DNA ploidy pattern and amplification of ERBB and ERBB2 genes in human gastric carcinomas

The DNA ploidy pattern and amplification of ERBB and ERBB2 genes were examined in paraffin-embedded tissue from gastric carcinomas using flow cytometry and a slot-blot hybridization technique. The incidence of aneuploidy in well differentiated adenocarcinomas (56%) was significantly higher (p less than 0.05) than that in poorly differentiated adenocarcinomas (21%). The DNA ploidy pattern was not remarkably different between the primary tumors and metastatic deposits in lymph nodes. Of the nine specimens having an aneuploid stem cell line in the primary tumor and/or in metastases, three showed ERBB2 gene amplification and one showed ERBB gene amplification. The incidence of epidermal growth factor (EGF) immunoreactivity in tumor cells showed no difference between diploid and aneuploid tumors. These findings indicate that aneuploidy is frequently associated with amplification of ERBB and ERBB2 genes.     

DNA ploidy pattern and amplification of ERBB and ERBB2 genes in human gastric carcinomas

The DNA ploidy pattern and amplification of ERBB and ERBB2 genes were examined in paraffinembedded tissue from gastric carcinomas using flow cytometry and a slot-blot hybridization technique. The incidence of aneuploidy in well differentiated adenocarcinomas (56%) was significantly higher (p<0.05) than that in poorly differentiated adenocarcinomas (21%). The DNA ploidy pattern was not remarkably different between the primary tumors and metastatic deposits in lymph nodes. Of the nine specimens having an aneuploid stem cell line in the primary tumor and/or in metastases, three showed ERBB2 gene amplification and one showed ERBB gene amplification. The incidence of epidermal growth factor (EGF) immunoreactivity in tumor cells showed no difference between diploid and aneuploid tumors. These findings indicate that aneuploidy is frequently associated with amplification of ERBB and ERBB2 genes.     

Alterations of class I HLA genes in human colon cancers

Summary Alterations of HLA class I genes were found in 3 of 12 human colon cancers. Rearrangements in HLA class I genes were observed in 2 cancers and amplification of HLA-coding genes was observed in 1 cancer. All 3 cancers were at an advanced stage. No examples of amplification or rearrangement in the HLA genes were found in 10 other tumours of diverse types. No alterations in the ₂-microgubulin gene were observed in 22 human solid tumours included in this study. The association between alterations in HLA genes and proto-oncogenes in these tumours is discussed.     

ERBB2 overexpression suppresses stress-induced autophagy and renders ERBB2-induced mammary tumorigenesis independent of monoallelic Becn1 loss

Defective autophagy has been implicated in mammary tumorigenesis, as the gene encoding the essential autophagy regulator BECN1 is deleted in human breast cancers and Becn1^+/? mice develop mammary hyperplasias. In agreement with a recent study, which reports concurrent allelic BECN1 loss and ERBB2 amplification in a small number of human breast tumors, we found that low BECN1 mRNA correlates with ERBB2-overexpression in breast cancers, suggesting that BECN1 loss and ERBB2 overexpression may functionally interact in mammary tumorigenesis. We now report that ERBB2 overexpression suppressed autophagic response to stress in mouse mammary and human breast cancer cells. ERBB2-overexpressing Becn1^+/+ and Becn1^+/? immortalized mouse mammary epithelial cells (iMMECs) formed mammary tumors in nude mice with similar kinetics, and monoallelic Becn1 loss did not alter ERBB2- and PyMT-driven mammary tumorigenesis. In human breast cancer databases, ERBB2-expressing tumors exhibit a low autophagy gene signature, independent of BECN1 mRNA expression, and have similar gene expression profiles with non-ERBB2-expressing breast tumors with low BECN1 levels. We also found that ERBB2-expressing BT474 breast cancer cells, despite being partially autophagy-deficient under stress, can be sensitized to the anti-ERBB2 antibody trastuzumab (tzb) by further pharmacological or genetic autophagy inhibition. Our results indicate that ERBB2-driven mammary tumorigenesis is associated with functional autophagy suppression and ERBB2-positive breast cancers are partially autophagy-deficient even in a wild-type BECN1 background. Furthermore and extending earlier findings using tzb-resistant cells, exogenously imposed autophagy inhibition increases the anticancer effect of trastuzumab on tzb-sensitive ERBB2-expressing breast tumor cells, indicating that pharmacological autophagy suppression has a wider role in the treatment of ERBB2-positive breast cancer.     

