Research in the Castro’s group

Our research is focused on identification and characterization of signaling mechanisms that control cell processes, such as growth and proliferation, and how deregulation of these mechanisms contributes to cancer. There are three main ongoing projects:

Interrupted Rheb-to-AMPK feedback signaling with cytoplasmic p27 retention: mechanisms and biological consequences (funded by FONDECYT Regular/CONICYT 1120923)
A complex of two tumor suppressor proteins, known as TSC1/2 tumor suppressor complex is the main controller of cell growth. The central role of this complex in cell growth is revealed by its inactivation and contribution to the development of Tuberous Sclerosis Complex (TSC) disease, a hamartoma syndrome characterized by development of benign tumors in several organs. In addition, inactivation of TSC1/2 occurs downstream of several pathways known to be deregulated in other hamartoma syndromes and cancer. The primary consequence of loss of TSC1/2 is the activation of the small GTPase Rheb, which in turn activates mTORC1, a kinase involved in promoting cell growth. We previously demonstrated that TSC2 functions as a GTPase-activating protein (GAP) for the inactivation of Rheb and thereby mTORC1, indicating that Rheb is the molecular link between TSC1/2 and mTORC1. Consistent with this, we recently showed that activated mTORC1 is reversed by depletion of Rheb in Tsc2-null cells, and Rheb deficiency reduced their tumorigenic potential in nude mice. In addition, we demonstrated that Rheb, independently of its regulation of mTORC1, induces cell proliferation through inhibition of the cell cycle inhibitor p27Kip1 (p27). However, how Rheb regulates p27 remains elusive. In correlation with the regulation of p27, we found that Rheb also induces mTORC1-independent AMPK activation, which is known to phosphorylate p27 and, consequently, decreases p27 nuclear accumulation and increases cytoplasmic retention of p27. There is evidence suggesting that AMPK activation and/or cytoplasmic p27 retention contribute to autophagy-mediated tumor cell survival under cancer-related stresses (see proposed model below). The general goal of this project is to identify the mechanism by which aberrant Rheb activation affects AMPK/p27 function, and to gain insights into the potential biological consequences of this regulation.



Model of proposed mechanism of Rheb regulation of p27 function and biological consequences of the mTORC1-independent Rheb functions. In the absence of TSC2 activity, aberrant Rheb activation induces mTORC1 and AMPK activation. We propose that Rheb-induced AMPK activation phosphorylates p27 resulting in decrease of nuclear p27 and

Molecular Mechanisms of Cancer Cell Resistance to Metabolic Stress: the role of NUAK1 signaling. (funded by FONDECYT Regular/CONICYT 1160731)
Cancer is a devastating disease that develops through accumulation of molecular alterations that change the genetic program driving the behavior of cells. Although there has been a significant advance in identifying some of these alterations, it is still poorly defined how cancer cells adapt to different microenvironments. In particular, it is still unclear how cancer cells survive to metabolic stress conditions caused by highly proliferative cells creating a tumor mass with a microenvironment initially far from blood vessels, resulting in shortage supply of nutrients and/or oxygen (see figure below). NUAK1 is a serine/threonine kinase that has been involved in cancer cell tolerance to metabolic stress. However, the mechanism of NUAK1 function on cancer cell survival and how this function affects tumor progression are poorly understood. In addition, it is unclear how NUAK1 is regulated during metabolic stress. Evidence indicates that NUAK1 promotes maintenance of intracellular ATP levels and cell survival under metabolic stress in a p53–independent manner. This is of particular relevance because p53 is the most frequently inactivated gene in cancer. Regarding NUAK1 regulation, we found that phosphorylation of NUAK1 increases during metabolic stress. Thus, the general goal of this project is to identify molecular mechanisms involved in NUAK1 regulation and function on cancer cell survival in response to metabolic stress.



Regulation of protein synthesis during cellular stress.
Translation of mRNA is the basic phenomenon of life that allows protein synthesis, relying in a constant expend of energy. Translational control is crucial for the correct physiological function of an organism, and is frequently disrupted in cancer cells. This phenomenon is acutely controlled by several signaling pathways, sustaining proper cell function at different conditions. Global inhibition of translation is a common response of eukaryotic cells to stressful conditions like temperature fluctuations, UV light and oxidative stress. This adaptive response allows selective and efficient synthesis of proteins relevant to the unfavorable conditions. Actively translated mRNAs form polysomes, ribonucleoprotein (RNP) complexes in which several ribosomes synthesize protein from the same mRNA molecule. However, when global translation is inhibited, stress granules (SGs) are formed. SGs are membrane-free cytoplasmic foci where components of the small ribosome subunits, untranslated mRNAs and initiation factors are sequestered and maintained inactive during cellular stress. Using different genetic, biochemical and imaging approaches, we are currently studying translational control by DISC1, a protein encoded from a gene whose mutations predispose humans to suffer schizophrenia and other mental disorders. There is evidence suggesting that DISC1 affects protein synthesis during oxidative stress, a function that could explain DISC1 association with mental disorders. In addition, DISC1 has been associated to the formation of SGs. Thus, our goal is to understand how DISC1 is involved in translational control during stress conditions.

