Peter C. Lucas

A critical role of RICK/RIP2 polyubiquitination in Nod-induced NF-κB activation

Nod1 and Nod2 are intracellular proteins that are involved in host recognition of specific bacterial molecules and are genetically associated with several inflammatory diseases. Nod1 and Nod2 stimulation activates NF‐κB through RICK, a caspase‐recruitment domain‐containing kinase. However, the mechanism by which RICK activates NF‐κB in response to Nod1 and Nod2 stimulation is unknown. Here we show that RICK is conjugated with lysine‐63‐linked polyubiquitin chains at lysine 209 (K209) located in its kinase domain upon Nod1 or Nod2 stimulation and by induced oligomerization of RICK. Polyubiquitination of RICK at K209 was essential for RICK‐mediated IKK activation and cytokine/chemokine secretion. However, RICK polyubiquitination did not require the kinase activity of RICK or alter the interaction of RICK with NEMO, a regulatory subunit of IκB kinase (IKK). Instead, polyubiquitination of RICK was found to mediate the recruitment of TAK1, a kinase that was found to be essential for Nod1‐induced signaling. Thus, RICK polyubiquitination links TAK1 to IKK complexes, a critical step in Nod1/Nod2‐mediated NF‐κB activation.

Wnt10b Inhibits Development of White and Brown Adipose Tissues

Wnt is a family of secreted signaling proteins that regulate diverse developmental processes. Activation of canonical Wnt signaling by Wnt10b inhibits differentiation of preadipocytes in vitro. To determine whether Wnt signaling blocks adipogenesis in vivo, we created transgenic mice in which Wnt10b is expressed from the FABP4 promoter. Expression of Wnt10b in adipose impairs development of this tissue throughout the body, with a decline of ∼50% in total body fat and a reduction of ∼60% in weight of epididymal and perirenal depots. FABP4-Wnt10b mice resist accumulation of adipose tissue when fed a high fat diet. Furthermore, transgenic mice are more glucose-tolerant and insulin-sensitive than wild type mice. Expression of Wnt10b from the FABP4 promoter also blocks development of brown adipose tissue. Interscapular tissue of FABP4-Wnt10b mice has the visual appearance of white adipose tissue but expresses neither brown (e.g. uncoupling protein 1) nor white adipocyte markers. Transgenic mice are unable to maintain a core body temperature when placed in a cold environment, providing further evidence that Wnt10b inhibits development of brown adipose tissue. Although food intake is not altered in FABP4-Wnt10b mice, oxygen consumption is decreased. Thus, FABP4-Wnt10b mice on a chow diet gain more weight than controls, largely because of an increase in weight of skin. In summary, inhibition by Wnt10b of white and brown adipose tissue development results in lean mice without lipodystrophic diabetes.

Alpha-Methylacyl-CoA racemase: a novel tumor marker over-expressed in several human cancers and their precursor lesions.

&agr;-Methylacyl-CoA racemase (AMACR) is a mitochondrial and peroxisomal enzyme involved in the metabolism of branched-chain fatty acid and bile acid intermediates. Recently, AMACR has been demonstrated to be over-expressed in localized and metastatic prostate cancer, suggesting that it may be an important tumor marker. This study examines AMACR expression in a variety of human cancers and their precursor lesions. A survey of online Expressed Sequence Tags (ESTs) and Serial Analysis of Gene Expression (SAGE) databases revealed that AMACR was over-expressed in multiple cancers. The findings were confirmed by AMACR immunohistochemistry performed on several tissue microarrays containing common human tumors, including prostate, colon, and breast. Based on prior work, AMACR protein expression was divided into two categories: negative (negative to weak staining intensity) and positive (moderate to strong staining intensity). AMACR protein over-expression was found in a number of cancers, including colorectal, prostate, ovarian, breast, bladder, lung, and renal cell carcinomas, lymphoma, and melanoma. Greatest over-expression was seen in colorectal and prostate cancer with positive staining in 92% and 83% cases, respectively. AMACR over-expression was present in 44% of breast cancer cases. AMACR was also over-expressed in precursor lesions. Sixty-four percent of high-grade prostatic intraepithelial neoplasia and 75% colonic adenomas demonstrated positive AMACR protein expression. Reverse transcriptase-polymerase chain reaction for AMACR using laser capture microdissected prostate tissue confirmed gene over-expression at the mRNA level. In conclusion, our study suggests that AMACR is potentially an important tumor marker for several cancers and their precursor lesions, especially those linked to high-fat diets.

