Current and future diagnostic and treatment strategies for patients with invasive lobular breast cancer.

BACKGROUND: Invasive lobular breast cancer (ILC) is the second most common type of breast cancer after invasive breast cancer of no special type (NST), representing up to 15% of all breast cancers. DESIGN: Latest data on ILC are presented, focusing on diagnosis, molecular make-up according to the European Society for Medical Oncology Scale for Clinical Actionability of molecular Targets (ESCAT) guidelines, treatment in the early and metastatic setting and ILC-focused clinical trials. RESULTS: At the imaging level, magnetic resonance imaging-based and novel positron emission tomography/computed tomography-based techniques can overcome the limitations of currently used imaging techniques for diagnosing ILC. At the pathology level, E-cadherin immunohistochemistry could help improving inter-pathologist agreement. The majority of patients with ILC do not seem to benefit as much from (neo-)adjuvant chemotherapy as patients with NST, although chemotherapy might be required in a subset of high-risk patients. No differences in treatment efficacy are seen for anti-human epidermal growth factor receptor 2 (HER2) therapies in the adjuvant setting and cyclin-dependent kinases 4 and 6 inhibitors in the metastatic setting. The clinical utility of the commercially available prognostic gene expression-based tests is unclear for patients with ILC. Several ESCAT alterations differ in frequency between ILC and NST. Germline BRCA1 and PALB2 alterations are less frequent in patients with ILC, while germline CDH1 (gene coding for E-cadherin) alterations are more frequent in patients with ILC. Somatic HER2 mutations are more frequent in ILC, especially in metastases (15% ILC versus 5% NST). A high tumour mutational burden, relevant for immune checkpoint inhibition, is more frequent in ILC metastases (16%) than in NST metastases (5%). Tumours with somatic inactivating CDH1 mutations may be vulnerable for treatment with ROS1 inhibitors, a concept currently investigated in early and metastatic ILC. CONCLUSION: ILC is a unique malignancy based on its pathological and biological features leading to differences in diagnosis as well as in treatment response, resistance and targets as compared to NST.

Minimal ProtoHox cluster inferred from bilaterian and cnidarian Hox complements.

Bilaterian animals have a Hox gene cluster essential for patterning the main body axis, and a ParaHox gene cluster. Comparison of Hox and ParaHox genes has led workers to postulate that both clusters originated from the duplication of an ancient cluster named ProtoHox, which contained up to four genes with at least the precursors of anterior and posterior Hox/ParaHox genes. However, the way in which genes diversified within the ProtoHox, Hox and ParaHox clusters remains unclear because no systematic study of non-bilaterian animals exists. Here we characterize the full Hox/ParaHox gene complements and genomic organization in two cnidarian species (Nematostella vectensis and Hydra magnipapillata), and suggest a ProtoHox cluster simpler than originally thought on the basis of three arguments. First, both species possess bilaterian-like anterior Hox genes, but their non-anterior genes do not appear as counterparts of either bilaterian central or posterior genes; second, two clustered ParaHox genes, Gsx and a gene related to Xlox and Cdx, are found in Nematostella vectensis; and third, we do not find clear phylogenetic support for a common origin of bilaterian Cdx and posterior genes, which might therefore have appeared after the ProtoHox cluster duplication. Consequently, the ProtoHox cluster might have consisted of only two anterior genes. Non-anterior genes could have appeared independently in the Hox and ParaHox clusters, possibly after the separation of bilaterians and cnidarians.

A molecular snapshot of the metazoan 'Eve'.

A description of the molecular make-up of the ancestral multicellular animal is emerging from the growing availability of molecular biological and biochemical data gleaned from the study of modern members of ancient groups of animals. We use the distributions of classes of transcription factors, signal transduction systems and other molecular innovations among metazoan phyla to infer some of the characteristics of the first animals.

The induction of core pluripotency master regulators in cancers defines poor clinical outcomes and treatment resistance.

