Enrica Martinelli

Class III β-Tubulin Overexpression Is a Marker of Poor Clinical Outcome in Advanced Ovarian Cancer Patients

Purpose: Overexpression of β III tubulin has been involved in paclitaxel resistance in several experimental models. We investigated the role of β III tubulin as predictor of clinical outcome in ovarian cancer patients given platinum/paclitaxel treatment. We also investigated whether β III tubulin expression could be modified after the selective pressure represented by chemotherapy in vivo. Experimental Design: The study was designed to include a series of consecutive ovarian cancer patients with unresectable disease at time of first surgery, who underwent interval debulking surgery with pathologic assessment of response to treatment with platinum/paclitaxel chemotherapy. Immunostaining was done on formalin-fixed, paraffin-embedded tissue sections from pretreatment and posttreatment tissue biopsies by using the polyclonal rabbit anti–class III β-tubulin antibody. Results: β III Tubulin immunoreaction was observed in 51 of 62 (82.2%) cases. β III Tubulin positivity was neither associated with clinicopathologic variables nor with pathologic response to chemotherapy. Significantly lower percentages of β III tubulin positivity were observed in posttreatment (range, 5-80%; median, 20%) versus pretreatment (range 10-100%; median, 40%) tissue biopsies (P = 0.0011). Cases with high β III tubulin expression showed a worse overall survival with respect to cases with low β III tubulin expression (median overall survival, 25 versus 46 months; P = 0.002). Multivariate analysis showed that high content of β III tubulin remains independently associated with a worse prognosis. Conclusions: Assessment of β III tubulin could be useful to identify poor prognosis ovarian cancer patients candidates to more aggressive and/or targeted therapy.

Survivin expression in ovarian cancer and its correlation with clinico-pathological, surgical and apoptosis-related parameters

We investigated the association of survivin expression with prognosis and other apoptosis-related biological factors in 110 primary ovarian cancer patients admitted to the Division of Gynecologic Oncology, Catholic University of Rome. Immunohistochemistry was performed on formalin-fixed, paraffin-embedded sections by using polyclonal antibody ab469 for survivin, and mouse monoclonal antibodies (clone 124 and DO-7), for bcl-2 and p53, respectively. Cytoplasmic survivin immunoreaction was observed in 84.5% cases, while nuclear survivin immunostaining was observed in 29.1% cases. We failed to find any relationship between cytoplasmic survivin positivity rate and any of the parameters examined. Serous tumours showed a lower percentage of nuclear survivin positivity with respect to other histotypes (20.5 vs 48.6%, respectively; P-value=0.004). The percentage of nuclear survivin positivity was higher in cases subjected to primary tumour cytoreduction (43.5%), with respect to patients subjected to exploratory laparotomy (20%) (P=0.024). Bcl-2 and p53 were, respectively, expressed in 27.3 and 60.0% of the cases and their expression was not correlated with survivin status. During the follow-up period, progression and death of disease were observed in 68 (61.8%) and 53 (48.2%) cases, respectively. There was no difference in time to progression and overall survival according to survivin status in ovarian cancer patients. In conclusion, in our experience, the immunohistochemical assessment of survivin status does not seem to be helpful in the prognostic characterisation of ovarian cancer. A more in depth investigation of the complex physiology of divergent survivin variants is needed in order to clarify the biological and the clinical role of differentially located survivin isoforms.

CD133 antigen expression in ovarian cancer

BackgroundMuch attention has been recently focused on the role of cancer stem cells (CSCs) in the initiation and progression of solid malignancies. Since CSCs are able to proliferate and self-renew extensively, thus sustaining tumor growth, the identification of CSCs through their antigenic profile might have relevant clinical implications. In this context, CD133 antigen has proved to be a marker of tumor cells with stemness features in several human malignancies.The aim of the study was to investigate the clinical role of the immunohistochemically assessed expression of CD133 in a large single Institution series of ovarian cancer patients.MethodsThe study included 160 cases admitted to the Gynecologic Oncology Unit, Catholic University of Campobasso and Rome. CD133 antigen was identified by the monoclonal mouse anti-CD133-1 antibody (clone CD133 Miltenyi biotec).ResultsIn the overall series CD133 positive tumor cells were observed in 50/160 (31.2%) cases. A diffuse cytoplasmic pattern was identified in 30/50 (60.0%), while an apical cytoplasmic pattern was found in 20/50 (40.0%) of CD133 positive tumors.As of September 2008, the median follow up was 37 months (range: 2–112). During the follow up period, progression and death of disease were observed in 123 (76.9%), and 88 (55.0%) cases, respectively. There was no difference in TTP between cases with negative (median TTP = 23 months) versus positive CD133 expression (median TTP = 24 months) (p value = 0.3). Similar results were obtained for OS. When considering the TTP and OS curves according to the pattern of CD133 expression, a trend to a worse prognosis for cases with diffuse cytoplasmic versus the apical cytoplasmic pattern was documented, although the statistical significance was not reached.ConclusionThe immunohistochemical assessment of CD133 expression seems not to provide additional prognostic information in ovarian cancer patients. The role of the different pattern of CD133 immunoreaction deserves further investigation in a larger series.

