The nucleocrine pathway comes of age.

More than 20 years ago, it was initially predicted that hormones and growth factors might promote cell growth by binding and thereby inactivating tumor suppressors, as exemplified by the proposed complex formation between insulin and retinoblastoma protein (RB). This mainly intracellular/nuclear growth-regulatory circuit was termed "the nucleocrine pathway" and the physical interaction between insulin and RB was subsequently proven through several methods, primarily by immunofluorescence and co-immunoprecipitation. Meanwhile, additional nucleocrine pairs have emerged through further experimental studies, specifically the FGF1-p53 and angiogenin-p53 heterodimers. Moreover, first experimental clues have been obtained as to the intranuclear presence of the previously surmised heterodimer between the EGF precursor and the p130 tumor suppressor. In addition, RB-binding motifs have recently been discovered in interleukin-6 (IL-6) and cellular apoptosis susceptibility (CAS) protein. These findings point to a more general significance of the nucleocrine pathway in cell growth regulation and as a particularly useful target in cancer therapy.

One hundred years of tumor suppressor research: crucial achievements and unique perspectives.

Tumor suppressors constitute the body's primary defense line against malignant transformation. Since Theodor Boveri's initial insight one century ago, a huge amount of knowledge on these molecules has been generated. However, the final step of application of this profound understanding in the clinical setting, i.e., the treatment of cancer patients with tumor suppressors and their derivatives, is still ahead. Nevertheless, the important success achieved with similar biomimetic approaches in the therapy of other diseases suggests that tumor suppressor-based antineoplastic interventions should be accomplished soon as they may be equally rewarding.

The practical utility of synthetic tumor suppressor peptides: a personal retrospective on 25 years.

In this brief survey, I look back upon a quarter of a century of my personal experience with synthetic peptides. Thereby, I focus on major steps in the design and experimental exploration of peptides that I have derived from the retinoblastoma tumor suppressor protein (RB). Along this way, both Merrifield's solid phase peptide synthesis method and collaborations with established investigators in the peptide and cancer research fields have played an important role.

From the RB tumor suppressor to MCR peptides.

About twenty years ago, the search for a peptide mimetic of the crucial 928-amino acid human retinoblastoma tumor suppressor protein (RB) has led to the identification of a potential active site in RB spanning 6 amino acids. Subsequent studies revealed that this hexapeptide needs to be coupled to a cellular internalization sequence in order to be biologically active. The prototype molecule resulting therefrom has been the synthetic 22-amino acid peptide MCR-4 that was proven through several investigations to exert both in vitro and in vivo anti-cancer activity both resembling and going beyond the antiproliferative effects of its template RB. This was followed by the elaboration of a distinct series of MCR peptides designed to also interfere with target structures located on the surface of cells and known to be involved in triggering cell proliferation such as the insulin receptor. For this second generation group, the prototype peptide has been another 22-amino acid peptide coined MCR-14 and equally demonstrated to display in vitro and in vivo anti-tumor effects. Finally, a miniaturization of the structure-function relationships intrinsic to the already small MCR-4 peptide has led to the development of the third generation 17-amino acid peptide MCR-15 which in turn was also shown to inhibit in vitro and in vivo malignant cell reproduction. Given more recent conceptual advances, it is warranted to claim that MCR peptides are likely candidate therapeutics not only for the treatment of neoplasias, but also of viral and bacterial infections, type 2 diabetes as well as hypertension.

Oncoprotein metastasis: an expanded topography.

In this survey, the initial insights on the sub- and transcellular process of oncoprotein metastasis (OPM) are linked to recent observations and advances in related fields. The six proteins described here, i.e. insulin, osteopontin, interleukin-6, anterior gradient-2 protein, cellular apoptosis susceptibility protein and hepatoma-derived growth factor, as well as distinct peptide fragments thereof might henceforth serve as pivotal biomarkers for OPM in clinical chemistry, molecular morphology, pathology and oncology and, as a result, guide as potential targets future structure-based interventions in cancer treatment.

Intracellular insulin in human tumors: examples and implications.

Insulin is one of the major metabolic hormones regulating glucose homeostasis in the organism and a key growth factor for normal and neoplastic cells. Work conducted primarily over the past 3 decades has unravelled the presence of insulin in human breast cancer tissues and, more recently, in human non-small cell lung carcinomas (NSCLC). These findings have suggested that intracellular insulin is involved in the development of these highly prevalent human tumors. A potential mechanism for such involvement is insulin's binding and inactivation of the retinoblastoma tumor suppressor protein (RB) which in turn is likely controlled by insulin-degrading enzyme (IDE). This model and its supporting data are collectively covered in this survey in order to provide further insight into insulin-driven oncogenesis and its reversal through future anticancer therapeutics.

Oncoprotein metastasis and its suppression revisited.

The past two decades have witnessed an increasing appreciation of the role of the tumor microenvironment, of genetic and epigenetic alterations in normal cells adjacent to tumors and of the migration of normal cells with aberrant intrinsic properties in cancer pathophysiology. Aside from these insights, a novel concept termed "oncoprotein metastasis" (OPM) has recently been advanced and proposed to reflect protein-based neoplastic phenomena that might occur even before any modifications relating to the morphology, location or (epi)genetic outfit of cells during the malignant process. Here, evidence is presented that supports the OPM perception and thus should contribute not only to further rethink the definition of a normal cell, but also the treatment of cancer disease in the years to come.