Stem cell characteristics have been associated with treatment resistance and poor prognosis across many cancer types. The ability to induce and regulate the pathways that sustain these characteristic hallmarks of lethal cancers in a novel in vitro model would greatly enhance our understanding of cancer progression and treatment resistance. In this work, we present such a model, based simply on applying standard pluripotency/embryonic stem cell media alone. Core pluripotency stem cell master regulators (OCT4, SOX2 and NANOG) along with epithelial-mesenchymal transition (EMT) markers (Snail, Slug, vimentin and N-cadherin) were induced in human prostate, breast, lung, bladder, colorectal, and renal cancer cells. RNA sequencing revealed pathways activated by pluripotency inducing culture that were shared across all cancers examined. These pathways highlight a potential core mechanism of treatment resistance. With a focus on prostate cancer, the culture-based induction of core pluripotent stem cell regulators was shown to promote survival in castrate conditions-mimicking first line treatment resistance with hormonal therapies. This acquired phenotype was shown to be mediated through the upregulation of iodothyronine deiodinase DIO2, a critical modulator of the thyroid hormone signalling pathway. Subsequent inhibition of DIO2 was shown to supress expression of prostate specific antigen, the cardinal clinical biomarker of prostate cancer progression and highlighted a novel target for clinical translation in this otherwise fatal disease. This study identifies a new and widely accessible simple preclinical model to recreate and explore underpinning pathways of lethal disease and treatment resistance.

Correction: The induction of core pluripotency master regulators in cancers defines poor clinical outcomes and treatment resistance.

The original version of this article contained an error in Fig. 5a where the colours of the labels representing the Hinge and LBD of the AR were incorrect and did not match the corresponding exons. The corrected version of this Figure now appears in the article. The conclusions of this paper were not affected. The authors apologise for this error and any confusion caused.

Biochemical and cytological changes associated with expression of deregulated pp60src in Xenopus oocytes.

We have examined the effects of Xenopus pp60c-src with constitutive kinase activity on the morphology and maturation of Xenopus laevis oocytes. When RNA encoding this deregulated variant was injected into stage VI oocytes, we observed a gross alteration in the cortex of the oocyte. This alteration involved aggregation of pigment and invagination of the cortex in a large area proximal to the site of injection. This phenomenon was not seen in oocytes injected with RNA encoding wild-type pp60c-src. We have correlated this phenomenon with the tyrosine phosphorylation of 84- and 100-kDa proteins. These phosphorylated proteins colocalized with the alteration in the oocyte cortex when assayed by both biochemical and immunocytochemical methods. Neither the pigment aggregation nor phosphorylation of the 84- and 100-kDa proteins was observed in oocytes expressing a nonmyristoylated version of the deregulated pp60c-src. Expression of deregulated Xenopus fyn, a src-family member, resulted in a phenotype similar to that seen with deregulated src. However, in the fyn-injected oocytes, many more proteins were phosphorylated on tyrosine than in the src-injected oocytes. Progesterone stimulation of oocytes expressing deregulated pp60c-src resulted in an increase in the number of tyrosine-phosphorylated proteins. This change may represent the response of pp60src to the resumption of the cell cycle in maturing oocytes. These data suggest that the oocyte may be a particularly useful system for investigating the role of pp60c-src in the regulation of cytoskeletal structure and in the regulation of events associated with the cell cycle.

Plasmids containing mouse rDNA do not recombine with cellular ribosomal genes when introduced into cultured mouse cells.

We have examined the fate of plasmids containing a segment of a mouse rDNA repeat after they were introduced by transfection into cultured mouse cells. In addition to the rDNA segment, the plasmids contained the thymidine kinase gene from herpes simplex virus 1 to allow for selection of the plasmid after transfection into thymidine kinase-deficient mouse cells. Thus far, no cases of homologous recombination between transfected plasmid DNAs and host cell sequences have been documented. We reasoned that the high repetition frequency of the rRNA genes in the mouse genome (200 copies per diploid cell) might create a favorable situation for obtaining homologous recombination events between the plasmids containing rDNA and host cell rDNA sequences. The plasmids were introduced into cells in both the presence and the absence of carrier DNA and both as covalently closed circles and linear molecules. The sites of plasmid integration in the genomes of various cell lines were examined by DNA restriction digests and hybridization, molecular cloning, and nuclear fractionation. In the seven cell lines examined, there was no evidence that the plasmids had integrated into the rRNA gene clusters of the cell. Thus, the apparent absence of site-specific integration of cloned DNAs introduced into mammalian cells does not appear to be due simply to the small target presented by most host cell sequences.

Structure and expression of STK, a src-related gene in the simple metazoan Hydra attenuata.

