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HMGB1 inhibits cell death in yeast and mammalian cells and is abundantly expressed in human breast carcinoma¹

MARIE-LUISE BREZNICEANU^*,2, KIRSTEN VÖLP^*,2, SUSANNE BÖSSER^*, CHRISTINE SOLBACH, PETER LICHTER, STEFAN JOOS and MARTIN ZÖRNIG^*,3

^* Chemotherapeutisches Forschungsinstitut, Georg-Speyer-Haus, 60596 Frankfurt, Germany;
Klinik für Gynäkologie und Geburtshilfe der Johann Wolfgang Goethe-Universität, 60590 Frankfurt, Germany; and
Deutsches Krebsforschungszentrum DKFZ, D-69120 Heidelberg, Germany

³Correspondence: Chemotherapeutisches Forschungsinstitut, Georg-Speyer-Haus, Paul-Ehrlich-Strasse 42-44, 60596 Frankfurt, Germany. E-mail: zoernig{at}em.uni-frankfurt.de

SPECIFIC AIM

We used a functional yeast survival screen in S. pombe to select for new anti-apoptotic mammalian genes. We present evidence that one of the genes we identified (HMGB1) is able to suppress cell death induced by the proapoptotic Bcl-2 family member Bak in yeast and to protect against apoptosis initiated by different stimuli in mammalian cells. We show high HMGB1 protein levels in human primary breast carcinoma and reduced Hmgb1 mRNA amounts in the lactating and involuting mouse mammary gland. These data suggest that HMGB1 may participate in the regulation of mammary gland apoptosis and that strong HMGB1 expression might promote tumor growth because of its anti-apoptotic properties.

PRINCIPAL FINDINGS

1. High mobility group-1 protein (HMGB1) protects against Bak-induced cell death in S. pombe
We used an inducible Bak expression system in S. pombe to screen a cDNA library for new anti-apoptotic mammalian genes. One of the S. pombe colonies surviving Bak expression after library transformation contained a truncated deletion mutant of the mouse Hmgb1 gene lacking the carboxyl-terminal 33 amino acids. HMGB1 still contains the two HMG-boxes identified in the full-length HMGB1 whereas the highly acidic carboxyl-terminal tail is deleted. Full-length HMGB1 and HMGB1 efficiently inhibit Bak killing in yeast.

The largely increased amount of Bak in Hmgb1 cotransformed yeast cells indicates that a protective mechanism other than transcriptional down-regulation of the Bak yeast expression construct is used by HMGB1. We did not obtain experimental evidence for direct binding of Bak to HMGB1 and HMGB1.

2. HMGB1 overexpression inhibits apoptosis in mammalian cells
Transient transfection of the human colon carcinoma cell line RKO, the human embryonic kidney cell line 293T, and the normal rat kidney cell line NRK1 with bak alone induced apoptosis, which was blocked by cotransfection of either Hmgb1 or full-length Hmgb1 (Fig. 1 ). HMGB1 and HMGB1 also protect against apoptosis initiated by Bax- or Casp-8-overexpression and against UV-, CD95-, and TRAIL-induced cell death.

View larger version (32K): [in this window] [in a new window]   Figure 1. HMGB1 and HMGB1 inhibit apoptosis induced in mammalian cells by different stimuli. RKO, 293T, and NRK1 cells were seeded in 10 cm plates at 1 x 106 cells/plate. The next day, 1.3 µg gfp-bak, casp-8 or bax cDNA together with 4.0 µg of empty vector pcDNA3.1, Hmgb1, or Hmgb1 cDNA were introduced using the PEI transfection method. 16 h later, cells were collected (including floating cells) and the % of dead cells was determined using the LIVE/DEAD viability/cytotoxicity kit from Molecular Probes. For UV, CD95L, and TRAIL killing, 5 µg of empty vector DNA, Hmgb1, or Hmgb1 cDNA was transfected. 24 h later the cells were treated with 10 s UV radiation, 10 ng/mL recombinant CD95L plus 1 µg/mL cycloheximide and 1 µg/mL anti-Flag antibody, or 10 ng/mL TRAIL plus 1 µg/mL cycloheximide. 16 h later, apoptosis was quantified using the LIVE/DEAD assay. All apoptotic stimuli were significantly inhibited by overexpression of either Hmgb1 or Hmgb1.