MiR-200c and HuR in ovarian cancer

BackgroundMicroRNAs in solid malignancies can behave as predictors of either good or poor outcome. This is the case with members of the miR-200 family, which are the primary regulators of the epithelial to mesenchymal transition and have been reported to act as both oncogenes and tumor suppressors. This study assessed the role of miR-200c as regulator of class III β-tubulin (TUBB3), a factor associated with drug-resistance and poor prognosis in ovarian cancer.MethodsExpression of miR-200c was assessed in a panel of ovarian cancer cell lines with inherent or acquired drug-resistance. Stable overexpression of miR-200c was obtained in A2780 and Hey cell lines. Crosslinking-coupled affinity purification method and ribonucleic-immunoprecipitation assay were used to characterise the complexes between miR-200c, HuR and 3′UTR region of TUBB3 mRNA. Nanofluidic technology and immunohistochemistry were used to analyze the expression of HuR, TUBB3 and miR-200c in 220 ovarian cancer patients.ResultsIn a panel of ovarian adenocarcinoma cell lines, we observed a direct correlation between miR-200c expression and chemoresistance. In A2780 cells miR-200c targeted TUBB3 3′UTR, while a positive correlation was observed between miR-200c and TUBB3 expression in most of the other cell lines. Through the analysis of 3′UTR-associated complexes, we found that the miR-200c can increase the association of the RNA binding protein HuR with TUBB3 mRNA, whereas HuR binding enhanced TUBB3 mRNA translation. Most importantly, in our analysis on 220 ovarian cancer patients we observed that overexpression of miR-200c correlated with poor or good outcome depending on the cellular localization of HuR.ConclusionThis study suggests a model for the combined regulatory activity of miR-200c and HuR on TUBB3 expression in ovarian cancer. When HuR is nuclear, high expression of miR-200c inhibits TUBB3 expression and results in a good prognosis, whereas when HuR occurs in cytoplasm, the same miRNA enhances TUBB3 expression and produces a poor outcome. These findings reveal the usefulness of multidimensional analysis in the investigation of the prognostic role of miRNA expression.

Class III β-tubulin and the cytoskeletal gateway for drug resistance in ovarian cancer.

The Class III β‐tubulin isotype (βIII‐tubulin) is a predictive biomarker in ovarian cancer and other solid tumor malignancies. We discovered that βIII‐tubulin function is linked to two GTPases: guanylate‐binding protein 1 (GBP1), which activates its function, and GNAI1, which inhibits it. This finding was demonstrated in a panel of ovarian cancer cells resistant to several chemotherapeutic agents. Using a protein microarray, we identified PIM1 as the downstream partner of GBP1, recruited into the cytoskeleton under hypoxic conditions. The clinical value of these observations was tested by performing an archive study of 98 ovarian cancer patients, which demonstrated that the βIII‐tubulin ‐/PIM1‐ cohort responded to treatment, exhibiting long overall survival (OS), while βIII‐tubulin +/PIM+ patients experienced poor outcomes and OS times similar to patients receiving palliation alone. βIII‐tubulin expression is commonly believed responsible for paclitaxel resistance due to its enhancement of the dynamic instability of microtubules, which counteracts the activity of taxanes. In contrast, our research reveals that βIII‐tubulin behaves as a gateway for prosurvival signals, such as PIM1, to move into the cytoskeleton. When cells are exposed to microenvironmental stressors, they activate this pathway by telling the cytoskeleton to incorporate PIM1 through GBP1 and βIII‐tubulin, which ultimately leads to drug resistance. This discovery reveals that βIII‐tubulin does not act alone but requires partners to play its role. The discovery of such protein:protein interactions underlying this prosurvival cascade makes feasible the development of therapeutic approaches using novel compounds that are capable of inhibiting the transmission of prosurvival signals into the cytoskeleton. J. Cell. Physiol. 227: 1034–1041, 2012.

Characterization of the pharmacologic profile of a standardized soy extract in the ovariectomized rat model of menopause: effects on bone, uterus, and lipid profile.