Loss of CDX2 Expression and Microsatellite Instability Are Prominent Features of Large Cell Minimally Differentiated Carcinomas of the Colon

Most large bowel cancers are moderately to well-differentiated adenocarcinomas comprised chiefly or entirely of glands lined by tall columnar cells. We have identified a subset of poorly differentiated colon carcinomas with a distinctive histopathological appearance that we term large cell minimally differentiated carcinomas (LCMDCs). These tumors likely include a group of poorly differentiated carcinomas previously described by others as medullary adenocarcinomas. To better understand the pathogenesis of these uncommon neoplasms, we compared molecular features of 15 LCMDCs to those present in 25 differentiated adenocarcinomas (DACs) of the colon. Tumors were examined for alterations commonly seen in typical colorectal carcinomas, including increased p53 and beta-catenin immunoreactivity, K-ras gene mutations, microsatellite instability, and loss of heterozygosity of markers on chromosomes 5q, 17p, and 18q. In addition, tumors were evaluated by immunohistochemistry for CDX2, a homeobox protein whose expression in normal adult tissues is restricted to intestinal and colonic epithelium. Markedly reduced or absent CDX2 expression was noted in 13 of 15 (87%) LCMDCs, whereas only 1 of the 25 (4%) DACs showed reduced CDX2 expression (P < 0.001). Nine of 15 (60%) LCMDCs had the high-frequency microsatellite instability phenotype, but only 2 of 25 (8%) DACs had the high-frequency microsatellite instability phenotype (P = 0.002). Our findings provide support for the hypothesis that the molecular pathogenesis of LCMDCs is distinct from that of most DACs. CDX2 alterations and DNA mismatch repair defects have particularly prominent roles in the development of LCMDCs.

Clinicopathologic features of metastasis in nonsentinel lymph nodes of breast carcinoma patients: A metaanalysis

In breast carcinoma patients with a positive sentinel lymph node (SN), the value of complete axillary lymph node dissection has been questioned. Multiple published reports have attempted to identify clinicopathologic characteristics of the primary tumor and SN that are associated with an increased likelihood of positive nonsentinel lymph nodes (NSN). Because of differences in lymph node evaluation techniques and limited patient numbers in each study, the authors performed a meta‐analysis to assess the regularity and relative strength of association between various characteristics and the risk of NSN metastasis.

CARMA3/Bcl10/MALT1-dependent NF-κB activation mediates angiotensin II-responsive inflammatory signaling in nonimmune cells

Angiotensin II (Ang II) is a peptide hormone that, like many cytokines, acts as a proinflammatory agent and growth factor. After injury to the liver, the hormone assists in tissue repair by stimulating hepatocytes and hepatic stellate cells to synthesize extracellular matrix proteins and secrete secondary cytokines and by stimulating myofibroblasts to proliferate. However, under conditions of chronic liver injury, all of these effects conspire to promote pathologic liver fibrosis. Much of this effect of Ang II results from activation of the proinflammatory NF-κB transcription factor in response to stimulation of the type 1 Ang II receptor, a G protein-coupled receptor. Here, we characterize a previously undescribed signaling pathway mediating Ang II-dependent activation of NF-κB, which is composed of three principal proteins, CARMA3, Bcl10, and MALT1. Blocking the function of any of these proteins, through the use of either dominant-negative mutants, RNAi, or gene targeting, effectively abolishes Ang II-dependent NF-κB activation in hepatocytes. In addition, Bcl10−/− mice show defective hepatic cytokine production after Ang II treatment. Evidence also is presented that this pathway activates NF-κB through ubiquitination of IKKγ, the regulatory subunit of the IκB kinase complex. These results elucidate a concrete series of molecular events that link ligand activation of the type 1 Ang II receptor to stimulation of the NF-κB transcription factor. These findings also uncover a function of the CARMA, Bcl10, and MALT1 proteins in cells outside the immune system.

α-Methylacyl-CoA Racemase: Expression Levels of this Novel Cancer Biomarker Depend on Tumor Differentiation

