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Tyro3, Axl, Mer (TAM) receptor tyrosine kinases reduce inflammatory, innate immune responses. We demonstrate that tumor-secreted protein S (Pros1), a Mer/Tyro3 ligand, decreased macrophage M1 cytokine expression in vitro and in vivo. In contrast, tumor cells with CRISPR-based deletion of Pros1 failed to inhibit M1 polarization. Tumor cell-associated Pros1 action was abrogated in macrophages from Mer- and Tyro3- but not Axl-KO mice. In addition, several other murine and human tumor cell lines suppressed macrophage M1 cytokine expression induced by IFN-γ and LPS. Investigation of the suppressive pathway demonstrated a role for PTP1b complexing with Mer. Substantiating the role of PTP1b, M1 cytokine suppression was also lost in macrophages from PTP1b-KO mice. Mice bearing Pros1-deficient tumors showed increased innate and adaptive immune infiltration, as well as increased median survival. TAM activation can also inhibit TLR-mediated M1 polarization. Treatment with resiquimod, a TLR7/8 agonist, did not improve survival in mice bearing Pros1-secreting tumors but doubled survival for Pros1-deleted tumors. The tumor-derived Pros1 immune suppressive system, like PD-L1, was cytokine responsive, with IFN-γ inducing Pros1 transcription and secretion. Inhibition of Pros1/TAM interaction represents a potential novel strategy to block tumor-derived immune suppression.

Purpose Rectal cancer is often clinically resistant to radiotherapy (RT) and identifying molecular markers to define the biologic basis for this phenomenon would be valuable. The nuclear factor κ-light chain-enhancer of activated B cells (NF-κB) is a potential anti-apoptotic transcription factor that has been associated with resistance to RT in model systems. The present study was designed to evaluate NF-κB activation in patients with rectal cancer undergoing chemoradiotherapy to determine whether NF-κB activity correlates with the outcome in rectal cancer patients. Methods and Materials A total of 22 patients underwent biopsy at multiple points in a prospective study and the data from another 50 were analyzed retrospectively. The pretreatment tumor tissue was analyzed for multiple NF-κB subunits by immunohistochemistry. Serial tumor biopsy cores were analyzed for NF-κB–regulated gene expression using reverse transcriptase polymerase chain reaction and for NF-κB subunit nuclear localization using immunohistochemistry. Results Several NF-κB target genes (Bcl-2, cellular inhibitor of apoptosis protein [cIAP]2, interleukin-8, and tumor necrosis factor receptor-associated-1) were significantly upregulated by a single fraction of RT at 24 h, demonstrating for the first time that NF-κB is activated by RT in human rectal tumors. The baseline NF-κB p50 nuclear expression did not correlate with the pathologic response to RT. However, an increasing baseline p50 level was prognostic for overall survival (hazard ratio, 2.15; p = .040). Conclusion NF-κB nuclear expression at baseline in rectal cancer was prognostic for overall survival but not predictive of the response to RT. Larger patient numbers are needed to assess the effect of NF-κB target gene upregulation on the response to RT. Our results suggest that NF-κB might play an important role in tumor metastasis but not to the resistance to chemoradiotherapy.

Unlike the proliferative action of other epidermal growth factor (EGF) receptor family members, HER4/ErbB4 is often associated with growth-inhibitory and differentiation signaling. These actions may involve HER4 two-step proteolytic processing by intramembraneous gamma-secretase, releasing the soluble, intracellular 80-kDa HER4 cytoplasmic domain, s80HER4. We demonstrate that pharmacological inhibition of either gamma-secretase activity or HER4 tyrosine kinase activity blocked heregulin-dependent growth inhibition of SUM44 breast cancer cells. We next generated breast cell lines stably expressing GFP-s80HER4 [green fluorescent protein (GFP) fused to the N terminus of the HER4 cytoplasmic domain, residues 676-1308], GFP-CT(HER4) (GFP fused to N terminus of the HER4 C-terminus distal to the tyrosine kinase domain, residues 989-1308), or GFP alone. Both GFP-s80HER4 and GFP-CTHER4 were found in the nucleus, but GFP-s80HER4 accumulated to a greater extent and sustained its nuclear localization. s80HER4 was constitutively tyrosine phosphorylated, and treatment of cells with a specific HER family tyrosine kinase inhibitor 1) blocked tyrosine phosphorylation; 2) markedly diminished GFP-s80HER4 nuclear localization; and 3) reduced signal transducer and activator of transcription (STAT)5A tyrosine phosphorylation and nuclear localization as well as GFP-s80HER4:STAT5A interaction. Multiple normal mammary and breast cancer cell lines, stably expressing GFP-s80HER4 (SUM44, MDA-MB-453, MCF10A, SUM102, and HC11) were growth inhibited compared with the same cell line expressing GFP-CTHER4 or GFP alone. The s80HER4-induced cell number reduction was due to slower growth because rates of apoptosis were equivalent in GFP-, GFP-CTHER4-, and GFP-s80HER4-expressing cells. Lastly, GFP-s80HER4 enhanced differentiation signaling as indicated by increased basal and prolactin-dependent beta-casein expression. These results indicate that surface HER4 tyrosine phosphorylation and ligand-dependent release of s80HER4 are necessary, and s80HER4 signaling is sufficient for HER4-dependent growth inhibition.

