The research program coordinated by Professor Guidalberto Manfioletti is centred mainly in basic aspects regarding the role of HMGA proteins in driving the process of neoplastic transformation. These architectural factors are able to assemble or modulate DNA/nucleoprotein macromolecular complexes thus participating in gene expression regulation, chromatin remodelling and dynamics, replication, and DNA repair. Being critical hubs in the chromatin network, they influence a variety of biological processes such as embryogenesis and differentiation and the deregulation of their activity leads to neoplastic transformation. A deep understanding of the pathways affected by HMGA1 could therefore point-out new potential targets for cancer therapy.

All the activities within this program are organized in two main lines of research:

Towards the definition of the global influence of HMGA1 proteins in cancer cells: an integrated genomic and proteomic approach

High-throughput genomic and proteomic methods are available to study molecular networks in different systems. The ability of these techniques to simultaneously interrogate thousands of transcripts/proteins has led to important advances in a number of biological problems. With these approaches we are exploring the influence of HMGA1 on gene transcription and protein expression.

Our current research is focused on breast cancer cells in which the silencing of HMGA1 causes a reversion of the tumoral phenotype resembling a mesenchymal-epithelial transition. We have evaluated the effect of HMGA1 silencing on the aggressiveness of breast cancer cells, demonstrating that HMGA1 is fundamental for controlling cell motility, invasiveness, and in maintaining stemness properties (Pegoraro et al. 2013).

Now we are facing at exploring the global contribution of HMGA1 in gene transcription and protein expression. 

a) Genomic approach

We are analyzing by DNA microarray and RNA-seq analyses the gene expression profile of breast cancer cells in which HMGA1 expression has been silenced. With the aid of bioinformatic tools we are dissecting the various pathways/factors tuned by HMGA1. The two techniques adopted provide different information, albeit RNA-seq offers a broader view since it circumvent the limitations of interrogating only the probes present on DNA microarray and extends also to the non-coding RNA world.

b) Proteomic approach

We aimed at dissecting by several different technical approaches (Label Free LC-MS/MS proteomic, 2D-DIGE proteomic, iTRAQ-based LC-MS/MS proteomic) the role of HMGA1 proteins in modulating the protein expression profile of cancer cells in order to find out cancer-associated molecules downstream HMGA1.

We are focusing both on global proteomic alterations and on alterations regarding secreted proteins because of their importance as potential cancer biomarkers. The data we have already collected show that HMGA proteins have a very large impact on protein expression regulation. As usual in genome- and proteome-wide screening many proteins with an already known specific role in cancer are fished out, but we are currently focusing onto the less characterized factors from a molecular oncology point of view because they could unravel new pathways involved in cancer development and, more interestingly, new potential therapeutic targets. As well as for the genomic approach we will take advantage of bioinformatic analyses by which we will compare HMGA1-linked protein alterations with different datasets derived from tissue of breast cancer patients that include gene expression and clinical outcome data.

The role of HMGA protein in influencing the epigenetic status of cancer cells.

In cancer cells HMGA are abundantly expressed and their levels are almost comparable to those of histone H1. Given that these proteins bind directly to DNA and they act as architectural transcription factors favoring the formation of stereospecific macromolecular regulatory complexes at the level of enhancers/promoters, in order to understand their function it is fundamental to shed light on the HMGA1 genome occupancy and to evaluate whether they contribute to epigenetic alterations occurring in cancer cells. We are accomplishing this task exploiting the cancer cellular model described above to evaluate the interaction of HMGA1 with core histones and testing whether key histone post-translational modifications involved in gene transcriptional regulation can modulate this association. The translational aspect of this research task regards the identification of the enzymes responsible for these epigenetic alterations that could represent new potential therapeutic targets.