3. HMGB1 protein is highly expressed in human breast carcinomas
HMGB1 protein levels were investigated in normal human breast and in primary human breast carcinomas by Western blot experiments (Fig. 2 ). In protein lysates from six different normal breast tissue samples, low amounts of HMGB1 protein could be detected (first six lanes). Of nine primary breast cell carcinomas tested, all expressed HMGB1, six in high amounts. We examined seven human breast carcinomas that had been passaged for a long time in nude mice. Such passaging of tumor cells in mice is regarded as an artificial model for tumor metastasis, i.e., genes important for metastasis become up-regulated. All seven passaged breast tumors expressed high HMGB1 protein levels.

View larger version (23K): [in this window] [in a new window]   Figure 2. HMGB1 protein overexpression in human mammary carcinomas. 40 µg of protein lysates from normal breast tissue (p7a, p9a, p11a, N1, N2, N3), primary breast tumors (83, 167, 249, 250, 256, 611, 876, 1096, 1132), and breast tumors passaged in nude mice (T13, T27, T48, T83, T130, T136, T236) was analyzed in a Western blot experiment with an anti-HMGB1 antibody (PharmMingen). As a loading control, the membrane was rehybridized with an anti-HSP27 antibody (Santa Cruz).

To screen a larger panel of breast carcinoma for HMGB1 expression, we analyzed a human breast tissue microarray with fixed tissue sections from 10 different normal breast samples and 50 different breast carcinomas. The intensity of the HMGB1 staining was quantified and on average threefold higher HMGB1 protein levels were detected in the tumor samples compared with normal tissue controls, consistent with the Western blot results described above.

4. Hmgb1 mRNA levels are regulated during different stages of mouse mammary gland development
A Northern blot with mouse mammary gland mRNA preparations from various stages before and during pregnancy and from lactating and involuting glands was hybridized with a Hmgb1 cDNA probe. Hmgb1 expression levels were normalized to the mRNA amounts of either of the two housekeeping genes ß-actin or gapdh. Whereas significant levels of Hmgb1 RNA are detected in the glands of nonpregnant and pregnant mice, Hmgb1 mRNA is down-regulated during lactation and in the involuting glands that undergo apoptosis. This result indicates that HMGB1 may regulate growth and involution of mammary gland tissue, possibly by influencing the apoptotic behavior of the cells.

CONCLUSIONS AND SIGNIFICANCE

Evidence is accumulating that a cell death machinery may exist in unicellular organisms like yeast.

Different apoptosis-inducing mammalian proteins kill yeast cells upon overexpression, possibly by triggering an intrinsic yeast cell death program. Among such proteins are proapoptotic members of the Bcl-2 family (Bax and Bak) and caspases (-3 and -8). The molecular mechanisms through which Bax and Bak induce yeast cell death are unknown. Our own overexpression studies with a functional bak-gfp construct in S. pombe were not suggestive of an exclusive mitochondrial localization of Bak-GFP in these cells.

Several groups have performed survival screens in S. cerevisiae to screen heterologous cDNA libraries for suppressors of Bax killing in yeast. We set up an inducible Bak expression system in S. pombe with the aim to screen human tumor-derived cDNA libraries to identify potential anti-apoptotic oncogenes. When we transformed Bak expressing S. pombe yeast cells in a first experiment with a cDNA library synthesized from mouse NIH 3T3 fibroblasts, we found the chromosomal protein HMGB1 as a protector against Bak-induced yeast cell death. The deletion mutant HMGB1 we isolated in the screen and that lacks the carboxyl-terminal 33 amino acids is even more potent in inhibiting Bak killing in yeast than the full-length protein.

If protected against Bak killing by overexpression of HMGB1, S. pombe cells grow exponentially while at the same time expressing a large amount of Bak (much higher than the amount of Bak necessary to kill wild-type yeast cells). These data rule out the possibility that HMGB1 down-regulates Bak transcription from the nmt-1 promoter used to express Bak in the yeast. We can also dismiss a scenario where HMGB1/HMGB1 directly bind to Bak thereby neutralizing Bak’s killing activity, similar to how Bcl-x_L may prevent Bak-induced cell death. Our localization studies and our negative results in a yeast two-hybrid-analysis and in coimmunoprecipitation experiments to prove direct Bak-HMGB1-interactions do not support such a binding model.