The function of the epidermal growth factor receptor (EGFR) family member HER4 remains unclear because its activating ligand, heregulin, results in either proliferation or differentiation. This variable response may stem from the range of signals generated by HER4 homodimers versus heterodimeric complexes with other EGFR family members. The ratio of homo- and heterodimeric complexes may be influenced both by a cell's EGFR family member expression profile and by the ligand or even ligand isoform used. To define the role of HER4 in mediating antiproliferative and differentiation responses, human breast cancer cell lines were screened for responses to heregulin. Only cells that expressed HER4 exhibited heregulin-dependent antiproliferative responses. In-depth studies of one line, SUM44, demonstrated that the antiproliferative and differentiation responses correlated with HER4 activation and were abolished by stable expression of a kinase-inactive HER4. HB-EGF, a HER4-specific ligand in this EGFR-negative cell line, also induced an antiproliferative response. Moreover, introduction and stable expression of HER4 in HER4-negative SUM102 cells resulted in the acquisition of a heregulin-dependent antiproliferative response, associated with increases in markers of differentiation. The role of HER2 in these heregulin-dependent responses was examined through elimination of cell surface HER2 signaling by stable expression of a single-chain anti-HER2 antibody that sequestered HER2 in the endoplasmic reticulum. In the cell lines with either endogenously (SUM44) or exogenously (SUM102) expressed HER4, elimination of HER2 did not alter HER4-dependent decreases in cell growth. These results suggest that HER4 is both necessary and sufficient to trigger an antiproliferative response in human breast cancer cells.

NEDD8 is a novel ubiquitin-like protein that has been shown to conjugate to nuclear proteins in a manner analogous to ubiquitination and sentrinization. To identify proteins that are involved in the NEDD8-conjugation and de-conjugation pathway, the yeast two-hybrid system was used to screen a human heart cDNA library using NEDD8 as a bait. Seven strongly positive clones were found to contain a cDNA insert encoding the ubiquitin C-terminal hydrolase, UCH-L3. In vitro GST pull-down assay demonstrated that UCH-L3 bound to both NEDD8 and ubiquitin. In contrast, UCH-L3 did not bind to sentrin-1, sentrin-2, or sentrin-3. Recombinant UCH-L3, but not UCH-L1, was able to cleave the C-terminus of NEDD8. Thus, UCH-L3 can function as a C-terminal hydrolase for both NEDD8 and ubiquitin. UCH-L3 may play a physiologically significant role in the cleavage of the C-terminus of NEDD8, which is required for NEDD8 to conjugate to target proteins.

Sentrin is a ubiquitin-like molecule that has been shown to interact with the death domains of Fas and tumor necrosis factor receptor 1 (TNFR1), PML, Rad51, Rad52, and RanGAP1. We have reported previously that sentrin can be conjugated to other proteins in a manner analogous to protein ubiquitination (Kamitani, T., Nguyen, H. P., and Yeh, E. T. H. (1997) J. Biol. Chem. 272, 14001-14004). Furthermore, the conserved C-terminal Gly-Gly residues are required for sentrinization to occur. To identify enzymes which play a role in sentrinization, the yeast two-hybrid system was used to screen a human placenta cDNA library using sentrin as bait. A strong positive interacting clone was found to contain a cDNA insert encoding the ubiquitin-conjugating enzyme, Ubc9. The interaction between sentrin and Ubc9 required the ubiquitin domain and the C-terminal Gly-Gly residues of sentrin. This interaction appears to be specific because sentrin could only interact weakly with UbcH5B, but could not interact with HHR6B, UbcH6 nor E2-EPF. In vitro translated sentrin could be precipitated by a GST-Ubc9 fusion protein, but not by glutathione S-transferase. A beta-mercaptoethanol-sensitive Ubc9-sentrin conjugate could also be identified in the in vitro binding assay. Substitution of the conserved cysteine residue of Ubc9 by serine abolished the formation of the Ubc9-sentrin conjugate. Taken together, Ubc9 is a strong candidate to be the key conjugating enzyme in the sentrinization pathway.

