The protein disulfide isomerases PDIA4 and PDIA6 mediate resistance to cisplatin-induced cell death in lung adenocarcinoma.

Intrinsic and acquired chemoresistance are frequent causes of cancer eradication failure. Thus, long-term cis-diaminedichloroplatine(II) (CDDP) or cisplatin treatment is known to promote tumor cell resistance to apoptosis induction via multiple mechanisms involving gene expression modulation of oncogenes, tumor suppressors and blockade of pro-apoptotic mitochondrial membrane permeabilization. Here, we demonstrate that CDDP-resistant non-small lung cancer cells undergo profound remodeling of their endoplasmic reticulum (ER) proteome (>80 proteins identified by proteomics) and exhibit a dramatic overexpression of two protein disulfide isomerases, PDIA4 and PDIA6, without any alteration in ER-cytosol Ca(2+) fluxes. Using pharmacological and genetic inhibition, we show that inactivation of both proteins directly stimulates CDDP-induced cell death by different cellular signaling pathways. PDIA4 inactivation restores a classical mitochondrial apoptosis pathway, while knockdown of PDIA6 favors a non-canonical cell death pathway sharing some necroptosis features. Overexpression of both proteins has also been found in lung adenocarcinoma patients, suggesting a clinical importance of these proteins in chemoresistance.

PGC-1ß mediates adaptive chemoresistance associated with mitochondrial DNA mutations.

Primary mitochondrial dysfunction commonly leads to failure in cellular adaptation to stress. Paradoxically, however, nonsynonymous mutations of mitochondrial DNA (mtDNA) are frequently found in cancer cells and may have a causal role in the development of resistance to genotoxic stress induced by common chemotherapeutic agents, such as cis-diammine-dichloroplatinum(II) (cisplatin, CDDP). Little is known about how these mutations arise and the associated mechanisms leading to chemoresistance. Here, we show that the development of adaptive chemoresistance in the A549 non-small-cell lung cancer cell line to CDDP is associated with the hetero- to homoplasmic shift of a nonsynonymous mutation in MT-ND2, encoding the mitochondrial Complex-I subunit ND2. The mutation resulted in a 50% reduction of the NADH:ubiquinone oxidoreductase activity of the complex, which was compensated by increased biogenesis of respiratory chain complexes. The compensatory mitochondrial biogenesis was most likely mediated by the nuclear co-activators peroxisome proliferator-activated receptor gamma co-activator-1α (PGC-1α) and PGC-1ß, both of which were significantly upregulated in the CDDP-resistant cells. Importantly, both transient and stable silencing of PGC-1ß re-established the sensitivity of these cells to CDDP-induced apoptosis. Remarkably, the PGC-1ß-mediated CDDP resistance was independent of the mitochondrial effects of the co-activator. Altogether, our results suggest that partial respiratory chain defects because of mtDNA mutations can lead to compensatory upregulation of nuclear transcriptional co-regulators, in turn mediating resistance to genotoxic stress.

Restoration of the immunogenicity of cisplatin-induced cancer cell death by endoplasmic reticulum stress.

In contrast to other cytotoxic agents including anthracyclins and oxaliplatin (OXP), cisplatin (CDDP) fails to induce immunogenic tumor cell death that would allow to stimulate an anticancer immune response and hence to amplify its therapeutic efficacy. This failure to induce immunogenic cell death can be attributed to CDDP's incapacity to elicit the translocation of calreticulin (CRT) from the lumen of the endoplasmic reticulum (ER) to the cell surface. Here, we show that, in contrast to OXP, CDDP is unable to activate the protein kinase-like ER kinase (PERK)-dependent phosphorylation of the eukaryotic translation initiation factor 2α (eIF2α). Accordingly, CDDP also failed to stimulate the formation of stress granules and macroautophagy, two processes that only occur after eIF2α phosphorylation. Using a screening method that monitors the voyage of CRT from the ER lumen to the cell surface, we identified thapsigargin (THAPS), an inhibitor of the sarco/ER Ca(2+)-ATPase as a molecule that on its own does not stimulate CRT exposure, yet endows CDDP with the capacity to do so. The combination of ER stress inducers (such as THAPS or tunicamycin) and CDDP effectively induced the translocation of CRT to the plasma membrane, as well as immunogenic cell death, although ER stress or CDDP alone was insufficient to induce CRT exposure and immunogenic cell death. Altogether, our results underscore the contribution of the ER stress response to the immunogenicity of cell death.

