DIRAS3 regulates the autophagosome initiation complex in dormant ovarian cancer cells.

DIRAS3 is an imprinted tumor suppressor gene that is downregulated in 60% of human ovarian cancers. Re-expression of DIRAS3 at physiological levels inhibits proliferation, decreases motility, induces autophagy, and regulates tumor dormancy. Functional inhibition of autophagy with choroquine in dormant xenografts that express DIRAS3 significantly delays tumor regrowth after DIRAS3 levels are reduced, suggesting that autophagy sustains dormant ovarian cancer cells. This study documents a newly discovered role for DIRAS3 in forming the autophagosome initiation complex (AIC) that contains BECN1, PIK3C3, PIK3R4, ATG14, and DIRAS3. Participation of BECN1 in the AIC is inhibited by binding of BECN1 homodimers to BCL2. DIRAS3 binds BECN1, disrupting BECN1 homodimers and displacing BCL2. Binding of DIRAS3 to BECN1 increases the association of BECN1 with PIK3C3 and ATG14, facilitating AIC activation. Amino acid starvation of cells induces DIRAS3 expression, reduces BECN1-BCL2 interaction and promotes autophagy, whereas DIRAS3 depletion blocks amino acid starvation-induced autophagy. In primary ovarian cancers, punctate expression of DIRAS3, BECN1, and the autophagic biomarker MAP1LC3 are highly correlated (P<0.0001), underlining the clinical relevance of these mechanistic studies. Punctate expression of DIRAS3 and MAP1LC3 was detected in only 21-23% of primary ovarian cancers but in 81-84% of tumor nodules found on the peritoneal surface at second-look operations following primary chemotherapy. This reflects a 4-fold increase (P<0.0001) in autophagy between primary disease and post-treatment recurrence. We suggest that DIRAS3 not only regulates the AIC, but induces autophagy in dormant, nutrient-deprived ovarian cancer cells that remain after conventional chemotherapy, facilitating their survival.

Stathmin expression and its relationship to microtubule-associated protein tau and outcome in breast cancer.

BACKGROUND: Microtubule-associated proteins (MAPs) endogenously regulate microtubule stability. Here, the prognostic value of stathmin, a destabilizing protein, was assessed in combination with MAP-tau, a stabilizing protein, in order to evaluate microtubule stabilization as a potential biomarker. METHODS: Stathmin and MAP-tau expression levels were measured in a breast cancer cohort (n = 651) using the tissue microarray format and quantitative immunofluorescence (AQUA) technology, then correlated with clinical and pathological characteristics and disease-free survival. RESULTS: Univariate Cox proportional hazard models indicated that high stathmin expression predicts worse overall survival (hazard ratio [HR] = 1.48; 95% confidence interval [CI] = 1.119-1.966; P = .0061). Survival analysis showed 10-year survival of 53.1% for patients with high stathmin expression versus 67% for low expressers (log-rank, P < .003). Cox multivariate analysis showed high stathmin expression was independent of age, menopausal status, nodal status, nuclear grade, tumor size, and estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 expression (HR = 1.19; 95% CI = 1.03-1.37; P = .01). The ratio of MAP-tau to stathmin expression showed a positive correlation to disease-free survival (HR = 0.679; 95% CI = 0.517-0.891; P = .0053) with a 10-year survival of 65.4% for patients who had a high ratio of MAP-tau to stathmin versus 52.5% 10-year survival rate for those with a low ratio (log-rank, P = .0009). Cox multivariate analysis showed the ratio of MAP-tau to stathmin was an independent predictor of overall survival (HR = 0.609; 95% CI = 0.422-0.879; P = .008). CONCLUSIONS: Low stathmin and high MAP-tau are associated with increased microtubule stability and better prognosis in breast cancer.

Evaluation of prognostic and predictive value of microtubule associated protein tau in two independent cohorts.

INTRODUCTION: Microtubule associated proteins (MAPs) endogenously regulate microtubule stabilization and have been reported as prognostic and predictive markers for taxane response. The microtubule stabilizer, MAP-tau, has shown conflicting results. We quantitatively assessed MAP-tau expression in two independent breast cancer cohorts to determine prognostic and predictive value of this biomarker. METHODS: MAP-tau expression was evaluated in the retrospective Yale University breast cancer cohort (n = 651) using tissue microarrays and also in the TAX 307 cohort, a clinical trial randomized for TAC versus FAC chemotherapy (n = 140), using conventional whole tissue sections. Expression was measured using the AQUA method for quantitative immunofluorescence. Scores were correlated with clinicopathologic variables, survival, and response to therapy. RESULTS: Assessment of the Yale cohort using Cox univariate analysis indicated an improved overall survival (OS) in tumors with a positive correlation between high MAP-tau expression and overall survival (OS) (HR = 0.691, 95% CI = 0.489-0.974; P = 0.004). Kaplan Meier analysis showed 10-year survival for 65% of patients with high MAP-tau expression compared to 52% with low expression (P = .006). In TAX 307, high expression was associated with significantly longer median time to tumor progression (TTP) regardless of treatment arm (33.0 versus 23.4 months, P = 0.010) with mean TTP of 31.2 months. Response rates did not differ by MAP-tau expression (P = 0.518) or by treatment arm (P = 0.584). CONCLUSIONS: Quantitative measurement of MAP-tau expression has prognostic value in both cohorts, with high expression associated with longer TTP and OS. Differences by treatment arm or response rate in low versus high MAP-tau groups were not observed, indicating that MAP-tau is not associated with response to taxanes and is not a useful predictive marker for taxane-based chemotherapy.

Genomic analysis of estrogen cascade reveals histone variant H2A.Z associated with breast cancer progression.

We demonstrate an integrated approach to the study of a transcriptional regulatory cascade involved in the progression of breast cancer and we identify a protein associated with disease progression. Using chromatin immunoprecipitation and genome tiling arrays, whole genome mapping of transcription factor-binding sites was combined with gene expression profiling to identify genes involved in the proliferative response to estrogen (E2). Using RNA interference, selected ERalpha and c-MYC gene targets were knocked down to identify mediators of E2-stimulated cell proliferation. Tissue microarray screening revealed that high expression of an epigenetic factor, the E2-inducible histone variant H2A.Z, is significantly associated with lymph node metastasis and decreased breast cancer survival. Detection of H2A.Z levels independently increased the prognostic power of biomarkers currently in clinical use. This integrated approach has accelerated the identification of a molecule linked to breast cancer progression, has implications for diagnostic and therapeutic interventions, and can be applied to a wide range of cancers.