Both cDNA clones and a genomic DNA clone encoding a 509-amino-acid protein that is 64% similar to chicken pp60c-src were isolated from the simple metazoan Hydra attenuata. We have designated this gene STK, for src-type kinase. Features of the amino acid sequence of the protein encoded by the STK gene suggest that it is likely to be myristoylated and regulated by phosphorylation in a manner similar to that found for pp60c-src. The genomic sequence encoding the protein was found to be interrupted by at least two introns, one of which was located in a position identical to that of one of the introns in the chicken src gene. The STK gene was expressed during early development of H. attenuata and at high levels in the epithelial cells of adult polyps. Probing of Hydra proteins with an antibody to phosphotyrosine indicated that the major phosphotyrosine-containing protein in H. attenuata may be the STK protein itself. H. attenuata is the simplest organism from which a protein-tyrosine kinase gene has been isolated. The presence of such a gene in the evolutionarily ancient phylum Cnidaria suggests that protein-tyrosine kinase genes arose concomitantly with or shortly after the appearance of multicellular organisms.

The Src/Csk regulatory circuit arose early in metazoan evolution.

We have identified a gene encoding a member of the Csk family of non-receptor protein-tyrosine kinases (PTKs) in the early-diverging metazoan Hydra. In situ hybridization analysis of the distribution of RNA from the Hydra Csk gene indicates that it is expressed in most of the epithelial cells of the adult polyp and in gametogenic cells. Comparison of the expression pattern of Hydra Csk with that of STK, the Hydra Src gene orthologue, reveals that the two genes are largely co-expressed. Such co-expression is consistent with a role for Hydra Csk in regulation of STK activity. This possibility was tested directly by coexpressing Hydra Csk with STK in yeast. Co-expression suppressed the growth inhibition seen when STK alone is expressed in yeast. Suppression was dependent on the presence of the putative regulatory tyrosine in the carboxyl-terminal tail of STK. Phosphotyrosine immunoblot analysis confirmed that expression of Csk resulted in suppression of STK kinase activity. Taken together these data indicate that the regulatory circuit involving Src and Csk PTKs was established prior to the divergence of the phylum Cnidaria from the rest of the metazoans.

Structure and expression of fyn genes in Xenopus laevis.

By low stringency screening of a Xenopus laevis oocyte cDNA library with a probe prepared from the yes oncogene of the avian retrovirus Y73, we have isolated clones encoding a 537 amino acid protein with 96% amino acid identity to the protein product of the human fyn proto-oncogene. In addition to the high degree of conservation at the amino acid sequence level, we also find a surprisingly high degree of sequence conservation between portions of the 5' untranslated regions of the frog and human genes. Probing of total DNA from a homozygous diploid frog with a fragment from one of the cDNA clones gave a hybridization pattern which is consistent with the presence of two fyn genes in the haploid Xenopus laevis genome. Hybridization analysis revealed that the ovary contains two different sized fyn RNAs, one 3.8 kb in length and the other 3.1 kb in length. Our finding of fyn transcripts brings to three the number of protein-tyrosine kinases of the src family whose mRNAs are found in X. laevis oocytes. This suggests that multiple protein-tyrosine kinases of the src family are required for processes in oogenesis or early development.

Structural organization of a src gene from Xenopus laevis.

By sequence analysis of genomic clones, the exon-intron structure of one of the two src genes from Xenopus laevis has been determined. The coding region of the gene is interrupted by 10 introns whose locations are identical to the introns in the coding regions of the src genes of human and chicken. The 5' untranslated region is contained on a separate exon with no sequence conservation relative to the corresponding region of the chicken gene. The 5' untranslated region of the Xenopus gene contains a G + C-rich stem-loop sequence and two ATG triplets. A 1.4-kb fragment containing the 5' untranslated region and sequences upstream of it acts as a promoter when introduced in the correct orientation into X. laevis cell lines. The DNA sequence of this fragment lacks the typical arrangement of TATA and CCAAT sequences but contains the ATGCAAAT octamer sequence and a (TA)39 sequence.

Identification of a gene encoding a novel protein-tyrosine kinase containing SH2 domains and ankyrin-like repeats.