Suppression of the DNA damage response in acute myeloid leukemia versus myelodysplastic syndrome.

The molecular mechanisms responsible for the evolution from the preleukemic entities of low-risk myelodysplastic syndrome (MDS) to the less favorable forms of high-risk MDS, as well as those enabling transformation to acute myeloid leukemia (AML), are still incompletely understood. Abundant evidence from solid tumors demonstrates that preneoplastic lesions activate signaling pathways of a DNA damage response (DDR), which functions as an 'anticancer barrier' hindering tumorigenesis. Testing the hypothesis that subgroups of MDS and AML differ with respect to DDR, we first assessed markers of DDR (phosphorylation of ATM, Chk-1, Chk-2 and H2AX) in cell lines representing different entities of MDS (P39, MOLM-13) and AML (MV4-11, KG-1) before and after gamma-irradiation. Although gamma-irradiation induced apoptosis and G(2)/M arrest and a concomitant increase in the phosphorylation of ATM, Chk-1 and H2AX in MDS-derived cell lines, this radiation response was attenuated in the AML-derived cell lines. It is noteworthy that KG-1, but not P39 cells exhibit signs of an endogenous activation of the DDR. Similarly, we found that the frequency of P-ATM(+) cells detectable in bone marrow (BM) biopsies increased in samples from patients with AML as compared with high-risk MDS samples and significantly correlated with the percentage of BM blasts. In contrast, the frequency of gamma-H2AX(+) cells was heterogeneous in all subgroups of AML and MDS. Whereas intermediate-1 MDS samples contained as little P-Chk-1 and P-Chk-2 as healthy controls, staining for both checkpoint kinases increased in intermediate-2 and high-risk MDS, yet declined to near-to-background levels in AML samples. Thus the activation of Chk-1 and Chk-2 behaves in accord with the paradigm established for solid tumors, whereas ATM is activated during and beyond transformation. In conclusion, we demonstrate the heterogeneity of the DDR response in MDS and AML and provide evidence for its selective suppression in AML because of the uncoupling between activated ATM and inactive checkpoint kinases.

Disruption of the hexokinase-VDAC complex for tumor therapy.

Unlike mitochondria from most normal tissues, cancer cell mitochondria demonstrate an association between the glycolytic enzyme hexokinase (HK) and the voltage-dependent anion channel (VDAC). This provides a therapeutic opportunity, as the association appears to protect tumor cells from mitochondrial outer membrane permeabilization (MOMP), an event that marks the point of no return in multiple pathways leading to cell death. In this issue of Oncogene, the plant hormone methyl jasmonate (MJ) is shown to disrupt the interaction between human HK and VDAC, causing the inhibition of glycolysis and the induction of MOMP. MJ has already been shown to have selective anticancer activity in preclinical studies, and this finding may stimulate the development of a novel class of small anticancer compounds that inhibit the HK-VDAC interaction.

Hierarchical involvement of Bak, VDAC1 and Bax in cisplatin-induced cell death.

