Agustin Rodriguez-Gonzalez

Choline Kinase Activation Is a Critical Requirement for the Proliferation of Primary Human Mammary Epithelial Cells and Breast Tumor Progression

Breast cancer is still one of the most important tumors among women in industrialized countries. Improvement in both understanding the molecular events associated with the disease and the development of new additional treatments is still an important goal to be achieved. Choline kinase (ChoK) is increased in human mammary tumors with high incidence, and this activation is associated with clinical variable indicators of greater malignancy. Here, we have investigated the role of ChoK in the development of breast cancer and found that ChoK is both necessary and sufficient for growth factor-induced proliferation in primary human mammary epithelial cells and an absolute requirement for the specific mitogenic response to heregulin in breast tumor-derived cells. These results demonstrate that ChoK plays an essential role in both normal human mammary epithelial cell proliferation and breast tumor progression. Furthermore, inhibition of ChoK shows a strong in vivo antitumor activity against human breast cancer xenografts. Thus, ChoK constitutes a novel bona fide molecular target for the treatment of breast cancer patients.

Choline kinase inhibition induces the increase in ceramides resulting in a highly specific and selective cytotoxic antitumoral strategy as a potential mechanism of action

Choline kinase (ChoK, E.C. 2.7.1.32) is involved in the synthesis of phosphatidylcholine (PC), and has been found to be increased in human tumors and tumor-derived cell lines. Furthermore, ChoK inhibitors have been reported to show a potent and selective antitumoral activity both in vitro and in vivo. Here, we provide the basis for a rational understanding of the antitumoral activity of ChoK inhibitors. In normal cells, blockage of de novo phosphorylcholine (PCho) synthesis by inhibition of ChoK promotes the dephosphorylation of pRb, resulting in a reversible cell cycle arrest at G0/G1 phase. In contrast, ChoK inhibition in tumor cells renders cells unable to arrest in G0/G1 as manifested by a lack of pRb dephosphorylation. Furthermore, tumor cells specifically suffer a drastic wobble in the metabolism of main membrane lipids PC and sphingomyelin (SM). This lipid disruption results in the enlargement of the intracellular levels of ceramides. As a consequence, normal cells remain unaffected, but tumor cells are promoted to apoptosis. Thus, we provide in this study the rationale for the potential clinical use of ChoK inhibitors.

Targeting steroid hormone receptors for ubiquitination and degradation in breast and prostate cancer

Proteolysis targeting chimeric molecules (Protacs) target proteins for destruction by exploiting the ubiquitin-dependent proteolytic system of eukaryotic cells. We designed two Protacs that contain the peptide ‘degron’ from hypoxia-inducible factor-1α, which binds to the Von –Hippel–Lindau (VHL) E3 ubiquitin ligase complex, linked to either dihydroxytestosterone that targets the androgen receptor (AR; Protac-A), or linked to estradiol (E2) that targets the estrogen receptor-α (ERα; Protac-B). We hypothesized that these Protacs would recruit hormone receptors to the VHL E3 ligase complex, resulting in the degradation of receptors, and decreased proliferation of hormone-dependent cell lines. Treatment of estrogen-dependent breast cancer cells with Protac-B induced the degradation of ERα in a proteasome-dependent manner. Protac-B inhibited the proliferation of ERα-dependent breast cancer cells by inducing G1 arrest, inhibition of retinoblastoma phosphorylation and decreasing expression of cyclin D1, progesterone receptors A and B. Protac-B treatment did not affect the proliferation of estrogen-independent breast cancer cells that lacked ERα expression. Similarly, Protac-A treatment of androgen-dependent prostate cancer cells induced G1 arrest but did not affect cells that do not express AR. Our results suggest that Protacs specifically inhibit the proliferation of hormone-dependent breast and prostate cancer cells through degradation of the ERα and AR, respectively.

