Arcangela De Nicolo

Efficacy of Neoadjuvant Cisplatin in Triple-Negative Breast Cancer

PURPOSE Cisplatin is a chemotherapeutic agent not used routinely for breast cancer treatment. As a DNA cross-linking agent, cisplatin may be effective treatment for hereditary BRCA1-mutated breast cancers. Because sporadic triple-negative breast cancer (TNBC) and BRCA1-associated breast cancer share features suggesting common pathogenesis, we conducted a neoadjuvant trial of cisplatin in TNBC and explored specific biomarkers to identify predictors of response. PATIENTS AND METHODS Twenty-eight women with stage II or III breast cancers lacking estrogen and progesterone receptors and HER2/Neu (TNBC) were enrolled and treated with four cycles of cisplatin at 75 mg/m(2) every 21 days. After definitive surgery, patients received standard adjuvant chemotherapy and radiation therapy per their treating physicians. Clinical and pathologic treatment response were assessed, and pretreatment tumor samples were evaluated for selected biomarkers. Results Six (22%) of 28 patients achieved pathologic complete responses, including both patients with BRCA1 germline mutations;18 (64%) patients had a clinical complete or partial response. Fourteen (50%) patients showed good pathologic responses (Miller-Payne score of 3, 4, or 5), 10 had minor responses (Miller-Payne score of 1 or 2), and four (14%) progressed. All TNBCs clustered with reference basal-like tumors by hierarchical clustering. Factors associated with good cisplatin response include young age (P = .001), low BRCA1 mRNA expression (P = .03), BRCA1 promoter methylation (P = .04), p53 nonsense or frameshift mutations (P = .01), and a gene expression signature of E2F3 activation (P = .03). CONCLUSION Single-agent cisplatin induced response in a subset of patients with TNBC. Decreased BRCA1 expression may identify subsets of TNBCs that are cisplatin sensitive. Other biomarkers show promise in predicting cisplatin response.

Centrosomal protein of 192 kDa (Cep192) promotes centrosome-driven spindle assembly by engaging in organelle-specific Aurora A activation

Centrosomes are primary microtubule (MT)-organizing centers (MTOCs). During mitosis, they dramatically increase their size and MT-nucleating activity and participate in spindle assembly from spindle poles. These events require the serine/threonine kinase, Aurora A (AurA), and the centrosomal protein of 192 kDa (Cep192)/spindle defective 2 (Spd-2), but the underlying mechanism remains unclear. We have found that Cep192, unlike targeting protein for Xklp2 (TPX2), a known MT-localizing AurA activator, is an AurA cofactor in centrosome-driven spindle assembly. Cep192, through a direct interaction, targets AurA to mitotic centrosomes where the locally accumulating AurA forms homodimers or oligomers. The dimerization of endogenous AurA, in the presence of bound Cep192, triggers potent kinase activation that, in turn, drives MT assembly. Depletion of Cep192 or specific interference with AurA-Cep192 binding did not prevent AurA oligomerization on MTs but abrogated AurA recruitment to centrosomes and its activation by either sperm nuclei or anti-AurA antibody (αAurA)-induced dimerization. In these settings, MT assembly by both centrosomes and αAurA-coated beads was also abolished or severely compromised. Hence, Cep192 activates AurA by a mechanism different from that previously described for TPX2. The Cep192-mediated mechanism maximizes AurA activity at centrosomes and appears essential for the function of these organelles as MTOCs.

Multimodal Assessment of Protein Functional Deficiency Supports Pathogenicity of BRCA1 p.V1688del

