SHP2 inhibitors maintain TGFß signalling through SMURF2 inhibition.

Despite the promising antitumor activity of SHP2 inhibitors in RAS-dependent tumours, overall responses have been limited by their narrow therapeutic window. Like with all MAPK pathway inhibitors, this is likely the result of compensatory pathway activation mechanisms. However, the underlying mechanisms of resistance to SHP2 inhibition remain unknown. The E3 ligase SMURF2 limits TGFß activity by ubiquitinating and targeting the TGFß receptor for proteosome degradation. Using a functional RNAi screen targeting all known phosphatases, we identify that the tyrosine phosphatase SHP2 is a critical regulator of TGFß activity. Specifically, SHP2 dephosphorylates two key residues on SMURF2, resulting in activation of the enzyme. Conversely, SHP2 depletion maintains SMURF2 in an inactive state, resulting in the maintenance of TGFß activity. Furthermore, we demonstrate that depleting SHP2 has significant implications on TGFß-mediated migration, senescence, and cell survival. These effects can be overcome through the use of TGFß-targeted therapies. Consequently, our findings provide a rationale for combining SHP2 and TGFß inhibitors to enhance tumour responses leading to improved patient outcomes.

TGF-ß, EMT, and resistance to anti-cancer treatment.

Transforming growth factor-ß (TGF-ß) signaling regulates cell-specific programs involved in embryonic development, wound-healing, and immune homeostasis. Yet, during tumor progression, these TGF-ß-mediated programs are altered, leading to epithelial cell plasticity and a reprogramming of epithelial cells into mesenchymal lineages through epithelial-to-mesenchymal transition (EMT), a critical developmental program in morphogenesis and organogenesis. These changes, in turn, lead to enhanced carcinoma cell invasion, metastasis, immune cell differentiation, immune evasion, and chemotherapy resistance. Here, we discuss EMT as one of the critical programs associated with carcinoma cell plasticity and the influence exerted by TGF-ß on carcinoma status and function. We further explore the composition of carcinoma and other cell populations within the tumor microenvironment, and consider the relevant outcomes related to the programs associated with cancer treatment resistance.

Mechanisms of Resistance to PI3K Inhibitors in Cancer: Adaptive Responses, Drug Tolerance and Cellular Plasticity.

The phosphatidylinositol-3-kinase (PI3K) pathway plays a central role in the regulation of several signalling cascades which regulate biological processes such as cellular growth, survival, proliferation, motility and angiogenesis. The hyperactivation of this pathway is linked to tumour progression and is one of the most common events in human cancers. Additionally, aberrant activation of the PI3K pathway has been demonstrated to limit the effectiveness of a number of anti-tumour agents paving the way for the development and implementation of PI3K inhibitors in the clinic. However, the overall effectiveness of these compounds has been greatly limited by inadequate target engagement due to reactivation of the pathway by compensatory mechanisms. Herein, we review the common adaptive responses that lead to reactivation of the PI3K pathway, therapy resistance and potential strategies to overcome these mechanisms of resistance. Furthermore, we highlight the potential role in changes in cellular plasticity and PI3K inhibitor resistance.

OTUD4 enhances TGFß signalling through regulation of the TGFß receptor complex.

Systematic control of the transforming growth factor-ß (TGFß) pathway is essential to keep the amplitude and the intensity of downstream signalling at appropriate levels. Ubiquitination plays a crucial role in the general regulation of this pathway. Here we identify the deubiquitinating enzyme OTUD4 as a transcriptional target of the TGFß pathway that functions through a positive feedback loop to enhance overall TGFß activity. Interestingly we demonstrate that OTUD4 functions through both catalytically dependent and independent mechanisms to regulate TGFß activity. Specifically, we find that OTUD4 enhances TGFß signalling by promoting the membrane presence of TGFß receptor I. Furthermore, we demonstrate that OTUD4 inactivates the TGFß negative regulator SMURF2 suggesting that OTUD4 regulates multiple nodes of the TGFß pathway to enhance TGFß activity.

Author Correction: c-Met activation leads to the establishment of a TGFß-receptor regulatory network in bladder cancer progression.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

c-Met activation leads to the establishment of a TGFß-receptor regulatory network in bladder cancer progression.

Treatment of muscle-invasive bladder cancer remains a major clinical challenge. Aberrant HGF/c-MET upregulation and activation is frequently observed in bladder cancer correlating with cancer progression and invasion. However, the mechanisms underlying HGF/c-MET-mediated invasion in bladder cancer remains unknown. As part of a negative feedback loop SMAD7 binds to SMURF2 targeting the TGFß receptor for degradation. Under these conditions, SMAD7 acts as a SMURF2 agonist by disrupting the intramolecular interactions within SMURF2. We demonstrate that HGF stimulates TGFß signalling through c-SRC-mediated phosphorylation of SMURF2 resulting in loss of SMAD7 binding and enhanced SMURF2 C2-HECT interaction, inhibiting SMURF2 and enhancing TGFß receptor stabilisation. This upregulation of the TGFß pathway by HGF leads to TGFß-mediated EMT and invasion. In vivo we show that TGFß receptor inhibition prevents bladder cancer invasion. Furthermore, we make a rationale for the use of combinatorial TGFß and MEK inhibitors for treatment of high-grade non-muscle-invasive bladder cancers.

