRC cells from death caused by hypoxia or anticancer agents. Taken collectively, our results support the notion that ras-induced anoikis resistance of CRC cells is essential for their ability to form tumors in vivo and thus represents a potential therapeutic target.

ras Oncogene triggers up-regulation of cIAP2 and XIAP in intestinal epithelial cells: epidermal growth factor receptor-dependent and -independent mechanisms of ras-induced transformation.

Detachment of normal epithelial cells from the extracellular matrix (ECM) triggers apoptosis, a phenomenon called anoikis. Conversely, carcinomas (cancers of epithelial origin) represent three-dimensional disorganized multicellular masses in which cells are deprived of adhesion to the ECM but remain viable. Resistance of cancer cells to anoikis is thought to be critical for tumor progression. However, the knowledge about molecular mechanisms of this type of resistance remains limited. Herein we report that ras oncogene, an established inhibitor of anoikis, triggers a significant upregulation of anti-apoptotic proteins cIAP2 and XIAP in intestinal epithelial cells. We also observed that the effect of ras on cIAP2 requires ras-induced autocrine production of transforming growth factor alpha (TGF-alpha), a ligand for epidermal growth factor receptor, whereas ras-triggered up-regulation of XIAP is TGF-alpha-independent. Moreover, overexpression of either cIAP2 or XIAP in nonmalignant intestinal epithelial cell was found to block anoikis. In addition, an established IAP antagonist Smac or Smac-derived cell-permeable peptide suppressed ras-induced anoikis resistance and subsequent anchorage-independent growth of ras-transformed cells. We conclude that ras-induced overexpression of cIAP2 and XIAP significantly contributes to the ability of ras-transformed intestinal epithelial cells to survive in the absence of adhesion to the ECM and grow in a three-dimensional manner.