NEDD4L protein levels are down in CRC.

<p>(A) NEDD4L levels were determined by western blotting of CRC (Ca) and adjacent normal (Nl) mucosa from twenty individuals. Levels were normalized to TUBA and Nl was compared to Ca. NEDD4L was significantly downregulated (∼42%) in CRC (*p<0.05). Data is represented in box and whisker plot format. The lines connecting data points show the relative NEDD4L levels in a given Nl-Ca matched pair. Red denotes decreased NEDD4L levels in Ca, while black denotes an increase. (B) Lysates generated from Nl-Ca matched pairs were blotted for NEDD4L and TUBA. Shown here are four representative pairs.</p

The NEDD4 family of E3 ubiquitin ligases.

<p>The nine members of the NEDD4 family are modular proteins. At their N-terminus, each has a C2 domain, which plays a role in proper cellular localization. The central portion of each protein contains two-to-four WW domains, which are responsible for target recognition. WW domains are 20–23 amino acid, Trp-based motifs that recognize PY motifs (PPXY, LPXY), as well as some phospho-Ser- or phospho-Thr-based motifs. At the C-terminus is the HECT domain, which directly conjugates an ubiquityl moiety to the target protein. The family members are grouped together by name and homology. ITCH is more closely homologous to WWP1 and 2 than to other family members.</p

Expression levels of the NEDD4 family in CRC.

<p>(A) The expression of all nine NEDD4 family members was examined in CRC by microarray profiling. Shown here is the average fold-change of all probes for each individual gene in Nl (N = 10), Ad (N = 6), Ca1 (N = 33), Ca2 (N = 76), Ca3 (N = 82), and Ca4 (N = 59). (B) <i>NEDD4</i> (213012_at) is the most highly upregulated member of the NEDD4 family in CRC. (C) <i>NEDD4L</i> (212445_s_at) is the most highly downregulated member of the NEDD4 family in CRC. Nl =  normal, Ad =  adenoma, and Ca1-4 = CRC, stage I-IV. *p<0.05.</p

The known targets of the NEDD4 family, the signaling pathways affected, and their expression levels in human cancers.

<p>In the “Signaling pathways affected” and “Expression in cancer” columns, ↓ indicates inhibition and downregulation, respectively, while ↑ indicates activation and upregulation. Patched (PTC), epithelial sodium channel (ENaC), cyclic nucleotide ras GEF (CNrasGEF), androgen receptor (AR), B-cell CLL/lymphoma 10 (Bcl10), potassium voltage-gated channel, shaker-related subfamily, member 3 (Kv1.3), sodium/glucose cotransporter 1 (SGLT1), sodium channel, voltage-gated, type V (NaV1.5), amyloid beta A4 precursor protein-binding family B member 1 (Fe65), dopamine transporter (DAT), inhibitor of growth 2 (ING2).</p

NEDD4L inhibits canonical WNT signaling at or below the level of β-catenin activation.

<p>(A) NEDD4L inhibits TOPFlash activity in HEK293 cells compared to empty vector (CTL) or catalytically inactive NEDD4L [NEDD4L (C>A)]. WNT3A (20 ng/ml) and R-spondin (100 ng/ml) were used to activate canonical WNT signaling. (B) When two different β-catenin constructs, β-catenin (wt) and β-catenin (ΔN89), are used to activate canonical WNT signaling a significant reduction in TOPFlash activity was observed in the presence of NEDD4L overexpression, as compared to empty vector or NEDD4L (C>A). All results are normalized to FOPFlash activity. *p<0.05.</p

Diminished TK1 protein levels correlate with attenuation of mTOR-PI3K pathway activity and upregulation of p27.

<p>(<b>A</b>) Western blot of DiFi cell lysates following cetuximab exposure (5 µg/mL) resulted in rapid attenuation of downstream MAPK targets including p-ERK, which remained well-below baseline levels to 24 hours. Paradoxically, TK1 protein levels increased from 1–12 hours until p27 protein levels rose, at which time TK1 fell bellow baseline. (<b>B</b>) DiFi cells treated with either 0.5 µg/mL or 5.0 µg/mL cetuximab resulted in a 50% reduction and full attenuation of p-ERK protein levels, respectively at 24 hours. At 0.5 µg/mL TK1 levels were unaffected despite a modest rise in p27 protein levels. However, 5.0 µg/mL cetuximab resulted in greatly decreased TK1 with a large increase in p27 protein levels. PI3K-mTOR activity, as measured by p-AKT Ser473, p-rpS6, and p-4E-BP1, was either maintained or elevated at 0.5 µg/mL cetuximab but was attenuated at 5.0 µg/mL cetuximab. (<b>C</b>) qRT-PCR analysis showed <i>TK1</i> mRNA was significantly reduced at 0.5 µg/mL (p = 0.0279) and 5.0 µg/mL (p = 0.0186), while no change in <i>p27</i> mRNA levels was observed at either dose. (<b>D</b>) Silencing mTORC1 facilitated upregulation of p27 and attenuated TK1 protein levels at 0.5 µg/mL cetuximab without affecting the anti-MAPK activity effects of cetuximab. Levels of p-AKT Ser473, p-rpS6, and p-4E-BP1 were reduced at 0.5 µg/mL cetuximab exposure with Raptor knockdown but not in scrambled siRNA control. (<b>E</b>) As evidence of the functionality of p27, <i>TK1</i> mRNA levels were similarly reduced at 0.5 µg/mL and 5.0 µg/mL with Raptor knockdown.</p