Adaptive Responses as Mechanisms of Resistance to BRAF Inhibitors in Melanoma.

: The introduction of v-raf murine sarcoma viral oncogene homolog B (BRAF) inhibitors in melanoma patients with BRAF (V600E) mutations has demonstrated significant clinical benefits. However, rarely do tumours regress completely. Frequently, the reason for this is that therapies targeting specific oncogenic mutations induce a number of intrinsic compensatory mechanisms, also known as adaptive responses or feedback loops, that enhance the pro-survival and pro-proliferative capacity of a proportion of the original tumour population, thereby resulting in tumour progression. In this review we will summarize the known adaptive responses that limit BRAF mutant therapy and discuss potential novel combinatorial therapies to overcome resistance.

MELK mediates the stability of EZH2 through site-specific phosphorylation in extranodal natural killer/T-cell lymphoma.

Oncogenic EZH2 is overexpressed and extensively involved in the pathophysiology of different cancers including extranodal natural killer/T-cell lymphoma (NKTL). However, the mechanisms regarding EZH2 upregulation is poorly understood, and it still remains untargetable in NKTL. In this study, we examine EZH2 protein turnover in NKTL and identify MELK kinase as a regulator of EZH2 ubiquitination and turnover. Using quantitative mass spectrometry analysis, we observed a MELK-mediated increase of EZH2 S220 phosphorylation along with a concomitant loss of EZH2 K222 ubiquitination, suggesting a phosphorylation-dependent regulation of EZH2 ubiquitination. MELK inhibition through both chemical and genetic means led to ubiquitination and destabilization of EZH2 protein. Importantly, we determine that MELK is upregulated in NKTL, and its expression correlates with EZH2 protein expression as determined by tissue microarray derived from NKTL patients. FOXM1, which connected MELK to EZH2 signaling in glioma, was not involved in mediating EZH2 ubiquitination. Furthermore, we identify USP36 as the deubiquitinating enzyme that deubiquitinates EZH2 at K222. These findings uncover an important role of MELK and USP36 in mediating EZH2 stability in NKTL. Moreover, MELK overexpression led to decreased sensitivity to bortezomib treatment in NKTL based on deprivation of EZH2 ubiquitination. Therefore, modulation of EZH2 ubiquitination status by targeting MELK may be a new therapeutic strategy for NKTL patients with poor bortezomib response.

Ubiquitination and adaptive responses to BRAF inhibitors in Melanoma.

Response to targeted therapies is limited by the activation or inhibition of feedback loops. Here we report the ubiquitin specific peptidase 28/F-box WD repeat-containing protein 7 (USP28/FBW7) complex functions as a negative regulator of mitogen-activated protein kinase (MAPK) pathway by targeting v-raf murine sarcoma viral oncogene homolog B (BRAF) for degradation, a process which is lost in a large proportion of BRAF mutant melanoma patients, resulting in resistance to BRAF inhibitor therapies.

Loss of USP28-mediated BRAF degradation drives resistance to RAF cancer therapies.

RAF kinase inhibitors are clinically active in patients with BRAF (V600E) mutant melanoma. However, rarely do tumors regress completely, with the majority of responses being short-lived. This is partially mediated through the loss of negative feedback loops after MAPK inhibition and reactivation of upstream signaling. Here, we demonstrate that the deubiquitinating enzyme USP28 functions through a feedback loop to destabilize RAF family members. Loss of USP28 stabilizes BRAF enhancing downstream MAPK activation and promotes resistance to RAF inhibitor therapy in culture and in vivo models. Importantly, we demonstrate that USP28 is deleted in a proportion of melanoma patients and may act as a biomarker for response to BRAF inhibitor therapy in patients. Furthermore, we identify Rigosertib as a possible therapeutic strategy for USP28-depleted tumors. Our results show that loss of USP28 enhances MAPK activity through the stabilization of RAF family members and is a key factor in BRAF inhibitor resistance.

Platforms for Investigating LncRNA Functions.