Using the polymerase chain reaction with primers corresponding to conserved regions in the kinase domain of protein-tyrosine kinases, we amplified segments of several protein-tyrosine kinase genes from Hydra vulgaris, a member of the ancient metazoan phylum Cnidaria. Characterization of cDNA clones for one of these genes, HTK16, revealed that it encodes a non-receptor protein-tyrosine kinase with two SH2 domains but no SH3 domain. In this regard the predicted HTK16 protein resembles two mammalian non-receptor protein-tyrosine kinases, the products of the ZAP-70 and syk genes. However, the HTK16 protein contains five ankyrin-like repeats, a structural motif which has not previously been found in protein-tyrosine kinases. The HTK16 protein also contains a potential tyrosine phosphorylation site in its carboxyl-terminal tail which resembles the phosphorylation site in members of the src family. RNA hybridization analysis indicates that the HTK16 gene is expressed in epithelial cells, cells which also express the Hydra homologue of the src protein. Our finding of the HTK16 gene in Hydra indicates that diversification of genes encoding non-receptor protein-tyrosine kinases was a very early event in metazoan evolution.

Single step purification of biologically active recombinant rat basic fibroblast growth factor by immobilized metal affinity chromatography.

The construction and use of a plasmid which allows the expression and single step purification of recombinant rat basic fibroblast growth factor (bFGF) is described. A cDNA encoding rat bFGF was subcloned into the expression plasmid pQE-9 (Qiagen) in such a way that the bFGF which is produced from the resulting construct contains 6 histidine residues near the amino terminus. The resulting plasmid, pQE-9-bFGF, was expressed in the E. coli strain M15[pREP4] and the 6 x His-bFGF was purified to homogeneity from the soluble fraction of the bacterial cell lysate in a single step by affinity chromatography on a nickel chelate resin. About 5 mg of 6 x His-bFGF was obtained from the soluble fraction from one liter of bacterial cell culture. Testing of the 6 x His-bFGF in a PC12 cell differentiation assay showed that its activity was comparable to the activities for native bFGF and recombinant bFGF purified by multistep methods.

Integrated concentrations of luteinizing hormone and puberty.

Integrated serum concentrations of luteinizing hormone have been compared among 30-minute collections from 10 boys (6-18 years old) and 5 girls (5-11 years old). This study suggests that perpubertal as well as pubertal boys have greater mean integrated concentrations of LH during sleep than during waking. One of two pubertal girls had greater concentrations of LH during sleep, while three prepubertal girls did not.

A gene whose major transcript encodes only the substrate-binding domain of a protein-tyrosine kinase.

We have identified a novel protein-tyrosine kinase gene family in the simple multicellular animal Hydra vulgaris that consists of at least three members. Two of the genes encode receptor protein-tyrosine kinases. The third member of the family is unusual in that in non-sexual animals, the only transcripts that it produces encode polypeptides lacking all or nearly all of the ATP-binding lobe. Characterization of multiple cDNA clones and hybridization mapping of genomic DNA indicate that the gene, which we have termed Hinterteil (Hint), undergoes alternative cis-splicing, alternative trans-splicing, and alternative polyadenylation. In-situ hybridization analysis shows that expression of the gene is upregulated during spermatogenesis. Sexual males also produce an additional Hint transcript that is larger than the transcript seen in non-sexual animals, but still not large enough to encode a receptor.

Appearance and disappearance of Syk family protein-tyrosine kinase genes during metazoan evolution.

Syk family protein-tyrosine kinases are essential components of immunoreceptor signaling in mammalian lymphocytes. The absence of Syk genes from the Caenorhabditis elegans genome suggests that this kinase family is of recent evolutionary origin. Surprisingly, we have found that Hydra vulgaris, a member of the early diverging animal phylum Cnidaria, contains a gene encoding a Syk kinase. Phylogenetic analysis indicates that a single Syk family gene was present in animals prior to the gene duplication that gave rise to Syk and ZAP-70, the two mammalian Syk family genes. C. elegans also lacks a Shark protein-tyrosine kinase gene, which we show is a member of a sister group to the Syk family. We conclude that both Syk and Shark genes were lost from the genome of an ancestor of C. elegans. This natural gene knockout result indicates that neither Syk nor Shark kinases are essential for processes held in common between the nematode and other metazoans. The Hydra Syk gene is expressed in epithelial cells, a site consistent with a role for Hydra Syk in recognition of foreign cells.

Identification of potential antiatherosclerotic/hypolipidemic agents by their effect on hepatic conversion of androst-4-ene-3,17-dione to etiocholanolone and androsterone.