Unequivocal discrimination between neutral variants and deleterious mutations is crucial for appropriate counseling of individuals with a BRCA1 or BRCA2 sequence change. An increasing number of variants of uncertain significance (VUS) are being identified, the unclassified biological effect of which poses clinical concerns. A multifactorial likelihood-based approach recently suggested disease causality for BRCA1 p.V1688del, a VUS recurrent in Italian breast/ovarian cancer families. Whether and how this single amino acid deletion in the BRCA1 COOH terminus (BRCT) domain affects the function of the mutant protein (DeltaValBRCA1) has not been elucidated. We undertook comprehensive functional characterization of DeltaValBRCA1, comprising comparative structural modeling, analysis of protein stability and associations, and analysis of DNA repair function. Our model predicted BRCT domain destabilization and folding disruption caused by BRCA1 p.V1688del. Consistently, the recombinant DeltaValBRCA1 was less stable than wild-type BRCA1 and, unlike the latter, failed to associate with BRIP1, CtIP, and Rap80 and to relocalize to sites of DNA damage. Yeast two-hybrid analysis revealed a compromised interaction with FHL2 and KPNA2, which is likely responsible for improper subcellular localization of DeltaValBRCA1. In addition, we found four new breast/ovarian cancer families of Italian ancestry who carried this sequence alteration. These results provide the first evidence of the effect of BRCA1 p.V1688del on protein stability and function, supporting the view that it is a deleterious mutation. Multimodal analyses like ours could advance understanding of tumor suppression by BRCA1 and ultimately contribute to developing efficient strategies for screening and characterization of VUS.

Aurora-PLK1 cascades as key signaling modules in the regulation of mitosis

Precise spatiotemporal control enables the mitotic kinases Aurora and PLK1 to orchestrate distinct mitotic processes. Gloss Ever since its first observation under the microscope over a century ago, mitosis—the process wherein a cell divides into two genetically identical daughter cells—has captured the attention of biologists and physicians alike owing to its beauty, sophistication, and importance for the life cycle of eukaryotic organisms. The key factors that control mitosis primarily do so through protein phosphorylation and regulated degradation. In this review, which contains six figures, one table, and 432 references, we discuss the mechanisms by which only a handful of mitotic protein kinases control various mitotic events in distinct spatiotemporal contexts. We highlight the central role in this control of the two-tiered Aurora-PLK1 cascades that are organized by several multifunctional scaffold proteins and integrated within larger mitotic regulatory networks. Mitosis is controlled by reversible protein phosphorylation involving specific kinases and phosphatases. A handful of major mitotic protein kinases, such as the cyclin B–CDK1 complex, the Aurora kinases, and Polo-like kinase 1 (PLK1), cooperatively regulate distinct mitotic processes. Research has identified proteins and mechanisms that integrate these kinases into signaling cascades that guide essential mitotic events. These findings have important implications for our understanding of the mechanisms of mitotic regulation and may advance the development of novel antimitotic drugs. We review collected evidence that in vertebrates, the Aurora kinases serve as catalytic subunits of distinct complexes formed with the four scaffold proteins Bora, CEP192, INCENP, and TPX2, which we deem “core” Aurora cofactors. These complexes and the Aurora-PLK1 cascades organized by Bora, CEP192, and INCENP control crucial aspects of mitosis and all pathways of spindle assembly. We compare the mechanisms of Aurora activation in relation to the different spindle assembly pathways and draw a functional analogy between the CEP192 complex and the chromosomal passenger complex that may reflect the coevolution of centrosomes, kinetochores, and the actomyosin cleavage apparatus. We also analyze the roles and mechanisms of Aurora-PLK1 signaling in the cell and centrosome cycles and in the DNA damage response.

Assays to Study Mitotic Centrosome and Spindle Pole Assembly and Regulation.

Faithful chromosome segregation during cell division requires proper bipolar spindle assembly and critically depends on spindle pole integrity. In most animal cells, spindle poles form as the result of the concerted action of various factors operating in two independent pathways of microtubule assembly mediated by chromatin/RanGTP and by centrosomes. Mutation or deregulation of a number of spindle pole-organizing proteins has been linked to human diseases, including cancer and microcephaly. Our knowledge on how the spindle pole-organizing factors function at the molecular level and cooperate with one another is still quite limited. As the list of these factors expands, so does the need for the development of experimental approaches to study their function. Cell-free extracts from Xenopus laevis eggs have played an instrumental role in the dissection of the mechanisms of bipolar spindle assembly and have recently allowed the reconstitution of the key steps of the centrosome-driven microtubule nucleation pathway (Joukov et al., Mol Cell 55:578-591, 2014). Here we describe assays to study both centrosome-dependent and centrosome-independent spindle pole formation in Xenopus egg extracts. We also provide experimental procedures for the use of artificial centrosomes, such as microbeads coated with an anti-Aurora A antibody or a recombinant fragment of the Cep192 protein, to model and study centrosome maturation in egg extract. In addition, we detail the protocol for a microtubule regrowth assay that allows assessment of the centrosome-driven spindle microtubule assembly in mammalian cells.

