HER2 drives Mucin-like 1 to control proliferation in breast cancer cells.

Mucin-like 1 (MUCL1) was first identified as a breast-specific gene over a decade ago. Based on its highly restricted mRNA expression in breast tissue and continued expression during breast tumorigenesis and progression, MUCL1 is an attractive tumor-associated antigen and a potential therapeutic target. However, very little is known about the cellular location, biological functions and regulation of the MUCL1 protein, which will have a major impact on its druggability. Here we describe our efforts to fully characterize the cellular localization of MUCL1, investigate its regulation by key breast cancer oncogenes such as human epidermal growth factor receptor 2 (HER2) and discover its functional roles in breast cancer. Although some mucins are membrane bound, our data indicate that MUCL1 is secreted by some breast cancer cells, whereas others only express high levels of intracellular MUCL1. MUCL1 expression is highest in HER2-amplified breast tumors and inhibiting HER2 activity in tumor cells resulted in a decreased MUCL1 expression. In-depth investigation demonstrated that phosphoinositide3-kinase/Akt pathway, but not Ras/MEK pathway, controls MUCL1 expression downstream of HER2. Phenotypic assays revealed a strong dependence of HER2-positive cells on MUCL1 for cell proliferation. We further identified the mechanism by which MUCL1 regulates cell growth. Knockdown of MUCL1 induced a G1/S phase arrest concomitant with decreased cyclin D and increased p21 and p27 levels. Finally, we investigated the impact of MUCL1 loss on kinase signaling pathways in breast cancer cells through phospho-kinase array profiling. MUCL1 silencing abrogated phospho-focal adhesion kinase (FAK), Jun NH2-terminal kinase (JNK) and c-Jun signals, but not extracellular signal-regulated kinase or Akt pathway activities, thereby pointing to FAK/JNK pathway as the downstream effector of MUCL1 signaling. We are the first to identify an important role for MUCL1 in the proliferation of breast cancer cells, probably mediated via the FAK/JNK signaling pathway. Taken together, these data suggest a potential utility for therapeutic targeting of this protein in breast cancer.

A novel oncogenic role for the miRNA-506-514 cluster in initiating melanocyte transformation and promoting melanoma growth.

Malignant melanoma is the most aggressive form of skin cancer and its incidence has doubled in the last two decades. It represents only 4% of skin cancer cases per year, but causes as many as 74% of skin cancer deaths. Early detection of malignant melanoma is associated with survival rates of up to 90%, but later detection (stage III to stage IV) is associated with survival rates of only 10%. Dysregulation of microRNA (miRNA) expression has been linked to tumor development and progression by functioning either as a tumor suppressor, an oncogene or a metastasis regulator in multiple cancer types. To understand the role of miRNA in the pathogenesis of malignant melanoma and identify biomarkers of metastasis, miRNA expression profiles in skin punches from 33 metastatic melanoma patients and 14 normal healthy donors were compared. We identified a cluster of 14 miRNAs on the X chromosome, termed the miR-506-514 cluster, which was consistently overexpressed in nearly all melanomas tested (30-60 fold, P<0.001), regardless of mutations in N-ras or B-raf. Inhibition of the expression of this cluster as a whole, or one of its sub-clusters (Sub-cluster A) consisting of six mature miRNAs, led to significant inhibition of cell growth, induction of apoptosis, decreased invasiveness and decreased colony formation in soft agar across multiple melanoma cell lines. Sub-cluster A of the miR-506-514 cluster was critical for maintaining the cancer phenotype, but the overexpression of the full cluster was necessary for melanocyte transformation. Our results provide new insights into the functional role of this miRNA cluster in melanoma, and suggest new approaches to treat or diagnose this disease.

Axin-dependent phosphorylation of the adenomatous polyposis coli protein mediated by casein kinase 1epsilon.