Objective: This study was aimed to assess the effect of a standardized soy extract (SSE, Soyselect) in the ovariectomized rat model of menopause. Design: Ovariectomized rats were treated for 6 weeks with the soy extract (50 or 100 mg/kg/day − PO), vehicle (distilled water), or 17β-estradiol (0.5 mg/kg/day − PO). Tissue-specific estrogen agonist effects were examined using the endpoints bone mineral density, biochemical parameters of bone turnover, modulation of cytokines involved in the bone remodeling, uterine weight, uterine histology, uterine hormone receptor status, and serum lipid level. Results: The SSE produced a bone-sparing effect associated with a slowing down in the increased bone turnover observed after ovariectomy (as indicated by measurements of serum osteocalcin levels and excretion ratio of deoxypyridinoline); changes in serum interleukin-6 levels observed after SSE suggested that this bone-sparing effect could be partly attributed to the modulation of osteoclastogenesis induced by interleukin-6. Remarkably, organ weight data and histopathologic analysis did not show any stimulatory activity of the SSE on the uterus. Immunohistochemical analysis showed a significant down-regulation of estrogen receptor-α (ERα) in uterine epithelium after 17β-estradiol treatment, but not after treatment with the SSE; no significant differences among groups were observed in ER-α uterine stromal levels. After treatment with 17β-estradiol, estrogen receptor-β (ER-β) expression was not modulated in the stroma or epithelium, whereas the SSE induced an up-regulation of ER-β stromal expression. Collectively, these results suggest that the lack of stimulatory activity on the uterine epithelium using soy treatment could be due to a negligible stimulatory activity on estrogen receptor-α and/or to the enhanced expression observed in stromal ER-β, the latter being considered as a negative modulator of ERα-mediated uterine proliferation. 17β-estradiol, but not the SSE, down-regulated uterine epithelial progesterone receptor (PR), compared with ovariectomized rats. In the stromal compartment, progesterone receptor expression was fully up-regulated by 17β-estradiol treatment and, to a lesser extent, by SSE treatment. The minor increase in lipid levels induced by ovariectomy was not affected by SSE administration. Finally, the lack of stimulatory activity on uterus was also confirmed in an immature female rat model. Conclusions: The results of this study demonstrated that the tested extract has an interesting profile of tissue-specific response, in that it is efficacious in preventing experimental osteoporosis without causing stimulation in uterus at doses that are effective in bone.

Prognostic role of the ratio between cyclooxygenase-2 in tumor and stroma compartments in cervical cancer.

Purpose: The aim of this study was to analyze the clinical role of cyclooxygenase (COX)-2 in a large series of 175 cervical cancer patients. Experimental Design: Immunohistochemistry was performed on paraffin-embedded sections by using rabbit antiserum against COX-2. The tumor:stroma (T/S) ratio of COX-2 expression was used to define the overall COX-2 content in the tumor. Results: The T/S COX-2 ratio values ranged from 0.03 to 48.2 (mean ± SE, 3.7 ± 0.5). A total of 95 of 175 patients (54.3%) were scored as having a high (>1) T/S COX-2 ratio. In locally advanced cervical cancer patients who underwent neoadjuvant treatment, the percentage of cases showing a high T/S COX-2 ratio was greater in patients who did not respond to treatment (26 of 29 patients, 89.7%) than in patients with a partial (32 of 50 patients, 64.0%) or complete (19 of 44 patients, 43.2%) response (P = 0.0003). When logistic regression was applied, International Federation of Gynecologists and Obstetricians (FIGO) stage (χ2 = 11.3; P = 0.0008) and T/S COX-2 ratio (χ2 = 5.3; P = 0.021) retained an independent role in predicting a poor chance of response. Cases with a high T/S COX-2 ratio had a shorter overall survival (OS) [2-year OS, 61%(95% confidence interval 750–83)] than cases with a low T/S COX-2 ratio (2-year OS, 90%; 95% confidence interval, 81–99; P = 0.0001). In multivariate analysis, the status of T/S COX-2 IDV ratio, together with advanced stage, retained an independent negative prognostic role for OS. Conclusions: The assessment of COX-2 status in both tumor and stroma compartment could provide valuable information to identify cervical cancer patients endowed with a very poor chance of response to neoadjuvant treatment and unfavorable prognosis.

Gender Influences the Class III and V β-Tubulin Ability to Predict Poor Outcome in Colorectal Cancer