The aggresome pathway as a target for therapy in hematologic malignancies

Misfolded or unfolded proteins are often refolded with the help of chaperones or degraded by the 26S proteasome. An alternative fate of these proteins is the aggresome pathway. The microtubule-organizing center (MTOC) transports unfolded proteins to lysosomes and are degraded through autophagy. Histone deacetylase 6 (HDAC6) deacetylates alpha-tubulin, which is thought to be a component of the MTOC. Recently, two small molecule inhibitors of the aggresome pathway and HDAC6 have been described. One inhibitor, tubacin, prevents deacetylation of alpha-tubulin and produces accumulation of polyubiquitinated proteins and apoptosis. Tubacin acts synergistically with the proteasome inhibitor, bortezomib, to induce cytotoxicity in one type of hematologic malignancy, multiple myeloma. The other, LBH589, is a pan HDAC inhibitor and hydroxamic acid derivative that induces apoptosis of multiple myeloma cells resistant to conventional therapies. In this review, we summarize recent reports on targeting the aggresome pathway and HDAC6 in hematologic malignancies.

Tubacin suppresses proliferation and induces apoptosis of acute lymphoblastic leukemia cells

Over the past decade, histone deacetylase inhibitors have increasingly been used to treat various malignancies. Tubacin (tubulin acetylation inducer) is a small molecule that inhibits histone deacetylase 6 (HDAC6) and induces acetylation of α-tubulin. We observed a higher antiproliferative effect of tubacin in acute lymphoblastic leukemia (ALL) cells than in normal hematopoietic cells. Treatment with tubacin led to the induction of apoptotic pathways in both pre-B and T cell ALL cells at a 50% inhibitory concentration (IC50) of low micromolar concentrations. Acetylation of α-tubulin increases within the first 30 min following treatment of ALL cells with tubacin. We also observed an accumulation of polyubiquitinated proteins and poly(ADP-ribose) polymerase (PARP) cleavage. Furthermore, the signaling pathways activated by tubacin appear to be distinct from those observed in multiple myeloma. In this article, we demonstrate that tubacin enhances the effects of chemotherapy to treat primary ALL cells in vitro and in vivo. These results suggest that targeting HDAC6 alone or in combination with chemotherapy could provide a novel approach to treat ALL.

Beneficial Effects of Bioactive Phospholipids: Genomic Bases

Nutritional-related diseases such as obesity, hyperlipidemia, type 2 diabetes, cardiovascular disorders, hypertension or cancer are increasingly prevalent in industrialized countries. Dietary lipids have been found to play a significant role in the prevention and improvement of the management of several of these chronic diseases. Closely to 10% of the intake of total dietary lipids correspond to phospholipids (PLs), which have been recognized as important contributors to their beneficial effects in human health. Bioactive PLs have essential roles as signaling and regulatory molecules, being involved in major cell functions such as cell growth, death, senescence, adhesion, migration or cell integrity. The analysis of the mechanisms of how PLs are involved in these varied cell functions is necessary for understanding and profiting from bioactive PLs action. This review explores the genomic bases of the physiological functions and molecular actions of bioactive PLs, and their effect in the development of chronic diseases.

Proteolysis Targeting Chimeric Molecules

Protein degradation is one of the mechanisms employed by the cell for irreversibly destroying proteins. In eukaryotes, ATP-dependent protein degradation in the cytoplasm and nucleus is carried out by the 26S proteasome. Most proteins are targeted to the 26S proteasome by covalent attachment of a multiubiquitin chain. A key component of the enzyme cascade that results in attachment of the multiubiquitin chain to the target or labile protein is the E3 ubiquitin ligase that controls the specificity of the ubiquitination reaction. Defects in ubiquitin-dependent proteolysis have been shown to result in a variety of human diseases, including cancer, neurodegenerative diseases, and metabolic disorders. We have developed a novel approach to target proteins that cause cancer for ubiquitination and degradation. This technology, known as Protac, involves a chimeric molecule that could potentially recruit any cancer-causing protein to an E3 ligase for ubiquitination and subsequent degradation. In this chapter, we describe the development of this technology for cancer therapy.