Axin and the adenomatous polyposis coli protein (APC) interact to down-regulate the proto-oncogene beta-catenin. We show that transposition of an axin-binding site can confer beta-catenin regulatory activity to a fragment of APC normally lacking this activity. The fragment containing the axin-binding site also underwent hyperphosphorylation when coexpressed with axin. The phosphorylation did not require glycogen synthase kinase 3beta but instead required casein kinase 1epsilon, which bound directly to axin. Mutation of conserved serine residues in the beta-catenin regulatory motifs of APC interfered with both axin-dependent phosphorylation and phosphorylation by CKIepsilon and impaired the ability of APC to regulate beta-catenin. These results suggest that the axin-dependent phosphorylation of APC is mediated in part by CKIepsilon and is involved in the regulation of APC function.

Overexpression of the retinoic acid-responsive gene Stra6 in human cancers and its synergistic induction by Wnt-1 and retinoic acid.

Genetic defects in the Wnt-1 signaling pathway contribute to human tumor progression and are especially prevalent in colorectal cancer. We screened mouse C57MG cells to isolate mRNAs induced by Wnt-1 and identified Stra6, an mRNA known to be up-regulated by retinoic acid. Up-regulation of Stra6 mRNA was also observed in hyperplastic mammary tissue and mammary gland tumors from transgenic mice expressing Wnt-1 and in human tumors that frequently harbor defects in Wnt-1 signaling. Stimulation of C57MG cells with retinoic acid plus Wnt-1 resulted in expression of Stra6 transcript to levels greatly exceeding that observed with either stimulus alone. This synergy could be explained in part by the up-regulation of retinoic acid receptor-gamma that was observed in response to Wnt-1 signaling. Accordingly, treatment of human colorectal cancer cell lines with retinoic acid resulted in the up-regulation of Stra6 mRNA and accumulation of Stra6 protein at the cell membrane. The data support a model in which Wnt-1 signaling synergizes with retinoids to activate retinoic acid receptor-gamma-responsive genes in human cancers.

Src family kinases and HER2 interactions in human breast cancer cell growth and survival.

Evidence from murine fibroblast models and human breast cancer cells indicates that c-Src and human EGF receptor (HER1) synergize to enhance neoplastic growth of mammary epithelial cells. To investigate whether interactions between c-Src and other HER family members may also play a role in breast tumor progression, we characterized 13 human breast carcinoma cell lines and 13 tumor samples for expression of HER family members and c-Src and examined a subset of the cell lines for Src-dependent, heregulin (HRG)-augmented, anchorage-dependent and independent growth. By immunoblotting, we found that all cell lines overexpressed one or more HER family member, and 60% overexpressed c-Src. Seventy-five per cent of the tumor tissues overexpressed HER2, while 64% overexpressed c-Src. Colony formation in soft agar was enhanced by HRG in three of five cell lines tested, a response that correlated with the presence of a c-Src/HER2 heterocomplex. This result suggests that HRG may act through both HER2 and c-Src to facilitate anchorage-independent growth. In contrast, HRG had little effect on anchorage-dependent growth in any of the cell lines tested. PP1, a Src family kinase inhibitor, reduced or ablated HRG-dependent and independent soft agar growth or anchorage dependent growth, and triggered apoptosis in all cell lines tested. The apoptotic effect of PP1 could be partially or completely reversed by HRG, depending on the cell line. These results suggest that while Src family kinases may cooperate with HRG to promote the survival and growth of human breast tumor cells, they also function independently of HER2/HRG in these processes.

c-Src-mediated phosphorylation of the epidermal growth factor receptor on Tyr845 and Tyr1101 is associated with modulation of receptor function.

Accumulating evidence indicates that interactions between the epidermal growth factor receptor (EGFR) and the nonreceptor tyrosine kinase c-Src may contribute to an aggressive phenotype in multiple human tumors. Previous work from our laboratory demonstrated that murine fibroblasts which overexpress both these tyrosine kinases display synergistic increases in DNA synthesis, soft agar growth, and tumor formation in nude mice, and increased phosphorylation of the receptor substrates Shc and phospholipase gamma as compared with single overexpressors. These parameters correlated with the ability of c-Src and EGFR to form an EGF-dependent heterocomplex in vivo. Here we provide evidence that association between c-Src and EGFR can occur directly, as shown by receptor overlay experiments, and that it results in the appearance of two novel tyrosine phosphorylations on the receptor that are seen both in vitro and in vivo following EGF stimulation. Edman degradation analyses and co-migration of synthetic peptides with EGFR-derived tryptic phosphopeptides identify these sites as Tyr845 and Tyr1101. Tyr1101 lies within the carboxyl-terminal region of the EGFR among sites of receptor autophosphorylation, while Tyr845 resides in the catalytic domain, in a position analogous to Tyr416 of c-Src. Phosphorylation of Tyr416 and homologous residues in other tyrosine kinase receptors has been shown to be required for or to increase catalytic activity, suggesting that c-Src can influence EGFR activity by mediating phosphorylation of Tyr845. Indeed, EGF-induced phosphorylation of Tyr845 was increased in MDA468 human breast cancer cells engineered to overexpress c-Src as compared with parental MDA 468 cells. Furthermore, transient expression of a Y845F variant EGFR in murine fibroblasts resulted in an ablation of EGF-induced DNA synthesis to nonstimulated levels. Together, these data support the hypothesis that c-Src-mediated phosphorylation of EGFR Tyr845 is involved in regulation of receptor function, as well as in tumor progression.

Mechanism of biological synergy between cellular Src and epidermal growth factor receptor.

Overexpression of both cellular Src (c-Src) and the epidermal growth factor receptor (EGFR) occurs in many of the same human tumors, suggesting that they may functionally interact and contribute to the progression of cancer. Indeed, in murine fibroblasts, overexpression of c-Src has been shown to potentiate the mitogenic and tumorigenic capacity of the overexpressed EGFR. Potentiation correlated with the ability of c-Src to physically associate with the activated EGFR and the appearance of two unique in vivo phosphorylations on the receptor (Tyr-845 and Tyr-1101). Using stable cell lines of C3H10T1/2 murine fibroblasts that contain kinase-deficient (K-) c-Src and overexpressed wild-type EGFR, we show that the kinase activity of c-Src is required for both the biological synergy with the receptor and the phosphorylations on the receptor, but not for the association of c-Src with the receptor. In transient transfection assays, not only epidermal growth factor but also serum- and lysophosphatidic acid-induced DNA synthesis was ablated in a dominant-negative fashion by a Y845F mutant of the EGFR, indicating that c-Src-induced phosphorylation of Y845 is critical for the mitogenic response to both the EGFR and a G protein-coupled receptor (lysophosphatidic acid receptor). Unexpectedly, the Y845F mutant EGFR was found to retain its full kinase activity and its ability to activate the adapter protein SHC and extracellular signal-regulated kinase ERK2 in response to EGF, demonstrating that the mitogenic pathway involving phosphorylation of Y845 is independent of ERK2-activation. The application of these findings to the development of novel therapeutics for human cancers that overexpress c-Src and EGFR is discussed.

Overexpression of cellular Src in fibroblasts enhances endocytic internalization of epidermal growth factor receptor.

Previous studies have demonstrated a requirement for the nonreceptor tyrosine kinase, cellular Src (c-Src), in epidermal growth factor (EGF)-induced mitogenesis and a synergistic interaction between c-Src and EGF receptor (EGFR) in tumorigenesis. Although endocytic internalization of EGFR may be thought to attenuate EGF-stimulated signaling, recent evidence suggests that signaling through Ras can be amplified by repeated encounters of endosome-localized, receptor. Shc.Grb2.Sos complexes with the plasma membrane, where Ras resides almost exclusively. Based on these reports, we examined EGFR trafficking behavior in a set of single and double c-Src/EGFR C3H10T1/2 overexpressors to determine if c-Src affects basal receptor half-life, ligand-induced internalization, and/or recycling. Our results show that overexpression of c-Src causes no change in EGFR half-life but does produce an increase in the internalization rate constant of EGF.EGFR complexes when the endocytic apparatus is not stoichiometrically saturated; this effect of c-Src on EGFR endocytosis is negligible at high receptor occupancy in cells overexpressing the receptor. In neither case are EGFR recycling rate constants affected by c-Src. These data indicate a functional role for c-Src in receptor internalization, which in turn could alter some aspects of EGFR signaling related to mitogenesis and tumorigenesis.

Misplaced Greenfield filter: diagnosis by transesophageal echocardiography.

A case of accidental placement of a Greenfield filter in the right atrium is described. Transthoracic echocardiography demonstrated the filter but was unable to provide details regarding the exact location of the filter. Transesophageal echocardiography showed the filter to be fixed to the tricuspid anulus. This finding, which was confirmed during surgery, was used to guide the subsequent surgical management.

Complications due to cloth wear in cloth-covered Starr-Edwards aortic and mitral valve prostheses--and their management.

Five cases of complications due to cloth wear in cloth-covered composite-seat Starr-Edwards aortic and mitral valvular prostheses are described. The complications of cloth wear were recurrent systemic emboli in three patients, two with aortic and one with mitral prosthesis, and severe hemolytic anemia in two patients with aortic prosthesis. The over-all incidence of clinically significant complications due to cloth wear in aortic and mitral valve prosthesis was 2.5 per cent. The diagnosis of cloth wear is impossible before reoperation and it was made by exclusion of other causes of recurrent transient cerebral ischemic attacks or systemic emboli and by exclusion of other causes of hemolytic anemia. Clinical and laboratory findings suggestive of cloth wear are described. Aggressive management of complications of cloth wear by reoperation is likely to prevent disabling or lethal consequences. Porcine xenograft aortic and mitral bioprostheses were used in these patients to replace the cloth-covered valvular prostheses. The symptoms due to cloth wear were abolished in all patients by reoperation, and all patients are off anticoagulants postoperatively. The operative mortality rate for reoperation in this small group of patients was zero.

Fell tube insertion after orthograde dilatation of malignant esophageal obstruction: palliation and morbidity.

Twenty-eight patients with malignant esophageal obstruction had palliative treatment using the Fell endoesophageal tube. The usual plan for these patients, irradiation followed by resection, was not followed because of the presence of tracheoesophageal fistula, celiac or hepatic metastases, or marked debilitation which precluded major operation. Palliation was obtained in ten patients who were discharged in a mean of 16.5 days with the ability to swallow liquids or pureed or ground foods and had a mean survival of 116 days. There was a high incidence of tube-related symptoms in all patients, and increased dysphagia, tube regurgitation, and difficulties in initiating swallowing were noted in patients with lesions above 24 cm from the incisors. The high incidence of postoperative ocmplications was responsible for the poor palliation and low survival in the remaining 18 patients who had a mean survival of 41 days. It was concluded that orthograde dilatation or forced seating of the prosthesis through a malignant esophageal obstruction carries a significant risk of perforation of the esophagus which, if it occurs, negates the palliative aspects of the procedure, increases hospitalization, and decreases survival. It is recommended that this procedure be used selectively in patients not otherwise amenable to resectional therapy with lesions beyond 24 cm from the incisors.

Infective endocarditis. An analysis of 54 surgically treated patients.

One hundred seventy-seven patients were admitted to the New York University Medical Center from 1970 through 1975 with infective endocarditis. Fifty-four of these patients required surgical treatment. The over-all mortality rate was 28 per cent. Two thirds of the deaths were early (10 patients) and one third late (5 patients). The mortality rate was 90 per cent in 10 patients treated for 4 to 6 weeks in whom the infection was uncontrolled and the clinical condition was deteriorating. However of the 12 patients with uncontrolled infection who were operated upon promptly within 10 days, 83 per cent survived. The fact that fungal and gram-negative infections responded poorly to medical therapy suggests the need for prompt, early surgical intervention. The mortality rate in the 32 patients operated upon in whom the infection was controlled was 12.5 per cent. It is our conclusion that all patients with infective endocarditis who develop progressive congestive failure, recurrent embolization, or progressive sepsis, despite treatment, shold have prompt valve replacement within 7 days of the institution of appropriate antimicrobial therapy.

Operative risk in patients with previous coronary artery bypass.

The records of 141 patients who had had coronary artery bypass and myocardial revascularization were reviewed. Fifteen percent (20) of the patients in this series required a surgical procedure from three months to five years following coronary artery bypass. Twelve percent (16) of these patients had elective operations, and 3% had emergency operations. In the elective group there were no deaths. One patient had a proved myocardial infarction, and three patients had transient arrhythmias with no changes in myocardial enzymes. In the emergency group there was one death, from sepsis following splenectomy for splenic abscess. Although the series is small, the data suggest that patients with coronary artery disease who have had myocardial revascularization are acceptable risks for elective and emergency operations. Whether the risk is lower in this group as compared to that in other patients with coronary disease who have not had bypass surgery has not been demonstrated.

The effects of excision of left ventricular scars on the electrocardiogram.

The effects of resection of post-ischemic ventricular scars on the electrocardiogram have been studied in 29 patients. No change occurred in atrial rhythm, P wave morphology, and A-V conduction. Fascicular blocks developed postoperatively in five patients. These were attributed to surgical trauma. Changes in mean frontal plane QRS axis were noted in 25 of 29 patients. There was no correlation between postoperative shifts in QRS axis and preoperative QRS axis, calculated scar size, or ventricular end-diastolic volume. No significant postoperative change was observed in Q waves or S-T segment deviation. It is concluded that electrocardiographic changes following scar resection occur randomly in a minority of patients. The presence of several unquantified factors makes it difficult to evaluate the post-resection electrocardiogram.

Coronary artery bypass with freeze-preserved saphenous vein allografts.

Over the past 5 years, 13 patients had coronary artery bypass performed with freeze-preserved saphenous vein allografts. There were no operative deaths or significant morbidity. Six patients were studied postoperatively at 42, 37, 10, 7, 5, and 1 months. Six of 8 grafts were patent with good flow. There were four late deaths; two of these occurred in patients who had concomitant resection of a ventricular aneurysm. Of the 9 surviving patients, 6 (6/9) are asymptomatic and 2 (2/9) have occasional chest pains; the condition of 1 patient (1/9) is unchanged. This experience suggests that free-preserved saphenous vein allografts may be used successfully for coronary bypass when autologous veins and internal mammary arteries are unavailable or insufficient for multiple bypass.

Esophagogastrostomy. Analysis of 55 cases.

At the New York University Medical Center from January, 1969, through December, 1973, esophagogastrostomies were performed in 56 patients. In 30 (Group A), fundoplications were combined with the esophagogastrostomies; in the other 26 (Group B), esophagogastrostomies only were performed. These two groups have been compared in an effort to determine the effectiveness of fundoplication in preventing gastric reflux following esophagogastrostomy. The operative mortality rate (10 per cent) and the 3 year survival rate (20 per cent) were approximately equal in the two groups. Clinical evidence of reflux was noted in 10 per cent of Group A and 47 per cent of Group B patients, while complications of reflux were noted in 5 per cent of Group A and in 33 per cent of Group B patients. The results of the present study suggest that fundoplication, while not prolonging survival, does prevent the symptoms and complications of gastric reflux and improves the quality of survival in these unfortunate patients. This leads us to recommend the routine use of fundoplication with esophagogastrostomy in patients with carcinoma of the esophagus and gastric cardia.