No abstract

Background: Cancer cells evade death caused by extracellular matrix (ECM)-detachment to facilitate metastasis. Results: ErbB2-expressing cancer cells form aggregates during ECM-detachment that promote survival signaling through EGFR. Conclusion: Multicellular aggregation in ErbB2 positive cancer cells promotes survival by preventing EGFR degradation. Significance: Disrupting aggregation or inhibiting EGFR may be effective strategies to eliminate ErbB2-expressing cancer cells during ECM-detachment.

Mitochondria are vital organelles that coordinate cellular energy homeostasis and have important roles in cell death. Therefore, the removal of damaged or excessive mitochondria is critical for maintaining proper cellular function. The PINK1-Parkin pathway removes acutely damaged mitochondria through a well-characterized mitophagy pathway, but basal mitochondrial turnover occurs via distinct and less well-understood mechanisms. Here we report that the MEKK3-MEK5-ERK5 kinase cascade is required for mitochondrial degradation in the absence of exogenous damage. We demonstrate that genetic or pharmacological inhibition of the MEKK3-MEK5-ERK5 pathway increases mitochondrial content by reducing lysosome-mediated degradation of mitochondria under basal conditions. We show that the MEKK3-MEK5-ERK5 pathway plays a selective role in basal mitochondrial degradation but is not required for non-selective bulk autophagy, damage-induced mitophagy, or restraint of mitochondrial biogenesis. This illuminates the MEKK3-MEK5-ERK5 pathway as a positive regulator of mitochondrial degradation that acts independently of exogenous mitochondrial stressors.

Mitochondria are vital organelles that coordinate cellular energy homeostasis and have important roles in cell death. Therefore, the removal of damaged or excessive mitochondria is critical for maintaining proper cellular function. The PINK1-Parkin pathway removes acutely damaged mitochondria through a well-characterized mitophagy pathway, but basal mitochondrial turnover occurs via distinct and less well-understood mechanisms. Here we report that the MEKK3-MEK5-ERK5 kinase cascade is required for mitochondrial degradation in the absence of exogenous damage. We demonstrate that genetic or pharmacological inhibition of the MEKK3-MEK5-ERK5 pathway increases mitochondrial content by reducing lysosome-mediated degradation of mitochondria under basal conditions. We show that the MEKK3-MEK5-ERK5 pathway plays a selective role in basal mitochondrial degradation but is not required for non-selective bulk autophagy, damage-induced mitophagy, or restraint of mitochondrial biogenesis. This illuminates the MEKK3-MEK5-ERK5 pathway as a positive regulator of mitochondrial degradation that acts independently of exogenous mitochondrial stressors.

Normal epithelial cells require attachment to the extracellular matrix (ECM) in order to survive. When detached from the ECM, normal cells will undergo apoptosis through a process known as anoikis. The ErbB2 receptor tyrosine kinase is well known to antagonize anoikis although the precise molecular mechanisms are not entirely known. Interestingly, we have discovered that the overexpression of ErbB2 in non-malignant mammary epithelial cells (MCF-10A) results in substantial aggregation of cells that coincides with protection from anoikis. Therefore, we were interested in understanding if these aggregates could be involved in the evasion of anoikis induction. We found that the disruption of cell-cell contacts in ErbB2-overexpressing MCF-10A cells (using methylcellulose or by antagonizing the formation of adherens junctions) induces caspase activation. Furthermore, in cells that form large aggregates, ErbB2 can physically interact with E-cadherin and EGFR in a fashion that prevents the internalization of EGFR and ultimately its lysosome mediated degradation. The disruption of aggregation causes the ErbB2/EGFR/E-Cadherin complex to fall apart and subsequently results in EGFR degradation in the lysosome. This degradation of EGFR diminishes signaling through the MAPK pathway which prevents the induction of anoikis. In summary, these data suggest that oncogenic signaling through ErbB2 promotes the formation of cellular aggregates that function to prevent EGFR from degradation and subsequently to block the induction of anoikis. Citation Format: Raju Rayavarapu, Nicholas Pagani, Brendan Heiden, Zachary T. Schafer. The role of cell-cell contacts in the survival of extracellular matrix detached mammary epithelial cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1347. doi:10.1158/1538-7445.AM2014-1347

Hyperactivating mutations in Ras are found in a significant percentage of cancers, with a particularly high frequency in pancreatic and colon carcinomas. The hyperactivation of Ras drives a vast number of distinct downstream signaling pathways that play an important role in disease progression. One outcome of overactive oncogenic Ras signaling is the inhibition of anoikis, a form of apoptotic cell death caused by detachment from the extracellular matrix (ECM). It is well-established that one mechanism of anoikis inhibition by Ras involves impaired mitochondrial cytochrome c release. In addition, here, we have found that Ras-mediated anoikis inhibition can also occur downstream of mitochondrial cytochrome c release. Our data suggest that anoikis inhibition following cytochrome c release is not dependent on changes in the abundance of the Apaf-1, pro-caspase-9, or pro-caspase-3. Instead, our data suggest that Ras signaling leads to an inhibition in the ability of caspase-9 to bind Apaf-1 which thereby inhibits proper formation of the apoptosome and caspase activation. Interestingly, in stark contrast to the inhibition of cytochrome c-induced apoptosis in ECM-detached cells, the overexpression of oncogenic Ras in ECM-attached cells results in enhanced sensitivity to exogenous cytochrome c. This sensitization was found to be due to upregulation of apoptosomal proteins (e.g. Apaf-1, pro-caspase-9) in an ERK/MAPK dependent manner. In aggregate, our data suggest that in addition to inhibiting the release of cytochrome c in both attachment and detachment, oncogenic Ras drives additional mechanisms that prevent apoptosome formation and caspase activation in detachment. Furthermore, our data support a model whereby ECM-attached cells containing oncogenic Ras mutations could be selectively eliminated by cytochrome c or agents that mimic its action. Citation Format: Chelsea M. McCallister, Raju Rayavarapu, Nicholas Pagani, Brendan Heiden, Melissa Shaw, Sydeny Shuff, Zachary T. Schafer. Ras-mediated regulation of cytochrome c-induced caspase activation is dependent on the status of extracellular matrix attachment. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3563.

Normal epithelial cells require attachment to the extracellular matrix (ECM) in order to proliferate and survive. Binding to the ECM facilitates the activation of pathways responsible for ATP production and cell survival. When detached from the ECM, normal cells will upregulate apoptotic pathways and will be systematically removed. This detachment-induced apoptosis is known as anoikis. ECM detachment in normal epithelial cells disrupts cellular signaling derived from integrin-mediated basal attachment as well as cadherin assisted signaling from lateral cell contacts. Cadherins, primarily E-cadherin, have been shown to be classic tumor suppressor genes and can prevent the proliferation of cells that fail to assume normal epithelial morphology and cell-cell contacts. The loss of E-cadherin endows an increased invasion capacity to breast tumors and regulation of E-cadherin levels are directly mediated by factors (i.e. Slug) that promote epithelial-to-mesenchymal transition (EMT) and metastasis. Cadherin molecules have previously been reported to interact with other receptor tyrosine kinases, such as EGFR (epithelial growth factor receptor), in order to suppress the ability of the receptor to bind ligand, dimerize and elicit downstream signaling. HER2 is an orphan member of the EGFR family of tyrosine kinases and is a potent oncogene found in 30% of all breast cancer tissue. Previous studies in our laboratory have found a direct correlation between upregulation of survival pathways in ECM detached cells and expression of HER2. In this study, we find that the overexpression of HER2 in non-malignant mammary epithelial cells (MCF-10A) results in substantial aggregation of detached cells, the inhibition of caspase activation and a rescue of ATP production. These data suggest that HER2 plays a role in protecting detached cells from anoikis and this activity may be linked to cell-cell adhesion in suspension. We have found that disruption of cell-cell contacts in HER2-expressing MCF-10A cells utilizing methylcellulose can induce caspase activity and diminish global ATP levels. Preliminary studies also reveal a previously uncharacterized interaction between HER2 and E-cadherin that takes place when E-cadherin dimers are disrupted. Thus, the disruption of E-cadherin dimers in cellular aggregates may facilitate the binding of E-cadherin to HER2 in a fashion that compromises downstream survival signaling. These data together suggest a potential role for E-cadherin as a modulator of HER2 activity and further suggest that the loss of E-cadherin via mechanisms like EMT may function as a means to promote ECM-detached tumor cell survival. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2082. doi:1538-7445.AM2012-2082