Cancer Letters

Cancer Letters

Volume 317, Issue 2, 28 April 2012, Pages 157-164
Cancer Letters

Extracellular HSPA1A promotes the growth of hepatocarcinoma by augmenting tumor cell proliferation and apoptosis-resistance

https://doi.org/10.1016/j.canlet.2011.11.020Get rights and content

Abstract

Intracellular HSP70 has been implicated as a cytoprotective protein, whereas the effect of extracellular HSP70 on tumor cells has not been fully understood to date. Here we report that extracellular HSPA1A, a stress-inducible member of HSP70 family, could promote tumor growth. HSPA1A promoted the proliferation of H22 hepatocarcinoma cells through TLR2 and TLR4 signaling. The effect of HSPA1A was abolished by inhibiting NF-κB. HSPA1A also augmented the apoptosis-resistance of H22 cells by activating NF-κB, thus to promote the proliferation of H22 cells in presence of mitomycin C. Furthermore, the promoting effect of HSPA1A on tumor cell proliferation was existent after the removal of HSPA1A, which might involve HSPA1A-promoted upregulation of TLR4 expression in tumor cells and release of HMGB1 from tumor cells. These findings suggest that extracellular HSPA1A functions as endogenous ligand for TLR2 and TLR4 to facilitate tumor growth.

Introduction

The development of a tumor could lead to reciprocal alternations in the host and the tumor. In tumor microenvironment, the death of cells due to metabolic, genotoxic, or anoxic stress results in the releasing of intracellular molecules. Besides these, cellular stress, damage, and inflammation also cause the releasing of intracellular molecules from cells. Some of these molecules are inflammatory mediators and are referred to as damage-associated molecular pattern (DAMP) molecules [1]. DAMP molecules play critical roles in triggering immune responses and activating repair mechanism so to produce both antitumor and protumor effects [1]. In tumor progression, the presence of necrosis in tumor is almost always deemed as a poor prognostic indicator [2], [3], [4], [5] by clinical observations, implying that the protumor effect of DAMP molecules might predominant in the process of tumor development.

What DAMP molecules are has been a matter of conjecture for a decade. In recent years, several candidates have been revealed, among which HSP72 is included [6]. The stress-inducible HSP70 in the cells functions as a cytoprotective protein when cells are exposed to stressful stimuli. The tumorigenic role of stress-inducible HSP70 in cells has been associated to its ability to maintain cell survival [7]. However, stress-inducible HSP70 can also be passively released from necrotic cells [8] as well as actively released when tumor cells are suffered from exogenous stress [9]. The HSP70 family consists of several highly homologous members. HSPA1A (originally name as HSP70-1, HSP72, HSPA1) is a stress-inducible member of HSP70 family [10]. Extracellular HSPA1A has been found to bind to Toll-like receptors (TLRs) 2 and 4 on antigen-presenting cells, NK cells, and B cells, and therefore exerts immunoregulatory effects [11]. Therefore, HSPA1A may have the potential utilization in anticancer therapeutical approaches based on its immunological role. On the other hand, however, TLR2 and TLR4 are expressed not only on immune cells but also on tumor cells [12]. TLR4 on tumor cells plays important roles in tumor progression, one of which is that TLR4 signaling may allow tumor cells to negatively regulate immune system for their own survival and growth [13]. Several studies have demonstrated that activation of TLR4 and TLR2 signaling in tumor cells by pathogen-associated molecular pattern (PAMP) molecules such as lipopolysaccharide (LPS) and zymosan A can dampen the anti-tumor functions of infiltrating immune cells or elicit a direct effect on tumor cells, thereby facilitating tumor outgrowth [14], [15], [16]. However, it is still not clear whether extracellular HSPA1A, as a potential ligand of TLR2 and TLR4, may promote tumor progression by directly affecting tumor cells.

In this study, we focus on the direct effect of extracellular HSPA1A (eHSPA1A) on tumor cells. Our data showed that eHSPA1A promoted tumor cell proliferation by activating NF-κB via TLR signaling pathway. eHSPA1A also promoted the expression of TLR4 in tumor cells and the release of HMGB1 from tumor cells, resulting in a long-lasting effect of TLR4 signaling. The tumor-promoting effect was still existent after the removal of HSPA1A.

Section snippets

Animals and cells

BALB/c mice (6–8-week-old) were purchased from Center of Medical Experimental Animals of Hubei Province (Wuhan, China). BALB/c athymic nude (nu/nu) mice (6–8 weeks old) were purchased from SLAC Laboratory Animal Co. Ltd. (Shanghai, China). The mice were maintained in the accredited animal facility of Tongji Medical College, and used for studies approved by the Animal Care and Use Committee of Tongji Medical College. Murine H22, human BEL-7402, and human HepG2 hepatocarcinoma cell lines were

eHSPA1A has different effect on tumor growth in different stage of tumor progression