Proteomic Consequences of a Single Gene Mutation in a Colorectal Cancer Model

The proteomic effects of specific cancer-related mutations have not been well characterized. In colorectal cancer (CRC), a relatively small number of mutations in key signaling pathways appear to drive tumorigenesis. Mutations in adenomatous polyposis coli (APC), a negative regulator of Wnt signaling, occur in up to 60% of CRC tumors. Here we examine the proteomic consequences of a single gene mutation by using an isogenic CRC cell culture model in which wildtype APC expression has been ectopically restored. Using LC–MS/MS label free shotgun proteomics, over 5000 proteins were identified in SW480Null (mutant APC) and SW480APC (APC restored). We observed 155 significantly differentially expressed proteins between the two cell lines, with 26 proteins showing opposite expression trends relative to gene expression measurements. Protein changes corresponded to previously characterized features of the APCNull phenotype: loss of cell adhesion proteins, increase in cell cycle regulators, alteration in Wnt signaling related proteins, and redistribution of β-catenin. Increased expression of RNA processing and isoprenoid biosynthetic proteins occurred in SW480Null cells. Therefore, shotgun proteomics reveals proteomic differences associated with a single gene change, including many novel differences that fall outside known target pathways

Combined ^V600EBRAF and mTOR inhibition results in transcriptional control of TK1 protein levels in COLO 205 cells.

<p>(<b>A</b>) Western blot of COLO 205 cells treated with PP242 (250 nM) and increasing PLX4032. Similar to single agent PLX4032, p-MEK, but not p-ERK, was inhibited in a PLX4032-dependent manner. Consistent with mTORC1/mTORC2 inhibition, p-AKT Ser473, but not p-AKT Thr308, was inhibited. Unlike single agent PLX4032, which resulted in concentration-dependent activation of p-rpS6, combined treatment maintained p-rpS6 levels at essentially baseline levels except at the highest PLX4032 concentration. Similarly, DUSP6 levels were inversely related to p-ERK protein levels. With combined mTOR and ^V600EBRAF blockade, p27 and TK1 protein levels were inversely correlated and dramatically affected by PLX4032 exposure. (<b>B</b>) Similarly, <i>TK1</i> mRNA was significantly reduced at PLX4032 concentrations as low as 10 nM. (<b>C</b>) Despite elevated p27 protein levels, <i>p27</i> mRNA was unaffected by combined mTOR-^V600EBRAF inhibition.</p

PLX4720 exposure does not affect [¹⁸F]-FLT PET in COLO 205 xenografts, despite evidence of target inhibition and diminished [¹⁸F]-FDG uptake.

<p>(<b>A</b>) Representative transverse [¹⁸F]-FLT and [¹⁸F]-FDG PET images acquired after three daily treatments with vehicle or 60 mg/kg PLX4720 (tumor indicated by arrowhead). (<b>B</b>) Quantification of PET data illustrated similar [¹⁸F]-FLT uptake in vehicle-treated and PLX4720-treated tumors. Unlike [¹⁸F]-FLT PET, PLX4720 exposure elicited a significant reduction in [¹⁸F]-FDG uptake (p = 0.0006). (<b>C</b>) Western blot analysis of vehicle- and PLX4720-treated tumor tissue confirmed that PLX4720 had no effect on TK1 protein levels in agreement with [¹⁸F]-FLT PET. Target inhibition as measured by p-MEK levels was observed. However, similar to <i>in vitro</i> studies, PLX4720-treated COLO 205 xenografts exhibited elevated p-ERK and p-rpS6 protein levels relative to vehicle controls.</p

[¹⁸F]-FLT PET reflects BEZ235-dependent inhibition of PI3K/mTOR activity in PLX4720 treated COLO 205 xenografts.

<p> Xenograft-bearing mice were imaged with [¹⁸F]-FLT PET on treatment day 4. (<b>A</b>) [¹⁸F]-FLT uptake was diminished in the combination treatment cohort relative to vehicle (p = 0.0087), but not single agent PLX4720- or BEZ235-treated cohorts. (<b>B</b>) Western blot of xenograft tissue harvested immediately following imaging illustrated elevated p-ERK and p-rpS6 levels in PLX4720-treated mice. Combining PLX4032 with BEZ235 resulted in reduced p-ERK and p-rpS6 protein levels. (<b>C</b>) TK1 levels, as measured by IHC, were reduced only in the combination treatment group in agreement with [¹⁸F]-FLT PET. (<b>D</b>) Consistent with <i>in vitro</i> studies, diminished TK1 levels, and consequently [¹⁸F]-FLT PET, correlated with elevated p27 that was elevated only in the combination treated group.</p