ERBB2 overexpression suppresses stress-induced autophagy and renders ERBB2-induced mammary tumorigenesis independent of monoallelic Becn1 loss

Defective autophagy has been implicated in mammary tumorigenesis, as the gene encoding the essential autophagy regulator BECN1 is deleted in human breast cancers and Becn1^+/− mice develop mammary hyperplasias. In agreement with a recent study, which reports concurrent allelic BECN1 loss and ERBB2 amplification in a small number of human breast tumors, we found that low BECN1 mRNA correlates with ERBB2-overexpression in breast cancers, suggesting that BECN1 loss and ERBB2 overexpression may functionally interact in mammary tumorigenesis. We now report that ERBB2 overexpression suppressed autophagic response to stress in mouse mammary and human breast cancer cells. ERBB2-overexpressing Becn1^+/+ and Becn1^+/− immortalized mouse mammary epithelial cells (iMMECs) formed mammary tumors in nude mice with similar kinetics, and monoallelic Becn1 loss did not alter ERBB2- and PyMT-driven mammary tumorigenesis. In human breast cancer databases, ERBB2-expressing tumors exhibit a low autophagy gene signature, independent of BECN1 mRNA expression, and have similar gene expression profiles with non-ERBB2-expressing breast tumors with low BECN1 levels. We also found that ERBB2-expressing BT474 breast cancer cells, despite being partially autophagy-deficient under stress, can be sensitized to the anti-ERBB2 antibody trastuzumab (tzb) by further pharmacological or genetic autophagy inhibition. Our results indicate that ERBB2-driven mammary tumorigenesis is associated with functional autophagy suppression and ERBB2-positive breast cancers are partially autophagy-deficient even in a wild-type BECN1 background. Furthermore and extending earlier findings using tzb-resistant cells, exogenously imposed autophagy inhibition increases the anticancer effect of trastuzumab on tzb-sensitive ERBB2-expressing breast tumor cells, indicating that pharmacological autophagy suppression has a wider role in the treatment of ERBB2-positive breast cancer.     

DNA structural features on borders of ERBB2 amplicons in breast cancer

In 25–30% of cases of breast cancer tumors, the amplification of the chromosome fragment around ERBB2 underlies the increased expression of genes adjacent to ERBB2. The increased expression of genes within ERBB2-containing amplicons may impact not only the growth and development of the tumor, but also the sensitivity of the tumor to different types of anti-cancer therapies. The initial cause of the amplification and the exact borders of ERBB2-amplified chromosome fragment are still not completely characterized. No specific DNA sequences were found on the junction regions during intrachromosomal DNA amplification. We hypothesized that amplification borders can be specified by the structural peculiarities of DNA, rather than the particular DNA sequence. This study focused on the mapping of ERBB2 amplification borders in breast cancer and the search for unusual structural features of DNA at the borders of the identified amplicons. The copy number of ten genes adjacent to ERBB2 was evaluated by real time PCR in 162 breast cancer samples. Several ERBB2-containing amplicons of various lengths were revealed. In the majority of the analyzed samples, the borders of these amplicons were located within ZNFN1A3 and RARA genes. A bioinformatics analysis of the nucleotide sequence peculiarities around ERBB2 gene revealed the presence of AT-rich DNA regions with a high degree of flexibility. These regions were able to form stable secondary structures. Positions of these sites strongly coincide with the positions of the ERBB2-containing amplicon borders found in real time PCR experiments. Based on the obtained results, one can suppose that the structural features of DNA are involved in the formation of ERBB2-containing amplicon borders in breast cancer cells and the data are of importance for understanding the mechanisms of oncogene amplification.     