To investigate the effect of extracellular HSPA1A (eHSPA1A) on tumor growth, we first detected extracellular HSP70 (eHSP70) in tumor tissue using HSP70 antibody which detects HSPA1A and HSPA1L. As shown in Fig. 1a, eHSP70 was detectable in the tissue at site of tumor cell inoculation, and increased in local tissue along with the development of tumor. To verify the existence of eHSP70 in different types of tumors, we analyzed periphery tumor tissues from cancer patients. eHSP70 was detected in

Discussion

In this report, we provide evidence that extracellular HSPA1A can facilitate tumor growth by promoting proliferation and apoptosis-resistance of tumor cells. Stress-inducible HSP70 is abundantly expressed in various types of tumor cells, and regarded as a cancer relevant survival protein [26]. Once released into the extracellular milieu, HSPA1A could be an immunostimulatory factor which might stimulate antitumor immune response [11], [27]. Nevertheless, our data in this study showed a distinct

Conflict of interest statement

The authors declare no conflict of interest.

Acknowledgements

This work was supported by National Science Foundation of China (Nos. 30830095, 30771974, 30772589), Science Foundation of Ministry of Education of China (No. 20070487004), and National Development Program (973) For Key Basic Research of China (No. 2009CB521806).

References (37)

  • M.I. Koukourakis et al.

    Lactate dehydrogenase-5 (LDH-5) overexpression in non-small-cell lung cancer tissues is linked to tumour hypoxia, angiogenic factor production and poor prognosis

    Br. J Cancer

    (2003)
  • D.S. Wheeler et al.

    Extracellular Hsp72, an endogenous DAMP, is released by virally infected airway epithelial cells and activates neutrophils via Toll-like receptor (TLR)-4

    Respir. Res.

    (2009)
  • E. Schmitt et al.

    Intracellular and extracellular functions of heat shock proteins: repercussions in cancer therapy

    J. Leukoc. Biol.

    (2007)
  • S. Basu et al.

    Necrotic but not apoptotic cell death releases heat shock proteins, which deliver a partial maturation signal to dendritic cells and activate the NF-kappa B pathway

    Int. Immunol.

    (2000)
  • M.A. Bausero et al.

    Alternative mechanism by which IFN-gamma enhances tumor recognition: active release of heat shock protein 72

    J. Immunol.

    (2005)
  • A. Asea

    Initiation of the immune response by extracellular Hsp72: chaperokine activity of Hsp72

    Curr. Immunol. Rev.

    (2006)
  • Y. Sato et al.

    Cancer cells expressing Toll-like receptors and the tumor microenvironment

    Cancer Microenviron.

    (2009)
  • M.G. Kelly et al.

    TLR-4 signaling promotes tumor growth and paclitaxel chemoresistance in ovarian cancer

    Cancer Res.

    (2006)
  • Cited by (47)

    • Decoding the roles of heat shock proteins in liver cancer

      2024, Cytokine and Growth Factor Reviews
    • TLR2/4 ligand-amplified liver inflammation promotes initiation of autoimmune hepatitis due to sustained IL-6/IL-12/IL-4/IL-25 expression

      2018, Molecular Immunology
      Citation Excerpt :

      CCl4 was purchased from aladdin Biochemical Technology Co., Ltd (Shanghai, China). Plasmids pCYP2D6, pIL6, pIL12, psTLR2 and psTLR4 are expression vectors (pcDNA3.1, Invitrogen, Carlsbad, CA) carrying the cDNA encoding human CYP2D6, murine IL-6, murine IL-12, the extracellular domain of murine TLR2 and TLR4, respectively, which were constructed in our laboratory (Li et al., 2017; Yan et al., 2013; Liu et al., 2010; Wu et al., 2012). For in vivo gene transfection, plasmids were prepared and analyzed as described previously (Geng et al., 2006).

    • Sex difference in the heat shock response to high external load resistance training in older humans

      2017, Experimental Gerontology
      Citation Excerpt :

      Though basically intracellular proteins, a great variety of stimuli have been reported to prompt Hsp70 release from living cells, including exercise and inflammatory cytokines (Walsh et al., 2001; Barreto et al., 2003). Wu and colleagues showed that extracellular Hsp70 (eHsp70) can promote tumor cell proliferation and resistance to apoptosis (Wu et al., 2012). Moreover, the presence of Hsp70 in the extracellular milieu has often been associated with a poor prognosis of disease (Pockley et al., 2003).

    • A novel quinazoline-based analog induces G2/M cell cycle arrest and apoptosis in human A549 lung cancer cells via a ROS-dependent mechanism

      2017, Biochemical and Biophysical Research Communications
      Citation Excerpt :

      In this study, our interest was focused on 6-amino-4-(4-phenoxyphenylethylamino)quinazoline (QNZ), a quinazoline-containing compound, known for its excellent NF-κB inhibitory activity [14]. Additionally, QNZ also exhibits as a novel anti-cancer reagent [15]. However, the action mechanisms behind are still unclear, and there is as yet scarcely any research related to designing QNZ-inspired anticancer agents based on the ROS-promoting strategy.

    View all citing articles on Scopus
    View full text