Retinoblastoma protein co-purifies with proteasomal insulin-degrading enzyme: implications for cell proliferation control.

Previous investigations on proteasomal preparations containing insulin-degrading enzyme (IDE; EC 3.4.24.56) have invariably yielded a co-purifying protein with a molecular weight of about 110kDa. We have now found both in MCF-7 breast cancer and HepG2 hepatoma cells that this associated molecule is the retinoblastoma tumor suppressor protein (RB). Interestingly, the amount of RB in this protein complex seemed to be lower in HepG2 vs. MCF-7 cells, indicating a higher (cytoplasmic) protein turnover in the former vs. the latter cells. Moreover, immunofluorescence showed increased nuclear localization of RB in HepG2 vs. MCF-7 cells. Beyond these subtle differences between these distinct tumor cell types, our present study more generally suggests an interplay between RB and IDE within the proteasome that may have important growth-regulatory consequences.

Intracellular presence of insulin and its phosphorylated receptor in non-small cell lung cancer.

Insulin has been known for a long time to influence the growth and differentiation of normal and transformed cells. In order to delineate the role of insulin specifically in non-small cell lung cancer (NSCLC), we have now searched by immunohistochemistry (IHC) for the presence of insulin in NSCLC samples. Among the 112 samples we studied, 30 were found to contain insulin, which was detected in the form of intracytoplasmic granula. Moreover, its expression significantly correlated with (a) the morphological/histopathological subtype of NSCLC, being more frequent in adenocarcinomas; (b) the grade of tumor differentiation, displaying an increase in low-grade carcinomas; (c) tumor size, occurring predominantly in smaller tumors; (d) the presence of phosphorylated, activated insulin receptor; (e) the median patient age, being present in relatively younger individuals. Furthermore and interestingly, surrounding atypical adenomatous hyperplastic areas and normal alveolar pneumocytes scored insulin-positive in some of the insulin-negative tumors. In addition, PCR exploration for insulin transcripts in some samples positive for immunoreactive insulin was negative, indicating a possibly exogenous origin for the intracellular insulin in our NSCLC cohort. Taken together, our data suggest that an intracellular insulin activity is important for the progression of low-grade human lung adenocarcinomas.

One for all and all for one: RB defends the cell while IDE, PTEN and IGFBP-7 antagonize insulin and IGFs to protect RB.

Tumor suppressor proteins are the main cellular barrier opposing neoplastic transformation. Among these host molecules, retinoblastoma protein (RB) plays a central role. A novel insight is now advanced to suggest that various inhibitors of insulin and insulin-like growth factor (IGF) signalling such as the putative tumor suppressor insulin-degrading enzyme (IDE) as well as the anti-oncogenic proteins PTEN and insulin-like growth factor-binding protein 7 (IGFBP-7) serve the common goal of ensuring that RB remains active. Since, moreover, IDE and IGFBP-7 each potentially achieves RB protection through preventing both binding and inactivation of RB by insulin, IGF-1 or IGF-2, the present perception also vindicates the importance of previous findings on the physical interaction of any of these growth factors with RB for cell fate. Notably, the therapeutic counterpart of this natural principle for maintaining or restoring RB function through insulin/IGF neutralization is the innovative class of anti-cancer agents termed MCR peptides and developed over the past decade.

Immunohistochemical demonstration of the zinc metalloprotease insulin-degrading enzyme in normal and malignant human breast: correlation with tissue insulin levels.

Insulin is a hormone crucial to metabolism and an essential growth factor for normal and neoplastic tissues. We have now determined insulin in extracts of 23 primary breast cancer specimens and of non-neoplastic breast tissues by a chemiluminescent immunoassay. Remarkably, insulin was measured only in grade 3 tumors, whereas grade 2 carcinomas and the normal mammary gland were each insulin-negative. We also performed immunohistochemistry for insulin-degrading enzyme (IDE), a cytoplasmic zinc metalloprotease belonging to the inverzincin family and participating in insulin cleavage. IDE was detected in most insulin-positive grade 3 carcinomas, indicating that it might be dysfunctional in these anaplastic tumors. IDE was equally present in the insulin-negative grade 2 carcinomas. Moreover, five grade 3 carcinomas and one grade 2 carcinoma displayed a loss of heterozygosity in the 10q chromosomal region harboring the IDE gene, but, despite these alterations, IDE was detected immunohistochemically, indicating a retention of the second allele. Compared to the expression of IDE in 92% of the tumors examined, only 57% of 21 normal breast specimens stained positively for IDE. In contrast to this increase in IDE-positive epithelial cells in breast cancer vs. normal breast, additional immunohistochemical analysis of 17 node-positive breast carcinomas and corresponding tumor-bearing lymph nodes showed that IDE expression decreases from primary tumor to lymph node metastasis. Altogether, this study represents the first demonstration of IDE in normal and neoplastic human mammary tissues. Our present report should also provide an experimental starting point towards exploring a potential role of IDE in the control of tumor progression.