Following the screening of a battery of distinct small-interfering RNAs that target various components of the apoptotic machinery, we found that knockdown of the voltage-dependent anion channel 1 (VDAC1) was particularly efficient in preventing cell death induced by cisplatin (CDDP) in non-small cell lung cancer cells. Both the downregulation of VDAC1 and its chemical inhibition with 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid reduced the apoptosis-associated modifications induced by CDDP, including mitochondrial transmembrane potential dissipation and plasma membrane permeabilization. VDAC1 inhibition strongly reduced the CDDP-induced conformational activation of Bax, yet had no discernible effect on the activation of Bak, suggesting that VDAC1 acts downstream of Bak and upstream of Bax. Accordingly, knockdown of Bak abolished the activation of Bax, whereas Bax downregulation had no effect on Bak activation. In VDAC1-depleted cells, the failure of CDDP to activate Bax could be reversed by means of the Bcl-2/Bcl-X(L) antagonist ABT-737, which concomitantly restored CDDP cytotoxicity. Altogether, these results delineate a novel pathway for the induction of mitochondrial membrane permeabilization (MMP) in the course of CDDP-induced cell death that involves a hierarchical contribution of Bak, VDAC1 and Bax. Moreover, our data suggest that VDAC1 may act as a facultative regulator/effector of MMP, depending on the initial cytotoxic event.

Consequence of parvalbumin deficiency in the mdx mouse: histological, biochemical and mechanical phenotype of a new double mutant.

We tested the hypothesis whether the mild dystrophy in mdx mice could result from the contribution of the cytosolic calcium buffer parvalbumin in maintaining a normal cytosolic [Ca2+]i, in spite of an increased passive Ca2+ influx. By crossing mdx mice with parvalbumin-deficient mice, double mutant mice, lacking both dystrophin and parvalbumin, were obtained. Though resting cytosolic [Ca2+]i and total calcium content were similar to that of mdx muscles, this new animal model presented a slightly more severe phenotype than the mdx mouse. Muscle pseudo-hypertrophy, the density of myotubes and of centronucleated fibres as well as the loss of IIB fibres were all increased in parvalbumin-deficient mdx mice. Many of these deficits were overcome in late adulthood, albeit fibrosis was clearly more pronounced than in mdx muscles. At 90 days, parvalbumin-deficient mdx mice showed higher levels of creatine phosphokinase and lower muscle strength, in vivo, than mdx mice. Isometric tension of isolated muscle was reduced, but the susceptibility to eccentric contraction was not increased. The slight aggravation of muscle dystrophy observed in mdx mice deprived of parvalbumin cannot explain the severity of the affection observed in xmd dogs and Duchenne dystrophy patients where parvalbumin is constitutively not expressed.

Does site-specific labelling and individual processing of sextant biopsies improve the accuracy of prostate biopsy in predicting pathological stage in patients with T1c prostate cancer?

OBJECTIVE: To evaluate whether individual labelling and processing of the sextant of origin improves the accuracy of prostate biopsy in predicting the final pathological stage after radical prostatectomy in patients with T1c prostate cancer. PATIENTS AND METHODS: The charts of 386 patients treated for prostate cancer by radical prostatectomy between January 1996 and June 1999 were reviewed. In all, 124 patients fulfilled the following inclusion criteria: no abnormality on digital rectal examination (DRE) or transrectal ultrasonography, a prostate specific antigen (PSA) level before biopsy of < or = 20 ng/mL, and prostate cancer diagnosed after one set of random sextant biopsies, with the cores being submitted in six separate containers individually labelled for the sextant of origin. RESULTS: Within this series of patients with a low tumour burden, the preoperative PSA, biopsy Gleason score and unilateral vs bilateral involvement were not significant predictors of disease extension. The percentage of positive cores and the number and topography of positive sextants were both statistically significant predictors of organ-confined disease. Although these two variables appeared to be statistically equivalent on a first analysis in the overall series, a subgroup of patients was identified who benefited from the complete topographical information, i.e. those 52 (42%) patients with a Gleason score of < 7, 25-75% positive biopsies and < or =3 positive sextants. CONCLUSION: These results support the individual labelling of biopsy cores in selected patients with a normal DRE and a moderately elevated PSA, as it helps to better predict the final pathological stage. This substantial benefit outweighs the additional effort by the pathologist.