HMGA1 positively regulates the microtubule-destabilizing protein stathmin promoting motility in TNBC cells and decreasing tumour sensitivity to paclitaxel

High Mobility Group A1 (HMGA1) is an architectural chromatin factor involved in the regulation of gene expression and a master regulator in Triple Negative Breast Cancer (TNBC). In TNBC, HMGA1 is overexpressed and coordinates a gene network that controls cellular processes involved in tumour development, progression, and metastasis formation. Here, we find that the expression of HMGA1 and of the microtubule-destabilizing protein stathmin correlates in breast cancer (BC) patients. We demonstrate that HMGA1 depletion leads to a downregulation of stathmin expression and activity on microtubules resulting in decreased TNBC cell motility. We show that this pathway is mediated by the cyclin-dependent kinase inhibitor p27^kip1 (p27). Indeed, the silencing of HMGA1 expression in TNBC cells results both in an increased p27 protein stability and p27-stathmin binding. When the expression of both HMGA1 and p27 is silenced, we observe a significant rescue in cell motility. These data, obtained in cellular models, were validated in BC patients. In fact, we find that patients with high levels of both HMGA1 and stathmin and low levels of p27 have a statistically significant lower survival probability in terms of relapse-free survival (RFS) and distant metastasis-free survival (DMFS) with respect to the patient group with low HMGA1, low stathmin, and high p27 expression levels. Finally, we show in an in vivo xenograft model that depletion of HMGA1 chemo-sensitizes tumour cells to paclitaxel, a drug that is commonly used in TNBC treatments. This study unveils a new interaction among HMGA1, p27, and stathmin that is critical in BC cell migration. Moreover, our data suggest that taxol-based treatments may be more effective in reducing the tumour burden when tumour cells express low levels of HMGA1.Subject terms: Breast cancer, Breast cancer     

Conformational role for the C-terminal tail of the intrinsically disordered high mobility group A (HMGA) chromatin factors

The architectural factors HMGA are highly connected hubs in the chromatin network and affect key cellular functions. HMGA have a causal involvement in cancer development; in fact, truncated or chimeric HMGA forms, resulting from chromosomal rearrangements, lack the constitutively phosphorylated acidic C-terminal tail and display increased oncogenic potential, suggesting a functional role for this domain. HMGA belong to the intrinsically disordered protein category, and this prevents the use of classical approaches to obtain structural data. Therefore, we combined limited proteolysis, ion mobility separation-mass spectrometry (IMS-MS), and electrospray ionization-mass spectrometry (ESI-MS) to obtain structural information regarding full length and C-terminal truncated HMGA forms. Limited proteolysis indicates that HMGA acidic tail shields the inner portions of the protein. IMS-MS and ESI-MS show that HMGA proteins can assume a compact form and that the degree of compactness is dependent upon the presence of the acidic tail and its constitutive phosphorylations. Moreover, we demonstrate that C-terminal truncated forms and wild type proteins are post-translationally modified in a different manner. Therefore, we propose that the acidic tail and its phosphorylation could affect HMGA post-translational modification status and likely their activity. Finally, the mass spectrometry-based approach adopted here proves to be a valuable new tool to obtain structural data regarding intrinsically disordered proteins.     

A new and fast method for preparing high quality lambda DNA suitable for sequencing.

A method is described for the rapid purification of high quality lambda DNA. The method can be used from either liquid or plate lysates and on a small scale or a large scale. It relies on the preadsobtion of all polyanions present in the lysate to an "insoluble" anion-exchange matrix (DEAE or TEAE). Phage particles are then disrupted by combined treatment with EDTA/proteinase K and the resulting DNA is precipitated by the addition of the cationic detergent cetyl (or hexadecyl)-trimethyl ammonium bromide-CTAB ("soluble" anion-exchange matrix). The precipitated CTAB-DNA complex is then exchanged to Na-DNA and ethanol precipitated. The resultant purified DNA is suitable for enzymatic reactions and provides a high quality template for dideoxy-sequence analysis.     

Mass spectrometric analysis of the HMGY protein from Lewis lung carcinoma. Identification of phosphorylation sites.

The primary structure of the Lewis lung carcinoma protein HMGY belonging to the nuclear group of proteins HMGI (high mobility group I) was determined using electrospray and fast atom bombardment mass spectrometry. It was demonstrated that the sequence of the tumor protein corresponds to the amino acid sequence derived from the cDNA from cultured cells and that the N-terminal serine residue is N-acetylated. Moreover, the two high performance liquid chromatography-purified forms Y1 and Y2 of the protein HMGY were shown to differ at the level of serine phosphorylation, since they contain three phosphate and two phosphate groups, respectively, in the C-terminal region. No other modification was detected in the remaining part of the molecule.