Collective evidence supports neutrality of BRCA1 V1687I, a novel sequence variant in the conserved THV motif of the first BRCT repeat

Unambiguous classification of BRCA1 and BRCA2 variants of uncertain significance (VUS) is a challenging task that vexes health care providers and has profound implications for patients and their family members. Numerous VUS have been described to date, which await assessment of their functional, hence clinical, impact. As a result of a routine BRCA1/BRCA2 mutational screening, we identified a previously unreported BRCA1 sequence alteration [c.5178G>A (V1687I)] in a patient diagnosed with early onset triple negative breast cancer. The sequence alteration falls in the invariant THV motif of the BRCT domain. To investigate its significance, we applied an integrated approach that, in addition to genetic and histopathological data, included in silico analyses, comparative structural modeling and verification of BRCT-mediated interactions. In line with web-based algorithms that predicted the benign nature of BRCA1 V1687I, the three-dimensional model of the BRCA1 V1687I BRCT domain did not reveal any major structural changes relative to its wild-type counterpart, thus suggesting that BRCA1 V1687I has a negligible impact on both the local architecture and the overall stability of the protein. Consistently, the BRCA1 V1687I protein was properly expressed and localized to the nucleus, and it was still capable of binding three BRCT-interacting, DNA damage response, and repair partner proteins, namely BRIP1/FANCJ, CtIP, and Abraxas. Our collected evidence suggests that, although occurring in a highly conserved region, the BRCA1 V1687I variant is likely a benign sequence alteration.

BRAF Gene Copy Number and Mutant Allele Frequency Correlate with Time to Progression in Metastatic Melanoma Patients Treated with MAPK Inhibitors

Metastatic melanoma is characterized by complex genomic alterations, including a high rate of mutations in driver genes and widespread deletions and amplifications encompassing various chromosome regions. Among them, chromosome 7 is frequently gained in BRAF-mutant melanoma, inducing a mutant allele–specific imbalance. Although BRAF amplification is a known mechanism of acquired resistance to therapy with MAPK inhibitors, it is still unclear if BRAF copy-number variation and BRAF mutant allele imbalance at baseline can be associated with response to treatment. In this study, we used a multimodal approach to assess BRAF copy number and mutant allele frequency in pretreatment melanoma samples from 46 patients who received MAPK inhibitor–based therapy, and we analyzed the association with progression-free survival. We found that 65% patients displayed BRAF gains, often supported by chromosome 7 polysomy. In addition, we observed that 64% patients had a balanced BRAF-mutant/wild-type allele ratio, whereas 14% and 23% patients had low and high BRAF mutant allele frequency, respectively. Notably, a significantly higher risk of progression was observed in patients with a diploid BRAF status versus those with BRAF gains [HR, 2.86; 95% confidence interval (CI), 1.29–6.35; P = 0.01] and in patients with low percentage versus those with a balanced BRAF mutant allele percentage (HR, 4.54; 95% CI, 1.33–15.53; P = 0.016). Our data suggest that quantitative analysis of the BRAF gene could be useful to select the melanoma patients who are most likely to benefit from therapy with MAPK inhibitors. Mol Cancer Ther; 17(6); 1332–40. ©2018 AACR.

MelaNostrum: a consensus questionnaire of standardized epidemiologic and clinical variables for melanoma risk assessment by the melanostrum consortium

Many melanoma observational studies have been carried out across different countries and geographic areas using heterogeneous assessments of epidemiologic risk factors and clinical variables.