alpha-Methylacyl-CoA racemase (AMACR) has previously been shown to be a highly sensitive marker for colorectal and clinically localized prostate cancer (PCa). However, AMACR expression was down-regulated at the transcript and protein level in hormone-refractory metastatic PCa, suggesting a hormone-dependent expression of AMACR. To further explore the hypothesis that AMACR is hormone regulated and plays a role in PCa progression AMACR protein expression was characterized in a broad range of PCa samples treated with variable amounts and lengths of exogenous anti-androgens. Analysis included standard slides and high-density tissue microarrays. AMACR protein expression was significantly increased in localized hormone-naive PCa as compared to benign (P < 0.001). Mean AMACR expression was lower in tissue samples from patients who had received neoadjuvant hormone treatment but still higher compared to hormone-refractory metastases. The hormone-sensitive tumor cell line, LNCaP, demonstrated stronger AMACR expression by Western blot analysis than the poorly differentiated cell lines DU-145 and PC-3. AMACR protein expression in cells after exposure to anti-androgen treatment was unchanged, whereas prostate-specific antigen, known to be androgen-regulated, demonstrated decreased protein expression. Surprisingly, this data suggests that AMACR expression is not regulated by androgens. Examination of colorectal cancer, which is not hormone regulated, demonstrated high levels of AMACR expression in well to moderately differentiated tumors and weak expression in anaplastic colorectal cancers. Taken together, these data suggest that AMACR expression is not hormone-dependent but may in fact be a marker of tumor differentiation.

Cleavage of NIK by the API2-MALT1 Fusion Oncoprotein Leads to Noncanonical NF-κB Activation

An oncogenic fusion protein promotes lymphomagenesis by activating a noncanonical signaling pathway. Proper regulation of nuclear factor κB (NF-κB) transcriptional activity is required for normal lymphocyte function, and deregulated NF-κB signaling can facilitate lymphomagenesis. We demonstrate that the API2-MALT1 fusion oncoprotein created by the recurrent t(11;18)(q21;q21) in mucosa-associated lymphoid tissue (MALT) lymphoma induces proteolytic cleavage of NF-κB–inducing kinase (NIK) at arginine 325. NIK cleavage requires the concerted actions of both fusion partners and generates a C-terminal NIK fragment that retains kinase activity and is resistant to proteasomal degradation. The resulting deregulated NIK activity is associated with constitutive noncanonical NF-κB signaling, enhanced B cell adhesion, and apoptosis resistance. Our study reveals the gain-of-function proteolytic activity of a fusion oncoprotein and highlights the importance of the noncanonical NF-κB pathway in B lymphoproliferative disease.

NF-κB signaling in lymphocytes: a new cast of characters

Cell-surface antigen receptors on B and T lymphocytes are complex, multisubunit assemblies that must recruit several accessory proteins and activate multiple signaling pathways in order to illicit a proper immune response. One pathway culminates in the activation of specific protein kinase C (PKC) isoforms, which is necessary for the ultimate activation of the NF-κB transcription factor. Since NF-κB plays a crucial role in the adaptive immune response (e.g. in lymphocyte proliferation and cytokine production), it is important to understand the molecular mechanisms by which NF-κB is regulated. Nevertheless, the connection between PKC activation and NF-κB has remained a mystery that has now been at least partly solved. Recent findings implicate a new scaffolding protein, Bimp3/CARMA1/CARD11, as a key factor in bridging PKC activation with the downstream activation of Bcl10 and MALT1, which ultimately stimulates NF-κB. Since some of these signaling components are lymphocyte specific, therapeutic agents that block this pathway could blunt the inappropriate proliferation of lymphocytes associated with certain inflammatory and neoplastic disorders. Alternatively, agents that specifically augment this pathway, thereby enhancing immune function in immunodeficiency, may be developed.

AGTR1 overexpression defines a subset of breast cancer and confers sensitivity to losartan, an AGTR1 antagonist

Breast cancer patients have benefited from the use of targeted therapies directed at specific molecular alterations. To identify additional opportunities for targeted therapy, we searched for genes with marked overexpression in subsets of tumors across a panel of breast cancer profiling studies comprising 3,200 microarray experiments. In addition to prioritizing ERBB2, we found AGTR1, the angiotensin II receptor type I, to be markedly overexpressed in 10–20% of breast cancer cases across multiple independent patient cohorts. Validation experiments confirmed that AGTR1 is highly overexpressed, in several cases more than 100-fold. AGTR1 overexpression was restricted to estrogen receptor-positive tumors and was mutually exclusive with ERBB2 overexpression across all samples. Ectopic overexpression of AGTR1 in primary mammary epithelial cells, combined with angiotensin II stimulation, led to a highly invasive phenotype that was attenuated by the AGTR1 antagonist losartan. Similarly, losartan reduced tumor growth by 30% in AGTR1-positive breast cancer xenografts. Taken together, these observations indicate that marked AGTR1 overexpression defines a subpopulation of ER-positive, ERBB2-negative breast cancer that may benefit from targeted therapy with AGTR1 antagonists, such as losartan.