Purpose: Colorectal cancer is one of the deadliest diseases in Western countries. To predict the outcome of therapy, we assessed the role of class III (TUBB3) and class V β-tubulin (TUBB6) as predictive biomarkers. Experimental Design: Using immunohistochemistry and nanofluidics, the expression of TUBB3 and TUBB6 was assessed in two cohorts of 180 and 134 patients, respectively. The CYP17A1 RS743572 was genotyped to identify GG carriers with enhanced androgen levels. TUBB3 and TUBB6 were investigated in 22 colorectal cancer cell lines in basal conditions and after serum starvation, the latter serving as activator of this prosurvival pathway. To ascertain the role of androgen receptor (AR) in such regulation, we silenced AR and checked TUBB3 and TUBB6 expression and sensitivity to chemotherapy. Results: There was a link between poor survival, the expression of TUBB3/TUBB6, and AR only in females. Conversely, only in males carriers of the GG phenotype exhibited the worst outcome. Importantly, male cell lines were resistant to serum starvation and exhibited higher levels of TUBB6, thereby suggesting that the pathway is activated by androgens. In female cells this phenomenon was absent. In both genders, AR was the main driver of TUBB3/TUBB6 expression, as constitutive silencing of AR was associated with downregulation of TUBB3/TUBB6 expression and increased sensitivity to oxaliplatin and SN-38. Conclusions: The involvement of androgens in the TUBB3 pathway opens the way for clinical trials to assess the efficacy of antiandrogens for increasing the efficacy of chemotherapy in male colorectal cancer patients. Clin Cancer Res; 18(10); 2964–75. ©2012 AACR.

Polarisation of Tumor-Associated Macrophages toward M2 Phenotype Correlates with Poor Response to Chemoradiation and Reduced Survival in Patients with Locally Advanced Cervical Cancer.

Objective we investigate the prognostic role of pre-treatment ratio between Type 1 (M1) and Type 2 (M2) tumor-associated macrophages (TAMs) in locally advanced cervical cancer (LACC) patients treated with chemoradiation (CT/RT). Methods 84 consecutive LACC patients treated with cisplatin-based CT/RT for a total dose of 50.0 Gy, followed by radical surgery were analysed. Double-staining immunohistochemistry of CD163/p-STAT, CD68/pSTAT1, CD163/c-MAF, and CD68/c-MAF was performed on tumor samples taken at the time of diagnosis. TAMs with CD163+pSTAT1+, or CD68+pSTAT1+ were defined M1; CD163+c-MAF+ or CD68+c-MAF+ defined the M2 phenotype. The number of M1 and M2 cells was counted at low magnification by evaluating for each case the same tumour area. The ratio between M1 and M2 (M1/M2) was finally calculated. Results At diagnosis, we observed a direct correlation between the number of circulating monocytes and of TAMs (p-value = 0.001). Patients with high M1/M2 experienced more frequently complete pathologic response (no residual tumor) to CT/RT, compared to cases with low M1/M2 (55.0% Vs 29.5%; p-value = 0.029). At multivariate analysis M1/M2 (OR = 2.067; p-value = 0.037) emerged as independent predictor of pathologic response to CT/RT. Women with high M1/M2 showed a longer 5-yrs Disease-free (67.2% Vs. 44.3%; p-value = 0.019), and 5-yrs Overall (69.3% Vs. 46.9%; p-value = 0.037) survival, compared to cases with low M1/M2. The presence of a high M1/M2 ratio was independently associated with an unfavourable survival outcome in multivariate analysis. Conclusions polarisation of TAMs toward a M2 phenotype, as reflected by a lower M1/M2 ratio, is an independent predictor of poor response to CT/RT, and shorter survival in LACC.

Cyclo-oxygenase-2 (Cox-2) expression and resistance to platinum versus platinum/paclitaxel containing chemotherapy in advanced ovarian cancer

BackgroundCyclo-oxygenase-2 (COX-2), the key enzyme in the conversion of arachidonic acid to prostaglandins, is involved in critical steps of tumor onset and progression, and is a strong predictor of chemotherapy resistance and poor outcome in advanced ovarian cancer. To our knowledge, no data has been reported until now about the association between COX-2 status and response to different chemotherapy regimens.MethodsA retrospective study was performed to investigate the association of COX-2 with outcome and response to platinum versus platinum/paclitaxel in 68 primary ovarian cancer. COX-2 immunoreaction was performed on paraffin-embedded sections by using rabbit polyclonal antiserum against COX-2.ResultsIn the overall series, COX-2 positivity was found in a statistically significant higher percentage of not responding cases than in patients responding to chemotherapy (n = 15/21; 71.4% versus n = 17/47; 36.1%; p value = 0.0072). A higher percentage of COX-2 positivity was found in patients unresponsive (n = 11/13; 84.6%) versus patients responsive to platinum-based chemotherapy (n = 9/26; 34.6%). In cases administered platinum/paclitaxel, COX-2 positivity was found in 4 out of 8 (50%) of un responsive versus 8 out of 21 (38.1%) of responsive cases. Logistic regression analysis of parameters likely to affect response to treatment resulted in a p value = 0.17 for the interaction COX-2/type of treatment.ConclusionAlthough these findings need to be confirmed in a larger series, our study suggests a possible indication that there is a difference in the influence of COX-2 on response depending on treatment regimen.