Intermittent Induction of HIF-1α Produces Lasting Effects on Malignant Progression Independent of Its Continued Expression

<div><p>Dysregulation of hypoxia-inducible transcription factors HIF-1α and HIF-2α correlates with poor prognosis in human cancers; yet, divergent and sometimes opposing activities of these factors in cancer biology have been observed. Adding to this complexity is that HIF-1α apparently possesses tumor-suppressing activities, as indicated by the loss-of-function mutations or even homozygous deletion of <i>HIF1A</i> in certain human cancers. As a step towards understanding this complexity, we employed 8-week intermittent induction of a stable HIF-1α variant, HIF1α(PP), in various cancer cell lines and examined the effects on malignant progression in xenografts of immunocompromised mice in comparison to those of HIF2α(PP). Although 8-week treatment led to eventual loss of HIF1α(PP) expression, treated osteosarcoma U-2 OS cells acquired tumorigenicity in the subcutaneous tissue. Furthermore, the prior treatment resulted in widespread invasion of malignant glioma U-87 MG cells in the mouse brain and sustained growth of U-118 MG glioma cells. The lasting effects of HIF-1α on malignant progression are specific because neither HIF2α(PP) nor β-galactosidase yielded similar effects. By contrast, transient expression of HIF1α(PP) in U-87 MG cells or constitutive expression of HIF1α(PP) but not HIF2α(PP) in a patient-derived glioma sphere culture inhibited tumor growth and spread. Our results indicate that intermittent induction of HIF-1α produces lasting effects on malignant progression even at its own expense.</p></div

Intermittent induction of HIF1α(PP) promoted intracranial spread of U-87 MG cells.

<p>Bioluminescent imaging analysis of β-gal and HIF1α(PP)-derived intracranial tumors (A) and HIF2α(PP)-derived intracranial tumors (C). The respective tumor volumes were calculated based on the relative luminescent units (RLU) and plotted in a log scale (B and D). *, <i>p</i>-value < 0.05. (E) HIF1α(PP)-derived tumors had small yet numerous lesions invading the Ammon’s horn of the hippocampal region (XG026) and hindbrain and cerebellum (XG042), as indicated by arrowheads. (F) β-gal- and HIF2α(PP)-derived tumors were large and often singular in the cerebral cortex. Tumor lesions are demarcated in dash lines. Hematoxylin and eosin—stained images are presented at 25× and 200× magnifications, with scale bars of 1 mm and 100 μm, respectively.</p

Tetracycline regulation of HIF1α(PP) and HIF2α(PP) expression and transcriptional activity.

<p>(A) Tetracycline regulation is diagrammed where the addition of tetracycline (tet) results in dissociation of tetracycline repressor (TetR) from the tetracycline operon (TetO) and, in turn, gene activation. (B) Western blot analysis of transduced cell types, as indicated, for the expression of HIF1α(PP) and HIF2α(PP) after 2-day treatment with tetracycline. (C) Transcriptional activities of HIF1α(PP) and HIF2α(PP) were tested in a reporter assay in reference to β-galactosidase (β-gal). ***, <i>p</i>-value < 0.001. (D, E) The expression of HIF target genes (<i>PGK1</i>, <i>CA9</i>, <i>VEGFA</i>, and <i>LOX</i>) was analyzed in specified cell lines by using real-time PCR after 2-day treatment with tetracycline.</p

Intermittent induction resulted in loss of of HIF1α(PP) expression.

<p>(A) Intermittent induction involves the administration of tetracycline into cell culture each week on day 1 and removal on day 4 each week for a total of 8 weeks. Afterwards, cells were allowed to expand for further analyses and injections. (B) After intermittent induction (8W), different types of cells as indicated were induced again with tetracycline for 2 days and analyzed by Western blotting in reference to those without intermittent induction (0W). (C) Cell proliferation was determined by cell counting after intermittent induction. ***, <i>p</i>-value < 0.001.</p

U-2 OS cells acquired tumorigenicity after intermittent induction of HIF1α(PP).

<p>(A) Tumor incidence is shown in 6 NSG mice after bilateral, subcutaneous injections of the 8-week HIF1α(PP) and HIF2α(PP) cells. (B) Only injections of the HIF1α(PP) cells produced tumors, as indicated by arrowheads. Scale bar, 1 cm. (C) Tumor volume was calculated based on measurements and plotted as a function of time. ***, <i>p</i>-value < 0.001. (D) Hematoxylin and eosin staining of the tumor specimens reveals invasion of the dermal layer (<i>a</i>), numerous mitoses (arrowheads) (<i>b</i>), necrosis (N) (<i>c</i>), and invasion into the striated muscle layer (<i>d</i>). Scale bar, 100 μm.</p