Research in the Pincheira group

SUMMARY

Cancer is the second cause of death in Chile so it becomes relevant for our society to advance in the knowledge of this disease. What external and / or internal factors contribute to the generation of cancer? How does a benign tumor become malignant, or a primary tumor metastatic? Why do treatments fail? Why treatments work in some patients and not in others? These are some of the questions and challenges that make scientific research necessary. The long-term goal of our research is to advance in the knowledge of cancer biology, to identify participants, to know how they function and how they are related to each other. More specifically we are investigating the function of a protein called SALL2, whose levels of expression are altered in many cancers, that is compared to a normal person SALL2 levels are increased or decreased in the cancer patient. Why and how this occur, and what is the significance of this alteration in disease are some of our current questions.

WHO is SALL2?

Regulation of gene expression by transcription factors is one of the most important mechanisms governing cellular functions. Thus, it is not surprising that alteration of transcription factor function is a frequent cause of human diseases. Our laboratory has focused attention in a transcription factor named SALL2. SALL2 is a conserved member of the SAL/SPAL family of transcription factors involved in development. The gene and protein structures are shown below (from our recent review “Hermosilla V et al, Carcinogenesis, 2017”)


SALL2 gene and isoforms structure. (A) Human SALL2 gene located in chromosome 14 consists of exon E1, E1A and E2, and contains two alternative promoters; P1 and P2. The consensus sites for WT1, AP4 and p53 are represented by rectangles in the P1 and/or P2 promoters. (B) Human SALL2 encodes two protein isoforms, SALL2 E1 (1007 AA) and SALL2 E1A (1005 AA) as result of alternative promoters P1 and P2, respectively. Common to both SALL2 isoforms are the zinc finger domains represented by ovals and the P, Q, S and E- rich motifs represented by rectangles. Both isoforms contain the p75NTR interaction domain, DNA binding and transactivation (LT binding) domains and a putative nuclear export sequence (NES). SALL2-E1 contains a repressor domain and a putative nuclear localization signal (NLS) not present in SALL2-E1A.


WHY SALL2 is interesting?

SALL2 is a poorly characterized transcription factor that in humans has been associated with renal dysfunction, coloboma (a congenital eye disease), neurological disorders such as Alzheimer and Autism, but mainly with cancer. Unfortunately, SALL2 normal functions and the identity of the genes that it regulates are largely unknown.

SALL2 gene contains two promoters (P1 and P2) that can originate two protein isoforms; these isoforms could account for different cellular functions and might be differentially involved in disease. However, there is scarce information about SALL2 isoforms regulation, targets and function.

SALL2 is deregulated in cancers, but data is confusing; in some cancers SALL2 is found upregulated (synovial sarcoma, oral cancer, glioblastoma, wilms tumor, testicular cancer) while in others it is absent (ovarian cancer, breast cancer, leukemia). To understand SALL2 deregulation we need first to understand how SALL2 is normally regulated.

A few studies, including ours have associated SALL2 with key cancer-driver genes such as p53 and Myc. However, more studies are needed to understand and validate the functional relationship between SALL2 and these key cancer-related proteins.

It is proposed that SALL2 has tumor suppressor activity, mediated by its pro-apoptotic and cell cycle regulatory functions. In addition, SALL2 seems to play a role in cellular stemness. Several studies have associated SALL2 with stemness based on its expression levels, and its potential to interact with—or to be regulated by—key transcription factors for pluripotency maintenance. See model below (from Hermosilla V et al. Carcinogenesis 2017)

Model of SALL2 regulation and function. Stress conditions and neurotrophin signaling impact on SALL2 transcription factor activity. Depending on the cellular condition, SALL2 is transcriptionally regulated by factors such as p53, WT1 and AP4. Dashed lines indicate other putative transcriptional regulators of SALL2. On the other hand, Cul4-DDB1 negatively regulates SALL2 protein stability under certain cellular conditions. SALL2 regulates cell proliferation and apoptosis by targeting p21, p16, MYC, BAX and NOXA. In addition, SALL2 is involved in development, stemness and brain functions through unidentified targets. Deregulation of SALL2 and SALL2-dependent targets are implicated in tumorigenesis and the resistance of cancer cells to chemotherapy.

Some of our findings:
1. - SALL2 is a novel transcription factor for neurotrophin receptors and plays a role in neuronal function (Pincheira et al. EMBO J, 2009).