Hypoxia-inducible factors, mTOR, and astrin constitute an integrative regulatory network in colon cancer cells.

Astrin/SPAG5 is a mitotic spindle protein found to be overexpressed in several human cancers, functioning as an oncogene. The expression of Astrin has not been reported so far in colon cancer, nor has it been related to HIFs expression or action. Since mTOR, Astrin, and hypoxia-inducible factors (HIFs) are involved in promoting the growth and survival of cancer cells, we investigated the possible interaction between them in cultured colon cancer cells. Both Astrin and HIF-1α and HIF-2α protein levels were found only expressed in colon cancer cells compared with nonmalignant cells. Our data indicate that mTOR stimulates both Astrin and HIFs expression, but notably, mTORC activity seems to be independent of Astrin expression levels. However, while HIF-1α or HIF-2α stable knockdown increased Astrin expression, mTOR activity was affected in an opposite way by HIF-1α or HIF-2α silencing, indicating that HIF-1α inhibits mTOR while HIF-2α stimulates its activity. These data suggest that mTOR, Astrin, and HIFs compose an integrative network interacting to activate positive or negative regulatory loops probably to coordinate cancer cell growth, metabolism, and survival under oncogenic stress.

Protein phosphatase 2A B55ß limits CD8+ T cell lifespan following cytokine withdrawal.

How T cells integrate environmental cues into signals that limit the magnitude and length of immune responses is poorly understood. Here, we provide data that demonstrate that B55ß, a regulatory subunit of protein phosphatase 2A, represents a molecular link between cytokine concentration and apoptosis in activated CD8+ T cells. Through the modulation of AKT, B55ß induced the expression of the proapoptotic molecule Hrk in response to cytokine withdrawal. Accordingly, B55ß and Hrk were both required for in vivo and in vitro contraction of activated CD8+ lymphocytes. We show that this process plays a role during clonal contraction, establishment of immune memory, and preservation of peripheral tolerance. This regulatory pathway may represent an unexplored opportunity to end unwanted immune responses or to promote immune memory.

Functional Interaction of Hypoxia-Inducible Factor 2-Alpha and Autophagy Mediates Drug Resistance in Colon Cancer Cells.

Hypoxia and the accumulation of hypoxia-inducible factors (HIFs) in tumors have been associated with therapeutic resistance and with autophagy establishment. We examined the effects of stable knockdown of HIF-1α or HIF-2α expression on autophagy and drug resistance in colon cancer cells. We found that under normoxic conditions, malignant cells exhibit increased basal levels of autophagy, compared with non-malignant cells, in addition to the previously reported coexpression of HIF-1α and HIF-2α. Knockdown of HIF-1α or HIF-2α expression resulted in increased autophagic and apoptotic cell death, indicating that the survival of cells is HIF-dependent. Cytotoxic-induced cell death was significantly increased by knockdown of HIFs but not by autophagy inhibition. Strikingly, although malignancy-resistant cells were sensitized to death by nutrient stress, the combination with HIF-2α depletion, but not with HIF-1α depletion, induced severe cell death. Oxidative stress levels were significantly increased as a result of HIF-2α specific inhibition or silencing suggesting that this may contribute to sensitize cells to death. The in vitro results were confirmed in vivo using a xenograft mouse model. We found that coordinated autophagy and mTOR inhibition enhanced cell death and induced tumor remission only in HIF-2α-silenced cells. Finally, using a specific HIF-2α inhibitor alone or in combination with drugs in patient-derived primary colon cancer cells, overcame their resistance to 5-FU or CCI-779, thus emphasizing the crucial role played by HIF-2α in promoting resistance and cell survival.

Identification of Dp71e, a new dystrophin with a novel carboxy-terminal end.

Several dystrophin Dp71 isoforms have previously been described and can be grouped into two subfamilies (Dp71d or Dp71f) depending upon the splicing of exon 78. As a consequence of this splicing, each group has a carboxy-terminal end with a unique amino acid composition; this composition imparts specific characteristics with respect to subcellular localization and interactions with particular members of the dystrophin-associated proteins (DAPs) complex. We have discovered a new alternative splicing event at the 3' region of the Dp71 transcript. This spliced region has a unique sequence that codes for 10 amino acids and prevents the translation of exons 78 and 79. This novel Dp71 isoform is called Dp71e and is expressed in undifferentiated cells and during nerve growth factor-induced differentiation of PC12 cells. Interestingly, Dp71e mRNA and protein expression increase during PC12 cell differentiation mediated by NGF. This new Dp71 isoform is also expressed in rat organs and in human cell lines.