Clinical observations have shown that hypercholesterolemia is associated with abnormal androgen metabolism, viz. an increased excretion of etiocholanolone (E) relative to androsterone (A). Substances which restore the A/E ratio to normal likewise lower serum cholesterol. Postulating that the abnormal steroid and sterol metabolism may be either causally related or dependent on the same metabolic defect, we have developed in vitro and in vivo models to select drugs which favorably effect the ratio of A to E produced from [4-14C]androst-4-ene-3,17-dione [4-14C]A-dione). The in vitro model employs a mixture of rat liver microsomal delta 4-3-ketosteroid-5 alpha-reductase and cytosolic 3 alpha-hydroxysteroid dehydrogenase and delta 4-3-ketosteroid-5 beta-reductase. Kinetic and mechanistic studies have been performed on active compounds using this in vitro assay. The in vivo model employs i.v. injection of [4-14C]A-dione followed by collection of bile in anesthetized, hypophysectomized female rats. Many compounds preselected in the in vitro assay likewise reduced the A/E ratio in vivo. One of these compounds (CGS 10614A) also lowered serum cholesterol and reduced the incidence and severity of atherosclerotic lesions in aortas of cholesterol-fed rabbits.

Demonstration of potent lipid-lowering activity by a thyromimetic agent devoid of cardiovascular and thermogenic effects.

A potent lipid-lowering thyromimetic (CGS 26214) devoid of cardiac and thermogenic activity was identified based on its ability to preferentially access and bind the nuclear fraction of hepatocytes over that of myocytes in culture. The difference in access achieved with CGS 26214 was at least 100-fold better for hepatocytes than for myocytes. This in vitro hepatoselectivity resulted in a compound with unprecedented in vivo lipid-lowering potency with a minimal effective dose of 1 microgram/kg in rats and dogs (approximately 25x that of L-T3). At the same time, CGS 26214 was free of any cardiovascular effects up to the highest dose tested of 25 mg/kg and 100 micrograms/kg in rats and dogs, respectively.

Effects of altered endocrine function on biliary metabolites of [4-14C]androst-4-ene-3,17-dione in rats. Possible utility as a model for identifying anti-atherosclerotic agents.

Atherosclerosis, coronary artery disease and elevated serum cholesterol are frequently associated with an abnormal pattern of androgen metabolites, especially an elevation of etiocholanolone (E) and/or epiandrosterone (EA) relative to androsterone (A). Therapeutic correction of these metabolic defects may lower serum cholesterol. We have attempted to reproduce this metabolic syndrome in rats by altering their endocrine status. Intact male rats excreted very little A or E in their bile; more than 80% of the [4-14C]A-dione was excreted as unknown polar compounds. Adrenalectomy, thyroidectomy or streptozotocin diabetes induced little or no change in the excretion of both E and A and did not alter the A/E ratio. Hypophysectomy (hypox), however, resulted in a huge increase in E excretion and a 10-fold decrease in the A/E ratio. Treatment of hypophysectomized males with bovine growth hormone (bGH) but not testosterone or thyroxine restored the pattern of androgen metabolites to that of intact male rats. Intact female rats excreted mainly A, and this was decreased by ovariectomy. Hypophysectomy, however, resulted in a marked increase in E and a corresponding large decrease in A excretion. Treatment of hypox females with estradiol or triiodothyronine did not correct the metabolic defects in A and E production, whereas GH resulted in a pattern of A-dione metabolism resembling that of intact males; i.e., primarily polar metabolites with low A and E. Hypophysectomy thus results in a dramatic increase in 5 beta-reductase activity in male and female rats. GH therapy restores the metabolic pathway to that seen in intact males. Our objective had been to find a model capable of detecting substances which would increase A and decrease E production. The male rat (regardless of endocrine status) has little 5 alpha-reductase activity. The intact female rat, however, has high 5 alpha-reductase activity, and retains significant 5 alpha-reductase in the absence of the ovaries. In hypox females, 5 alpha-reductase was much reduced while 5 beta-reductase was increased. Furthermore, serum cholesterol was elevated in hypox females but could be lowered by exogenous androsterone. Thus the hypox female rat appears to offer the best model for identifying non-hormonal agents which could enhance the production of A and/or decrease the production of E. Such agents might favorably influence cholesterol metabolism.