Downregulation of 26S proteasome catalytic activity promotes epithelial-mesenchymal transition.

The epithelial-mesenchymal transition (EMT) endows carcinoma cells with phenotypic plasticity that can facilitate the formation of cancer stem cells (CSCs) and contribute to the metastatic cascade. While there is substantial support for the role of EMT in driving cancer cell dissemination, less is known about the intracellular molecular mechanisms that govern formation of CSCs via EMT. Here we show that β2 and β5 proteasome subunit activity is downregulated during EMT in immortalized human mammary epithelial cells. Moreover, selective proteasome inhibition enabled mammary epithelial cells to acquire certain morphologic and functional characteristics reminiscent of cancer stem cells, including CD44 expression, self-renewal, and tumor formation. Transcriptomic analyses suggested that proteasome-inhibited cells share gene expression signatures with cells that have undergone EMT, in part, through modulation of the TGF-β signaling pathway. These findings suggest that selective downregulation of proteasome activity in mammary epithelial cells can initiate the EMT program and acquisition of a cancer stem cell-like phenotype. As proteasome inhibitors become increasingly used in cancer treatment, our findings highlight a potential risk of these therapeutic strategies and suggest a possible mechanism by which carcinoma cells may escape from proteasome inhibitor-based therapy

Kindlins, Integrin Activation and the Regulation of Talin Recruitment to αIIbβ3

Talins and kindlins bind to the integrin β3 cytoplasmic tail and both are required for effective activation of integrin αIIbβ3 and resulting high-affinity ligand binding in platelets. However, binding of the talin head domain alone to β3 is sufficient to activate purified integrin αIIbβ3 in vitro. Since talin is localized to the cytoplasm of unstimulated platelets, its re-localization to the plasma membrane and to the integrin is required for activation. Here we explored the mechanism whereby kindlins function as integrin co-activators. To test whether kindlins regulate talin recruitment to plasma membranes and to αIIbβ3, full-length talin and kindlin recruitment to β3 was studied using a reconstructed CHO cell model system that recapitulates agonist-induced αIIbβ3 activation. Over-expression of kindlin-2, the endogenous kindlin isoform in CHO cells, promoted PAR1-mediated and talin-dependent ligand binding. In contrast, shRNA knockdown of kindlin-2 inhibited ligand binding. However, depletion of kindlin-2 by shRNA did not affect talin recruitment to the plasma membrane, as assessed by sub-cellular fractionation, and neither over-expression of kindlins nor depletion of kindlin-2 affected talin interaction with αIIbβ3 in living cells, as monitored by bimolecular fluorescence complementation. Furthermore, talin failed to promote kindlin-2 association with αIIbβ3 in CHO cells. In addition, purified talin and kindlin-3, the kindlin isoform expressed in platelets, failed to promote each other's binding to the β3 cytoplasmic tail in vitro. Thus, kindlins do not promote initial talin recruitment to αIIbβ3, suggesting that they co-activate integrin through a mechanism independent of recruitment

Functional mapping of auto-inhibitory sites in talin

Integrin activation by ìnside-out' signaling is a key process in cell migration and adhesion to extracellular matrix. Recent in-vivo and in-vitro studies have provided convincing evidence that the binding of talin, a cytoskeletal protein, to the integrin [beta] cytoplasmic tails is necessary and sufficient for integrin activation. The significance of its role in integrin signaling leads to the expectation that functions of talin are tightly controlled. Indeed, previous data have shown that cellular distribution of talin and its interaction with integrins is highly regulated; talin resides in the cytosol of platelets under resting conditions but, in response to intracellular signals, translocates to the membrane where it interacts with and activate integrins. However, despite intense interests and vigorous efforts, our understanding of molecular basis of talin regulation remains incomplete. In contrast to FL talin, the N-terminal THD binds to and strongly activates integrins without requiring additional signaling. Thus, we hypothesized that a region in the talin rod domain is involved in suppressing the functions of talin. Sequential C-terminal truncations of the talin rod domain and subsequent mutational analysis revealed that an [alpha]-helical bundle termed Domain E negatively regulates membrane recruitment of talin via its inter- domain interaction with the F3 domain of talin. However, increased membrane association of talin caused by disruption of the Domain E-F3 interaction is insufficient for plasma membrane localization of talin, for the talin- integrin interaction, or for integrin activation in cells or in an in-vitro nanodisc system. NMR analysis revealed an inter-domain interaction between another α- helical bundle in the talin rod called VBS1/2a and the F23 domain of talin, and truncation of VBS1/2a led to increased plasma membrane localization of talin. In addition, truncation analysis identified a short fragment within the linker region between THD and the rod domain that interferes with the talin-integrin interaction. In this study, we have mapped three auto-inhibitory sites within talin and have assigned a specific function to each one of them. These findings provide insights as to how talin is regulated and what may happen to talin during the final steps in integrin activatio

Functional mapping of auto-inhibitory sites in talin

Integrin activation by ìnside-out' signaling is a key process in cell migration and adhesion to extracellular matrix. Recent in-vivo and in-vitro studies have provided convincing evidence that the binding of talin, a cytoskeletal protein, to the integrin [beta] cytoplasmic tails is necessary and sufficient for integrin activation. The significance of its role in integrin signaling leads to the expectation that functions of talin are tightly controlled. Indeed, previous data have shown that cellular distribution of talin and its interaction with integrins is highly regulated; talin resides in the cytosol of platelets under resting conditions but, in response to intracellular signals, translocates to the membrane where it interacts with and activate integrins. However, despite intense interests and vigorous efforts, our understanding of molecular basis of talin regulation remains incomplete. In contrast to FL talin, the N-terminal THD binds to and strongly activates integrins without requiring additional signaling. Thus, we hypothesized that a region in the talin rod domain is involved in suppressing the functions of talin. Sequential C-terminal truncations of the talin rod domain and subsequent mutational analysis revealed that an [alpha]-helical bundle termed Domain E negatively regulates membrane recruitment of talin via its inter- domain interaction with the F3 domain of talin. However, increased membrane association of talin caused by disruption of the Domain E-F3 interaction is insufficient for plasma membrane localization of talin, for the talin- integrin interaction, or for integrin activation in cells or in an in-vitro nanodisc system. NMR analysis revealed an inter-domain interaction between another α- helical bundle in the talin rod called VBS1/2a and the F23 domain of talin, and truncation of VBS1/2a led to increased plasma membrane localization of talin. In addition, truncation analysis identified a short fragment within the linker region between THD and the rod domain that interferes with the talin-integrin interaction. In this study, we have mapped three auto-inhibitory sites within talin and have assigned a specific function to each one of them. These findings provide insights as to how talin is regulated and what may happen to talin during the final steps in integrin activatio

PPAR Agonists for the Prevention and Treatment of Lung Cancer

Lung cancer is the most common and most fatal of all malignancies worldwide. Furthermore, with more than half of all lung cancer patients presenting with distant metastases at the time of initial diagnosis, the overall prognosis for the disease is poor. There is thus a desperate need for new prevention and treatment strategies. Recently, a family of nuclear hormone receptors, the peroxisome proliferator-activated receptors (PPARs), has attracted significant attention for its role in various malignancies including lung cancer. Three PPARs, PPARα, PPARβ/δ, and PPARγ, display distinct biological activities and varied influences on lung cancer biology. PPARα activation generally inhibits tumorigenesis through its antiangiogenic and anti-inflammatory effects. Activated PPARγ is also antitumorigenic and antimetastatic, regulating several functions of cancer cells and controlling the tumor microenvironment. Unlike PPARα and PPARγ, whether PPARβ/δ activation is anti- or protumorigenic or even inconsequential currently remains an open question that requires additional investigation. This review of current literature emphasizes the multifaceted effects of PPAR agonists in lung cancer and discusses how they may be applied as novel therapeutic strategies for the disease

PPARs: Key Regulators of Airway Inflammation and Potential Therapeutic Targets in Asthma

Asthma affects approximately 300 million people worldwide, significantly impacting quality of life and healthcare costs. While current therapies are effective in controlling many patients' symptoms, a large number continue to experience exacerbations or treatment-related adverse effects. Alternative therapies are thus urgently needed. Accumulating evidence has shown that the peroxisome proliferator-activated receptor (PPAR) family of nuclear hormone receptors, comprising PPARα, PPARβ/δ, and PPARγ, is involved in asthma pathogenesis and that ligand-induced activation of these receptors suppresses asthma pathology. PPAR agonists exert their anti-inflammatory effects primarily by suppressing pro-inflammatory mediators and antagonizing the pro-inflammatory functions of various cell types relevant to asthma pathophysiology. Experimental findings strongly support the potential clinical benefits of PPAR agonists in the treatment of asthma. We review current literature, highlighting PPARs' key role in asthma pathogenesis and their agonists' therapeutic potential. With additional research and rigorous clinical studies, PPARs may become attractive therapeutic targets in this disease