Prior to the sequencing of the human genome, it was presumed that most of the DNA coded for proteins. However, with the advent of next-generation sequencing, it has now been recognized that most complex eukaryotic genomes are in fact transcribed into noncoding RNAs (ncRNAs), including a family of transcripts referred to as long noncoding RNAs (lncRNAs). LncRNAs have been implicated in many biological processes ranging from housekeeping functions such as transcription to more specialized functions such as dosage compensation or genomic imprinting, among others. Interestingly, lncRNAs are not limited to a defined set of functions but can regulate varied activities such as messenger RNA degradation, translation, and protein kinetics or function as RNA decoys or scaffolds. Although still in its infancy, research into the biology of lncRNAs has demonstrated the importance of lncRNAs in development and disease. However, the specific mechanisms through which these lncRNAs act remain poorly defined. Focused research into a small number of these lncRNAs has provided important clues into the heterogeneous nature of this family of ncRNAs. Due to the complex diversity of lncRNA function, in this review, we provide an update on the platforms available for investigators to aid in the identification of lncRNA function.

Deciphering the roles of lncRNAs in breast development and disease.

Breast cancer is the second leading cause of cancer related deaths in women. It is therefore important to understand the mechanisms underlying breast cancer development as well as raises the need for enhanced, non-invasive strategies for novel prognostic and diagnostic methods. The emergence of long non-coding RNAs (lncRNAs) as potential key players in neoplastic disease has received considerable attention over the past few years. This relatively new class of molecular regulators has been shown from ongoing research to act as critical players for key biological processes. Deregulated expression levels of lncRNAs have been observed in a number of cancers including breast cancer. Furthermore, lncRNAs have been linked to breast cancer initiation, progression, metastases and to limit sensitivity to certain targeted therapeutics. In this review we provide an update on the lncRNAs associated with breast cancer and mammary gland development and illustrate the versatility of such lncRNAs in gene control, differentiation and development both in normal physiological conditions and in diseased states. We also highlight the therapeutic and diagnostic potential of lncRNAs in cancer.

The roles of ubiquitin modifying enzymes in neoplastic disease.

The initial experiments performed by Rose, Hershko, and Ciechanover describing the identification of a specific degradation signal in short-lived proteins paved the way to the discovery of the ubiquitin mediated regulation of numerous physiological functions required for cellular homeostasis. Since their discovery of ubiquitin and ubiquitin function over 30years ago it has become wholly apparent that ubiquitin and their respective ubiquitin modifying enzymes are key players in tumorigenesis. The human genome encodes approximately 600 putative E3 ligases and 80 deubiquitinating enzymes and in the majority of cases these enzymes exhibit specificity in sustaining either pro-tumorigenic or tumour repressive responses. In this review, we highlight the known oncogenic and tumour suppressive effects of ubiquitin modifying enzymes in cancer relevant pathways with specific focus on PI3K, MAPK, TGFß, WNT, and YAP pathways. Moreover, we discuss the capacity of targeting DUBs as a novel anticancer therapeutic strategy.

USP26 regulates TGF-ß signaling by deubiquitinating and stabilizing SMAD7.

The amplitude of transforming growth factor-ß (TGF-ß) signal is tightly regulated to ensure appropriate physiological responses. As part of negative feedback loop SMAD7, a direct transcriptional target of downstream TGF-ß signaling acts as a scaffold to recruit the E3 ligase SMURF2 to target the TGF-ß receptor complex for ubiquitin-mediated degradation. Here, we identify the deubiquitinating enzyme USP26 as a novel integral component of this negative feedback loop. We demonstrate that TGF-ß rapidly enhances the expression of USP26 and reinforces SMAD7 stability by limiting the ubiquitin-mediated turnover of SMAD7. Conversely, knockdown of USP26 rapidly degrades SMAD7 resulting in TGF-ß receptor stabilization and enhanced levels of p-SMAD2. Clinically, loss of USP26 correlates with high TGF-ß activity and confers poor prognosis in glioblastoma. Our data identify USP26 as a novel negative regulator of the TGF-ß pathway and suggest that loss of USP26 expression may be an important factor in glioblastoma pathogenesis.

USP15 regulates SMURF2 kinetics through C-lobe mediated deubiquitination.

Ubiquitin modification of the TGF-ß pathway components is emerging as a key mechanism of TGF-ß pathway regulation. To limit TGF-ß responses, TGF-ß signaling is regulated through a negative feedback loop whereby the E3 ligase SMURF2 targets the TGF-ß receptor (TßR) complex for ubiquitin-mediated degradation. Counteracting this process, a number of deubiquitinating (DUBs) enzymes have recently been identified that deubiquitinate and stabilize the TßR. However the precise mechanism by which these DUBs act on TßR function remains poorly defined. Here, we demonstrate that apart from targeting the TßR complex directly, USP15 also deubiquitinates SMURF2 resulting in enhanced TßR stability and downstream pathway activation. Through proteomic analysis, we show that USP15 modulates the ubiquitination of Lys734, a residue required for SMURF2 catalytic activity. Our results show that SMURF2 is a critical target of USP15 in the TGF-ß pathway and may also explain how USP15 and SMURF2 target multiple complementary protein complexes in other pathways.