The human homeobox gene HOX7 maps to chromosome 4p16.1 and may be implicated in Wolf-Hirschhorn syndrome

Summary The gene encoding human 17beta-hydroxy-steroid dehydrogenase (17-HSD; EC 1.1.1.62) is assigned to chromosome 17 by Southern blotting analyses of panels of human x rodent somatic cell hybrids and independently to 17q12–q21 using chromosomal in situ hybridization. A search for physical linkage between 17-HSD and the proto-oncogenes, THRA1 and ERBB2 (both reported to be located in this region of chromosome 17) was performed by pulsed-field gel electrophoresis (PFGE) using several rare-cutting restriction endonucleases. Because all three genes hybridized to DNA fragments of different lengths it seems unlikely that the gene for 17-HSD is located very close to THRA1 and ERBB2. Further evidence for this assumption was obtained from the absence of any coamplification of the 17-HSD gene in 9 breast tumors with amplification of the ERBB2 gene. Analyses of Southern blots of ScaI-digested DNAs from unrelated individuals from Northern Finland revealed a relatively infrequent diallelic restriction fragment length polymorphism, the allele frequencies of which were 0.04 (A1) and 0.96 (A2).     

Contribution of PGAP3 co-amplified and co-overexpressed with ERBB2 at 17q12 involved poor prognosis in gastric cancer

The locus at 17q12 erb-b2 receptor tyrosine kinase 2 (ERBB2) has been heavily amplificated and overexpressed in gastric cancer (GC), but it remains to be elucidated about the clinical significance of the co-amplification and co-overexpression of PGAP3 gene located around ERBB2 in GC. The profile of PGAP3 and ERBB2 in four GC cell lines and tissue microarrays containing 418 primary GC tissues was assessed to investigate the co-overexpression and clinical significance of the co-amplified genes, and to evaluate the impact of the co-amplified genes on the malignancy of GC. Co-amplification of PGAP3 and ERBB2 accompanied with co-overexpression was observed in a haploid chromosome 17 of NCI-N87 cells with double minutes (DMs). PGAP3 and ERBB2 were overexpressed and positively correlated in 418 GC patients. Co-overexpression of the PGAP3 and ERBB2 was correlated with T stage, TNM stage, tumour size, intestinal histological type and poor survival proportion in 141 GC patients. In vitro, knockdown of the endogenous PGAP3 or ERBB2 decreased cell proliferation and invasion, increased G1 phase accumulation and induced apoptosis in NCI-N87 cells. Furthermore, combined silencing of PGAP3 and ERBB2 showed an additive effect on resisting proliferation of NCI-N87 cells compared with targeting ERBB2 or PGAP3 alone. Taken together, the co-overexpression of PGAP3 and ERBB2 may be crucial due to its significant correlation with clinicopathological factors of GC. Haploid gain of PGAP3 co-amplified with ERBB2 is sufficient to facilitate the malignancy and progression of GC cells in a synergistic way.     

Breast Cancer Anti-Estrogen Resistance 4 (BCAR4) Drives Proliferation of IPH-926 lobular Carcinoma Cells

Background

Most breast cancers depend on estrogenic growth stimulation. Functional genetic screenings in in vitro cell models have identified genes, which override growth suppression induced by anti-estrogenic drugs like tamoxifen. Using that approach, we have previously identified Breast Cancer Anti-Estrogen Resistance 4 (BCAR4) as a mediator of cell proliferation and tamoxifen-resistance. Here, we show high level of expression and function of BCAR4 in human breast cancer.

Methods

BCAR4 mRNA expression was evaluated by (q)RT-PCR in a panel of human normal tissues, primary breast cancers and cell lines. A new antibody raised against C78-I97 of the putative BCAR4 protein and used for western blot and immunoprecipitation assays. Furthermore, siRNA-mediated gene silencing was implemented to study the function of BCAR4 and its downstream targets ERBB2/3.

