Inhibition of HIF-1alpha activity by homeodomain-interacting protein kinase-2 correlates with sensitization of chemoresistant cells to undergo apoptosis.
BACKGROUND
Homeodomain-interacting protein kinase-2 (HIPK2), a transcriptional co-repressor with apoptotic function, can affect hypoxia-inducible factor 1 (HIF-1) transcriptional activity, through downmodulation of its HIF-1alpha subunit, in normoxic condition. Under hypoxia, a condition often found in solid tumors, HIF-1alpha is activated to induce target genes involved in chemoresistance, inhibition of apoptosis and tumor progression. Here, we investigated whether the HIPK2 overexpression could downregulate HIF-1alpha expression and activity in tumor cells treated with hypoxia-mimicking condition, and evaluated whether HIPK2-dependent downregulation of HIF-1alpha could sensitize chemoresistant tumor cells to adriamycin (ADR)-induced apoptosis.


METHODS
Tumor cell lines carrying wild-type p53, siRNA p53, or mutant p53 were overexpressed with HIPK2 (full length or catalytic inactive mutant) and treated with cobalt chloride (CoCl2) to mimic hypoxia, in the presence or absence of ADR treatment. HIF-1alpha expression was measured by semiquantitative reverse-transcriptase (RT)-PCR and Western immunoblotting and HIF-1 activity was evaluated by luciferase assay using reporter plasmid containing hypoxia response elements (HREs) upstream of luciferase gene. HIF-1 target genes, including multidrug resistance 1 (MDR1) and the antiapoptotic Bcl2 were determined by RT-PCR. Cell survival and apoptosis were measured by colony assay and cleavage of the caspase-3 substrate PARP, respectively.


RESULTS
Overexpression of HIPK2 resulted in downmodulation of cobalt-stabilized HIF-1alpha protein and HIF-1alpha mRNA levels, with subsequent inhibition of HIF-1 transcriptional activity. MDR1 and Bcl-2 gene expression was downmodulated by HIPK2 overexpression in cobalt-treated cells. Inhibition of HIF-1 transcriptional activity was dependent on HIPK2 catalytic activity. HIPK2 overexpression did not induce per se apoptosis of cobalt-treated cells, on the contrary it sensitized cobalt-treated cells to ADR-induced apoptosis, regardless of their p53 status.


CONCLUSIONS
The ability of HIPK2 to restore the apoptosis-inducing potential of chemotherapeutic drug in hypoxia-mimicking condition and therefore to sensitize chemoresistant tumor cells suggests that HIPK2 may induce fundamental alterations in cell signaling pathways, involving or not p53 function. Thus potential use of HIPK2 is promising for cancer treatment by potentiating cytotoxic therapies, regardless of p53 cell status.
Nardinocchi
Lavinia
L
Department of Experimental Oncology, Molecular Oncogenesis Laboratory, Regina Elena Cancer Institute, 00158 Rome, Italy. nardinocchi@ifo.it
Puca
Rosa
R
Sacchi
Ada
A
D'Orazi
Gabriella
G
D000903:Antibiotics, Antineoplastic / Q000494:pharmacology; D017209:Apoptosis / Q000502:physiology*; D015153:Blotting, Western; D002352:Carrier Proteins / Q000378:metabolism*; D015687:Cell Hypoxia / Q000187:drug effects / Q000502:physiology; D045744:Cell Line, Tumor; D004317:Doxorubicin / Q000494:pharmacology; D019008:Drug Resistance, Neoplasm / Q000502:physiology*; D015972:Gene Expression Regulation, Neoplastic / Q000502:physiology*; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism
D000903:Antibiotics, Antineoplastic; D002352:Carrier Proteins; C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D016159:Tumor Suppressor Protein p53; D004317:Doxorubicin; C115114:HIPK2 protein, human; D017346:Protein Serine-Threonine Kinases