Autocrine regulation of tumor cell repopulation by Hsp70-HMGB1 alarmin complex.

BACKGROUND: Cancer recurrence is regulated by a variety of factors, among which is the material of dying tumor cells; it is suggested that remaining after anti-cancer therapy tumor cells receive a signal from proteins called damage-associated molecular patterns (DAMPs), one of which is heat shock protein 70 (Hsp70). METHODS: Two models of tumor repopulation were employed, based on minimal population of cancer cells and application of conditioned medium (CM). To deplete the CMs of Hsp70 affinity chromatography on ATP-agarose and immunoprecipitation were used. Cell proliferation and the dynamics of cell growth were measured using MTT assay and xCELLigence technology; cell growth markers were estimated using qPCR and with the aid of ELISA for prostaglandin E detection. Immunoprecipitation followed by mass-spectrometry was employed to identify Hsp70-binding proteins and protein-protein interaction assays were developed to reveal the above protein complexes. RESULTS: It was found that CM of dying tumor cells contains tumor regrowth-initiating factors and the removal of one of them, Hsp70, caused a reduction in the relapse-activating capacity. The pull out of Hsp70 alone using ATP-agarose had no effect on repopulation, while the immunodepletion of Hsp70 dramatically reduced its repopulation activity. Using proteomic and immunochemical approaches, we showed that Hsp70 in conditioned medium binds and binds another abundant alarmin, the High Mobility Group B1 (HMGB1) protein; the complex is formed in tumor cells treated with anti-cancer drugs, persists in the cytosol and is further released from dying tumor cells. Recurrence-activating power of Hsp70-HMGB1 complex was proved by the enhanced expression of proliferation markers, Ki67, Aurka and MCM-10 as well as by increase of prostaglandin E production and autophagy activation. Accordingly, dissociating the complex with Hsp70 chaperone inhibitors significantly inhibited the pro-growth effects of the above complex, in both in vitro and in vivo tumor relapse models. CONCLUSIONS: These data led us to suggest that the abundance of the Hsp70-HMGB1 complex in the extracellular matrix may serve as a novel marker of relapse state in cancer patients, while specific targeting of the complex may be promising in the treatment of cancers with a high risk of recurrence.

Structural Characteristics of High-Mobility Group Proteins HMGB1 and HMGB2 and Their Interaction with DNA.

Non-histone nuclear proteins HMGB1 and HMGB2 (High Mobility Group) are involved in many biological processes, such as replication, transcription, and repair. The HMGB1 and HMGB2 proteins consist of a short N-terminal region, two DNA-binding domains, A and B, and a C-terminal sequence of glutamic and aspartic acids. In this work, the structural organization of calf thymus HMGB1 and HMGB2 proteins and their complexes with DNA were studied using UV circular dichroism (CD) spectroscopy. Post-translational modifications (PTM) of HMGB1 and HMGB2 proteins were determined with MALDI mass spectrometry. We have shown that despite the similar primary structures of the HMGB1 and HMGB2 proteins, their post-translational modifications (PTMs) demonstrate quite different patterns. The HMGB1 PTMs are located predominantly in the DNA-binding A-domain and linker region connecting the A and B domains. On the contrary, HMGB2 PTMs are found mostly in the B-domain and within the linker region. It was also shown that, despite the high degree of homology between HMGB1 and HMGB2, the secondary structure of these proteins is also slightly different. We believe that the revealed structural properties might determine the difference in the functioning of the HMGB1 and HMGB2 as well as their protein partners.

Genetic tool for fate mapping of Oct4 (Pou5f1)-expressing cells and their progeny past the pluripotency stage.

BACKGROUND: Methods based on site-specific recombinases are widely used in studying gene activities in vivo and in vitro. In these studies, constitutively active or inducible variants of these recombinases are expressed under the control of either lineage-specific or ubiquitous promoters. However, there is a need for more advanced schemes that combine these features with possibilities to choose a time point from which lineage tracing starts in an autonomous fashion. For example, the key mammalian germline gatekeeper gene Oct4 (Pou5f1) is expressed in the peri-implantation epiblast which gives rise to all cells within embryos. Thus the above techniques are hardly applicable to Oct4 tracing past the epiblast stage, and the establishment of genetic tools addressing such a limitation is a highly relevant pursuit. METHODS: The CRISPR/Cas9 tool was used to manipulate the genome of mouse embryonic stem cells (ESCs), and various cell culture technics-to maintain and differentiate ESCs to neural cell, lentivirus-based reprogramming technique-to generate induced pluripotent stem cells (iPSCs). RESULTS: In this paper, we have developed a two-component genetic system (referred to as O4S) that allows tracing Oct4 gene activity past the epiblast stage of development. The first component represents a knock-in of an ubiquitous promoter-driven inducible Cre, serving as a stop signal for downstream tdTomato. Upon activation of Cre activity with 4-hydroxytamoxifen (4-OHT) at any given time point, the recombinase excises a stop signal and poses the second component of the system-the FlpO recombinase, knocked into 3'UTR of Oct4, to be expressed upon activation of the latter gene. Oct4-driven expression of FlpO, in turn, triggers the tdTomato expression and thus, permanently marks Oct4+ cells and their progeny. We have validated the O4S system in cultured ESCs and shown that it is capable, for example, to timely capture an activation of Oct4 gene during the reprogramming of somatic cells into iPSCs. CONCLUSIONS: The developed O4S system can be used to detect Oct4 activation event, both permanent and transient, in somatic cell types outside the germline. The approach can be equally adjusted to other genes, provided the first component of the system is placed under transcriptional control of these genes, thus, making it a valuable tool for cell fate mapping in mice.