Results

Except for placenta, all human normal tissues tested were BCAR4-negative. In primary breast cancers, BCAR4 expression was comparatively rare (10%), but associated with enhanced proliferation. Relative high BCAR4 mRNA expression was identified in IPH-926, a cell line derived from an endocrine-resistant lobular breast cancer. Moderate BCAR4 expression was evident in MDA-MB-134 and MDA-MB-453 breast cancer cells. BCAR4 protein was detected in breast cancer cells with ectopic (ZR-75-1-BCAR4) and endogenous (IPH-926, MDA-MB-453) BCAR4 mRNA expression. Knockdown of BCAR4 inhibited cell proliferation. A similar effect was observed upon knockdown of ERBB2/3 and exposure to lapatinib, implying that BCAR4 acts in an ERBB2/3-dependent manner.

Conclusion

BCAR4 encodes a functional protein, which drives proliferation of endocrine-resistant breast cancer cells. Lapatinib, a clinically approved EGFR/ERBB2 inhibitor, counteracts BCAR4-driven tumor cell growth, a clinical relevant observation.     

The canine ERBB2 gene maps to a chromosome region frequently affected by aberrations in tumors of the dog (Canis familiaris)

The dog offers an increasingly important model for several human diseases, including cancer. Accordingly, the results of canine gene mapping studies will be of considerable significance. Herein, we have addressed the mapping of the canine gene ERBB2 (alias HER2, NEU). ERBB2 is a protooncogene encoding a tyrosine kinase receptor protein, the overexpression of which correlates with a more rapid progression and a worse prognosis in breast cancer. In addition, it apparently plays a role in the development of other tumors as well. By fluorescence in situ hybridization (FISH), we have mapped the canine ERBB2 to 1q13.1. Cytogenetic studies of canine tumors revealed that this region is very often affected by clonal chromosome aberrations in tumors of the dog.     

Genome-wide profiling of gene amplification and deletion in cancer

Accumulations of genetic changes in somatic cells induce phenotypic transformations leading to cancer. Among these genetic changes, gene amplification and deletion are most frequently observed in several kinds of cancers. Amplification of oncogene and/or deletion of tumor suppressor gene, together with dysfunction of the gene by point mutation, are the main causes of cancer. Genome-wide analysis of amplification and deletion of genes in cancers is basic to resolving the mechanisms of carcinogenesis. Comparative genomic hybridization (CGH) developed in 1992 has been utilized to identify DNA copy number abnormalities in various kind of cancers and several reports have shown its usefulness in screening of the genes involved in carcinogenesis, and also in the identification of prognostic factors in cancer. We have shown that 1q23 gain is associated with neuroblastomas that are resistant to aggressive treatment, and have poor prognosis, and 1q and 13q gains are possibly related to drug resistance in ovarian cancers. Recently, the "rough draft" of the human genome was reported and we are ready to utilize the vast information on genomic sequences in cancer research. Moreover, microarray technology enables us to analyze more than ten thousand genes at a time and revealed genetic abnormalities in cancers at a genome-wide level. By combination of microarray and CGH, a powerful screening method for oncogenes and tumor suppressor genes in cancers, called array-CGH, has been developed by several groups. In this article, we overview these genome-wide analytical methods, CGH and array-CGH, and discuss their potential in molecular characterization of cancers.     

Phenylhexyl isothiocyanate has dual function as histone deacetylase inhibitor and hypomethylating agent and can inhibit myeloma cell growth by targeting critical pathways

Background

Amplification of the ERBB2 (Her-2/neu) oncogene, which occurs in approximately 25% of breast carcinomas, is a known negative prognostic factor. Available data indicate that a variable number of nearby genes on chromosome 17q may be co-amplified or deleted, forming a continuous amplicon of variable size. In approximately 25% of these patients, the amplicon extends to the gene for topoisomerase II alpha (TOP2A), a target for anthracyclines. We sought to understand the significance of these associated genomic changes for breast cancer prognosis and predicting response to therapy.