A periplasmic arginine transport system that is a member of the ATP -dependent transport superfamily was identified in Pasteurella haemolytica. The gene encoding the periplasmic binding protein (lapT) was cloned and the protein overexpressed in Escherichia coli. LapT was purified to homogeneity using a modified osmotic shock procedure and anion-exchange column chromatography. Filter-binding assays established that LapT is an L-arginine-binding protein. Various amino acids were tested for their ability to inhibit L-arginine binding to LapT. When present in 100-fold excess, only L-arginine, D-arginine and citrulline competed with L-arginine for binding. Arginine transport in P. haemolytica whole cells was competitively inhibited by the same amino acids, suggesting that the LapT permease specifically transports L-arginine. The dissociation constant for the L-arginine-LapT complex was 170 nM and the stoichiometry of binding was approximately 0.8 mol L-arginine (mol LapT)-1. A polyclonal antibody raised against the purified protein permitted detection of LapT in P. haemolytica periplasmic fractions.

Standard therapy for stage II/III rectal cancer consists of a fluoropyrimidine and radiation therapy followed by surgery. Preclinical data demonstrated that bortezomib functions as a radiosensitizer in colorectal cancer models. The purpose of this study was to determine the maximum tolerated dose (MTD) of bortezomib in combination with chemotherapy and radiation.Patients with locally advanced rectal adenocarcinomas, as staged by endoscopic ultrasound, were eligible. Bortezomib was administered on days 1, 4, 8, and 11 every 21 days for 2 cycles with 5-fluorouracil at 225 mg/m2/day continuously and 50.4 Gy of radiation. Dose escalation of bortezomib was conducted via a standard 3 + 3 dose escalation design. A subset of patients underwent serial tumor biopsies for correlative studies.Nine patients in 2 dose cohorts were enrolled. Diarrhea was the principal dose-limiting toxicity and occurred at the 1.0-mg/m2 dose level. There was no clear evidence of suppression of nuclear factor-kappaB target gene expression in biopsy samples.The MTD of bortezomib in combination with chemotherapy and radiation may be below a clinically relevant dose, limiting the clinical applicability of this combination. Performing biopsies before and during irradiation for determining gene expression in response to radiation therapy is feasible.

In general, epidermal growth factor receptor family members stimulate cell proliferation. In contrast, at least one HER4 isoform, JM-a/Cyt1, inhibits cell growth after undergoing a two-step proteolytic cleavage that first produces a membrane-anchored 80-kDa fragment (m80(HER4)) and subsequently liberates a soluble 80-kDa fragment, s80(HER4). Here we report that s80(HER4) Cyt1 action increased the expression of WWP1 (for WW domain-containing protein 1), an E3 ubiquitin ligase, but not other members of the Nedd4 E3 ligase family. The HER4 Cyt1 isoform contains three proline-rich tyrosine (PY) WW binding motifs, while Cyt2 has only two. WWP1 binds to all three Cyt1 PY motifs; the interaction with PY2 found exclusively in Cyt1 was strongest. WWP1 ubiquitinated and caused the degradation of HER4 but not of EGFR, HER2, or HER3. The HER4-WWP1 interaction also accelerated WWP1 degradation. Membrane HER4 (full length and m80(HER4), the product of the first proteolytic cleavage) were the preferred targets of WWP1, correlating with the membrane localization of WWP1. Conversely s80(HER4), a poorer WWP1 substrate, was found in the cell nucleus, while WWP1 was not. Deletion of the C2 membrane association domain of WWP1 allowed more efficient s80(HER4) degradation, suggesting that WWP1 is normally part of a membrane complex that regulates HER4 membrane species levels, with a predilection for the growth-inhibitory Cyt1 isoform. Finally, WWP1 expression diminished HER4 biologic activity in MCF-7 cells. We previously showed that nuclear s80(HER4) is ubiquitinated and degraded by the anaphase-promoting complex, suggesting that HER4 ubiquitination within specific cellular compartments helps regulate the unique HER4 signaling capabilities.

HER4 expression in human breast cancers correlates with a positive prognosis. While heregulin inhibits the growth of HER4-positive breast cancer cells, it does so by undefined mechanisms. We demonstrate that heregulin-induced HER4 activity inhibits cell proliferation and delays G(2)/M progression of breast cancer cells. While investigating pathways of G(2)/M delay, we noted that heregulin increased the expression of BRCA1 in a HER4-dependent, HER2-independent manner. Induction of BRCA1 by HER4 occurred independently of the cell cycle. Moreover, BRCA1 expression was elevated in HER4-postive human breast cancer specimens. Heregulin stimulated c-Jun N-terminal kinase (JNK), and pharmacologic inhibition of JNK impaired heregulin-enhanced expression of BRCA1 and mitotic delay; inhibition of Erk1/2 did not. Knockdown of BRCA1 with small interfering RNA in a human breast cancer cell line interfered with HER4-mediated mitotic delay. Heregulin/HER4-dependent mitotic delay was examined further with an isogenic pair of mouse mammary epithelial cells (MECs) derived from mice harboring homozygous LoxP sites flanking exon 11 of BRCA1, such that one cell line expressed BRCA1 while the other cell line, after Cre-mediated excision, did not. BRCA1-positive MECs displayed heregulin-dependent mitotic delay; however, the isogenic BRCA1-negative MECs did not. These results suggest that heregulin-mediated growth inhibition in HER4-postive breast cancer cells requires BRCA1.

Efforts to conclusively establish that human epidermal receptor (HER)-2 overexpression is important to androgen-dependent carcinoma of the prostate (AD-CaP) or to progression to androgen independence (AI-CaP) have failed because of variability in tissue procurement, antibodies, immunostaining procedures, and assessment methods. However, because some in vitro and animal model data correlate HER-2 overexpression with progression to androgen independence, trials of agents that target the HER-2 receptor are under way. To clarify human tumor findings, we studied HER-2 expression at the gene (DNA), mRNA, and protein levels in well-characterized CaP specimens.Fifty AD-CaP and 25 AI-CaP specimens from similar numbers of Caucasian and African Americans were immunostained for HER-2 receptor. HER-2 mRNA levels were measured using real-time fluorescence quantitative PCR in patients for whom frozen specimens were available. HER-2 amplification was evaluated using fluorescent in situ hybridization.HER-2 receptor immunostained in 52% of androgen-dependent and one (4%) androgen-independent tumor. HER-2 immunostaining was not related to age, race, serum prostate-specific antigen levels, or pathologic stage and Gleason grade. HER-2 overexpression was not detected in AI-CaP at the mRNA or gene level. Mean HER-2 mRNA expression was higher (P0.05) in AD-CaP than AI-CaP (22,080 versus 15,496 HER-2 copies). HER-2 was not amplified in any of 20 AD-CaP or 19 AI-CaP specimens.HER-2 protein and message overexpression and HER-2 amplification were not found in AI-CaP.

To determine expression levels of annexin I (lipocortin I) in patient-matched benign prostatic epithelium (BPE), high-grade prostatic intraepithelial neoplasia (HGPIN), and prostate cancer (CaP). EXPERIMETNAL DESIGN: Annexin I protein expression was examined with a standard immunohistochemical protocol in 69 radical prostatectomy specimens, 45 of which also contained HGPIN. Immunostained sections were scored visually by a genitourinary pathologist and mean optical density was measured with digital image analysis. Real-time fluorescence quantitative PCR was used to measure expression levels of annexin I mRNA in patient-matched CaP and BPE from 14 snap-frozen, radical prostatectomy specimens.Annexin I protein expression was reduced in 91% (41/45) of HGPIN lesions and 94% (65/69) of invasive CaP compared with BPE in the same histological section when assessed visually. Mean absorbance was reduced significantly (P0.05) in 97.7% (44/45) of HGPIN lesions and 98.5% (68/69) of CaP glands compared with BPE. In 79% of cases (11/14; P0.05), mRNA expression was reduced in CaP as compared with patient-matched BPE. Annexin I mRNA and protein expression levels did not correlate with Gleason grade, pathological stage, or race.Down-regulation of annexin I protein expression is a common finding in HGPIN and CaP, suggesting that annexin I dysregulation may be an important early event in CaP initiation. Because mRNA levels are reduced in a high proportion of cases, one likely mechanism for annexin I dysregulation occurs at the level of gene transcription. Results of these studies support a valuable role for a molecular profiling approach to CaP research.

Yearly, 50,000 Americans die of colorectal cancer metastases to liver and lung. EGFR-targeted agents improve survival in a subset of metastatic colorectal cancer (mCRC) cases, and overexpression of the HER-family ligands amphiregulin (AREG) and epiregulin (EREG) correlates with EGFR-targeted agent susceptibility. Here we present evidence of association between EREG overexpression and disease aggression by showing that knockdown of EREG or HER3 impede anchorage independent growth in the HCT 116 CRC cell line. We also find that a HER3-EREG axis gene signature groups mCRC tumors into prognostically distinct groups. Snap-frozen CRC hepatic metastases (65) with patient matched normal liver, as well as 71 primary CRC tumors with patient matched normal colonic mucosa were analyzed via qRT-PCR for receptors (HER1-4) and ligands (AREG, and EREG). Among the mCRC cases HER2, HER3, AREG, and EREG were expressed at higher levels than unmatched normal mucosa (p50 um diameter) of 170 for HER3, and 285 for EREG as compared to 530 for the empty vector (p 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 4527. doi:1538-7445.AM2012-4527