We could show that HMGB1 and full-length HMGB1 are able to efficiently suppress apoptosis induced by several different stimuli in mammalian cells. The inhibition of apoptosis includes protection against Bak and Bax killing as well as prevention of apoptosis induced by overexpression of Casp-8, UV radiation, and triggering of the death receptors CD95 and the TRAIL receptors. HMGB1 is an abundant nuclear protein with roughly 1 x 10⁶ molecules per nucleus, yet despite its abundance the protein may be limiting within cells: transient overexpression of HMGB1 enhances transcriptional activity of factors such as P53 and steroid hormone receptors. Similarly, an increase of HMGB1 protein levels in mammalian cells by transfection leads to protection against Bak killing.

Inhibition of apoptosis is a key event at the onset of and during tumorigenesis. Increased resistance to the many apoptotic stimuli a tumor cell is exposed to can be achieved by loss-of-function mutations (resulting in inactivation of proapoptotic genes) or gain-of-function mutations (resulting in increased activity of anti-apoptotic genes). A prototypic example of the latter is represented by the t(14;18) translocation involving bcl-2, which leads to Bcl-2-overexpression in human follicular B cell lymphoma. We investigated HMGB1 expression in human tumors. Our analysis revealed profound HMGB1 protein levels in human primary breast carcinomas. We observed strong HMGB1 expression in human breast carcinomas transplanted into nude mice. In contrast, expression of HMGB1 in normal breast tissue was low. This suggests that HMGB1 is involved in the development of mammary carcinomas. Hmgb1 mRNA appears to be regulated during mouse mammary gland development. Its expression is lowest during lactation and involution, when the mammary ducts undergo apoptosis. This finding raises the possibility that HMGB1 participates in the cyclic regulation of mammary gland apoptosis.

Elevated HMGB1 mRNA levels have been reported in human gastrointestinal adenocarcinomas vs. corresponding noncancerous mucosa. The authors suggesting a correlation between HMGB1 RNA expression and differentiation/staging of the carcinomas. A strong intertumoral variation of HMGB1 mRNA expression within 13 breast cancer samples was published that corresponds nicely to our tissue microarray results. The authors argue that this variation may contribute to the different responses of estrogen receptor-positive breast tumors to endocrine therapy. This argument is based on the observation that HMGB1 increases binding of the estrogen receptor to its DNA target sequence.

Inhibition of apoptosis by HMGB1 protein has to the best of our knowledge not been reported. HMGB1 belongs to the high mobility group (HMG) of DNA binding proteins, which are abundant, heterogeneous, nonhistone components of chromatin. Members of the structurally distinct HMGA family are highly expressed during embryonic development and in proliferating cells but are rare in adult cells. As critical components of enhanceosomes, they participate in gene regulation and their overexpression has been strongly correlated with tumorigenesis.

In certain cells, HMGB1 can be observed at the cell surface, where it has been reported to contribute to cellular migration and tumor invasion. The molecule is secreted by activated monocytes and macrophages and released by necrotic (but not by apoptotic) cells. Under such conditions, HMGB1 acts as a cytokine and mediates inflammation.

HMGB proteins recognize and bind to altered DNA confirmations such as stem-loop, four-way junction, kinked, or underwound DNA. Although they possess little or no sequence preference, HMGB1 interacts with proteins like P53 or steroid hormone receptors and increases the apparent DNA binding affinity of these transcription factors. Such a role for HMGB1 as a regulator of transcription may influence the apoptotic behavior of a cell. It has been shown that HMGB1 inhibits P73/ß- and P53-dependent transactivation from the bax gene promoter in p53-deficient SAOS-2 cells.

Future studies will investigate whether by suppressing cell death, HMGB1 can act as an oncogene that might be considered for molecular tumor therapy (Fig. 3 ). Our data show that screening of mammalian cDNA libraries in yeast survival assays allows the isolation of new anti-apoptotic tumor-relevant molecules; we are using tumor-derived cDNA libraries to identify further cell death-inhibiting oncogenes.

View larger version (17K): [in this window] [in a new window]   Figure 3. HMGB1 inhibits cell death in yeast and in mammalian cells. Overexpression of HMGB1 in human breast carcinoma implies a role for HMGB1 as an anti-apoptotic protein supporting tumor development.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0621fje; doi: 10.1096/fj.02-0621fje

² The first two authors contributed equally to this work.

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