Methods and patients

Archival tissue samples from 63 breast cancer patients with ERBB2 amplification, stages 0–IV, were previously analyzed with FISH probes for genes located near ERBB2. In the present study, the clinical outcome data were determined for all patients presenting at stages I–III for whom adequate clinical follow up was available.

Results

Four amplicon patterns (Classes) were identified. These were significantly associated with the clinical outcome, specifically, recurrence of breast cancer. The Amplicon class IV with deleted TOP2A had 67% (6/9) cases with recurrence, whereas the other three classes combined had only 12% (3/25) cases (p-value = 0.004) at the time of last follow-up. TOP2A deletion was also significantly associated with time to recurrence (p-value = 0.0002). After adjusting for age in Cox regression analysis, the association between TOP2A deletion and time to recurrence remains strongly significant (p-value = 0.002) whereas the association with survival is marginally significant (p-value = 0.06).

Conclusion

TOP2A deletion is associated with poor prognosis in ERBB2-amplified breast carcinomas. Clarification of the mechanism of this association will require additional study.     

Identification of Nine Genomic Regions of Amplification in Urothelial Carcinoma,Correlation with Stage,and Potential Prognostic and Therapeutic Value

We performed a genome wide analysis of 164 urothelial carcinoma samples and 27 bladder cancer cell lines to identify copy number changes associated with disease characteristics, and examined the association of amplification events with stage and grade of disease. Multiplex inversion probe (MIP) analysis, a recently developed genomic technique, was used to study 80 urothelial carcinomas to identify mutations and copy number changes. Selected amplification events were then analyzed in a validation cohort of 84 bladder cancers by multiplex ligation-dependent probe assay (MLPA). In the MIP analysis, 44 regions of significant copy number change were identified using GISTIC. Nine gene-containing regions of amplification were selected for validation in the second cohort by MLPA. Amplification events at these 9 genomic regions were found to correlate strongly with stage, being seen in only 2 of 23 (9%) Ta grade 1 or 1–2 cancers, in contrast to 31 of 61 (51%) Ta grade 3 and T2 grade 2 cancers, p<0.001. These observations suggest that analysis of genomic amplification of these 9 regions might help distinguish non-invasive from invasive urothelial carcinoma, although further study is required. Both MIP and MLPA methods perform well on formalin-fixed paraffin-embedded DNA, enhancing their potential clinical use. Furthermore several of the amplified genes identified here (ERBB2, MDM2, CCND1) are potential therapeutic targets.     

Imaging of Endocytic Trafficking and Extracellular Vesicles Released Under Neratinib Treatment in ERBB2+ Breast Cancer Cells

Breast cancers (BCa) with ERBB2 amplification show rapid tumor growth, increased disease progression, and lower survival rate. Deregulated intracellular trafficking and extracellular vesicle (EVs) release are mechanisms that support cancer progression and resistance to treatments. Neratinib (NE) is a Food and Drug Administration–approved pan-ERBB inhibitor employed for the treatment of ERBB2⁺ BCa that blocks signaling and causes survival inhibition. However, the effects of NE on ERBB2 internalization, its trafficking to multivesicular bodies (MVBs), and the release of EVs that originate from these organelles remain poorly studied. By confocal and electron microscopy, we observed that low nanomolar doses of NE induced a modest ERBB2 internalization along with an increase of clathrin-mediated endocytosis and of the CD63+ MVB compartment in SKBR-3 cells. Furthermore, we showed in the culture supernatant two distinct EV subsets, based on their size and ERBB2 positivity: small (30–100 nm) ERBB2⁻ EVs and large (>100 nm) ERBB2⁺ EVs. In particular, we found that NE increased the overall release of EVs, which displayed a reduced ERBB2 positivity compared with controls. Taken together, these results provide novel insight into the effects of NE on ERBB2⁺ BCa cells that may lead to a reduction of ERBB2 potentially transferred to distant target cells by EVs: