Jen Jen Yeh and colleagues developed and validated a six-gene signature in patients with pancreatic ductal adenocarcinoma that may be used to better stage the disease in these patients and assist in treatment decisions.

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.The epidermal growth factor receptor (EGFR) family has been implicated in breast cancer pathogenesis and progression (reviewed in references 13 and 39). Aberrant expression of at least two of the family members, EGFR and HER2, has been associated with poor prognosis and differential response to therapy (21,28,31,44). Recently, treatment targeted against HER2 has demonstrated clinical efficacy, emphasizing the importance of members of this receptor family in breast cancer prognosis and therapy (10).The EGFR family consists of four known members: EGFR (HER1, erbB-1), HER2 (erbB-2), HER3 (erbB-3), and HER4 (erbB-4) (reviewed in references 13, 34, and 39). The four receptors form homodimers or heterodimers upon activation by two sets of ligands, the EGF and heregulin/neuregulin families. There are several possible hetero-and homodimeric receptor combinations, which theoretically result in differential activation of multiple downstream signal transduction pathways. Additional heterogeneity results from varying phenotypic responses, depending on cell type and the duration or intensity of downstream signaling, determined in part by differences in ligand affinity, recycling, and intracellular environment, as well as other factors th...

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 GlyGly 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 ␤-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.Sentrin is a 101-amino acid ubiquitin-like protein that interacts with the death domains of Fas and TNFR1 1 (1), with PML, a tumor suppressor implicated in the pathogenesis of promyelocytic leukemia (2), with Rad51 and Rad52, proteins that are involved in repairing double-stranded DNA breaks (3), and with RanGAP1, a GTPase-activating protein that is critically involved in nuclear protein transport (4,5). Using the COS cell expression system, we have shown that sentrin can be conjugated to other proteins in a manner analogous to protein ubiquitination (6). Moreover, sentrinized proteins appear to reside in the nucleus. We have also shown that the C-terminal four amino acids of sentrin, His-Ser-Thr-Val, were efficiently cleaved to allow the conjugation of sentrin to other proteins via the conserved Gly 97 residue. Thus, the biochemistry of sentrinization pathway appears to be remarkably similar to that of the ubiquitination pathway.Conjugation of ubiquitin to its target protein requires the initial activation of the conserved C-terminal Gly residue catalyzed by a specific ubiquitin-activating enzyme, E1 (7-11). An intermediate, ubiquitin adenylate, is formed by displacement of PP i from ATP and ubiquitin adenylate is then transferred to a thiol site in E1 with release of AMP. Through transacylation, ubiquitin is transferred to a ubiquitin-conjugating enzyme, E2, to form another thiol ester bond. Finally, ubiquitin is transferred from E2 to its target protein through an isopeptide linkage with the ⑀-amino group of the Lys residue of the target protein. The tr...

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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.

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.The epidermal growth factor receptor (EGFR) family of receptor tyrosine kinases (RTKs) consists of EGFR/HER1/ ErbB1, ErbB2/HER2, ErbB3/HER3, and ErbB4/HER4. All family members have a ligand-binding ectodomain, a single transmembrane segment, an intracellular tyrosine kinase domain, and a tyrosine-rich carboxy terminus. Ten ligands from two related ligand families, the epidermal growth factor (EGF) and heregulin/neuregulin families, bind to EGFR family RTKs. Ligand-dependent homo-and heterodimerization results in kinase activation and cross-phosphorylation. Although HER1 and HER4 are capable of both homo-and heterodimerization under most conditions, HER2 and HER3 are obligate heterodimeric partners, since HER2 does not bind any known ligand, and HER3 is devoid of intrinsic kinase activity. The activated receptor complexes exhibit multiple signaling capabilities including stimulation of canonical mitogenactivated protein kinase, phosphatidylinositol 3-kinase, and/or STAT signaling pathways (10,41,47).All four EGFR family members play a role in regulating cardiovascular, neuronal, and epithelial development. Null mutations of any of the EGFR family members result in embryonic lethality. Despite these similarities, HER4 exhibits unique biological activities that set it apart from the rest of the EGFR fam...

Cell proliferation, differentiation, and survival are highly coordinated processes during the development and maturation of the mammary gland, and control of these mechanisms is critical for the prevention of breast cancers (reviewed in reference 39). Aberrant regulation of the HER/ErbB family of receptor tyrosine kinases (RTKs) and their ligands is a common occurrence in many human cancers, including breast cancer (14,15,45). This family consists of four related members, HER1/ErbB1/EGFR (epidermal growth factor receptor), HER2/ErbB2/Neu, HER3/ErbB3, and HER4/ErbB4. Each protein is comprised of a large amino-terminal extracellular domain, a transmembrane domain, and a large intracellular domain with a tyrosine-rich carboxy-terminal region and a tyrosine kinase-like sequence (27,33,56). The tyrosine kinase activity of the ErbBs is induced upon ligand interaction, leading to receptor dimerization (homo-and heterodimerization) and subsequent receptor transphosphorylation. Although HER2 is the preferential heterodimeric partner, HER2 does not bind any conventional ligand within the two major families of ErbB ligands (EGF-like ligands and heregulin [HRG]/neuregulin-like ligands) and therefore relies on HER1, HER3, or HER4 for activation of its tyrosine kinase activity.The well-documented growth stimulatory effects of HER1 and HER2 have driven the investigation of ErbB signaling in breast cancer. HER1 is expressed in nearly all human carcinomas, including breast cancers, while nearly 20 to 25% of breast cancers overexpress HER2 and/or exhibit gene amplification at the her2 locus (26,37,40,48). Expression of either HER1 or HER2 in tumor specimens correlates with a shorter survival period, a higher grade of malignancy, and an overall poor prognosis (10, 12-14, 19, 24). Genetically engineered animal models of breast cancer confirm the role of HER1 and HER2 in driving proliferation of the mammary epithelium (reviewed in reference 39). Recent evidence suggests that increased expression of HER3 in breast cancers also correlates with a poor prognosis. HER3 is overexpressed in about 20% of all breast cancers and is frequently coexpressed with HER2 (2,5,10,23,31,52,53). This has generated the hypothesis that HER2/ HER3 heterodimers may function to simultaneously drive cellular proliferation and survival in breast cancer cells.In contrast, there is evidence that HER4 expression correlates with a more differentiated tumor grade, longer survival, and positive prognostic indicators, such as estrogen receptor expression (1,17,29,38,41,44). Women whose breast tumors express HER4 exhibit the lowest risk of death due to cancer compared to women whose tumors express HER1, HER2, or HER3. During breast development, HER4 expression and activity (measured by tyrosine phosphorylation) are lowest during phases of epithelial cell proliferation and highest during phases of differentiation (35). Mammary glands from mice that lack HER4 activity, either by Cre-Lox technology, cardiac-specific transgene rescue of a HER4 knockout (with the mammary ...

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.