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.

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Class III B-tubulin (TUBB3) overexpression has been reported in ovary, lung, breast, and gastric cancer patients. Currently, no clinical drugs are available for a specific targeting of TUBB3, whereas the investigational drug IDN5390 specifically interacts with TUBB3. To gain insight into the pathways leading to TUBB3 up-regulation, we did a human genome microarray analysis in A2780 cells made resistant to IDN5390 to identify selected pathways specifically disrupted in resistant cells. Using this approach, we discovered that semaphorin 6A (SEMA6A) is down-regulated not only in IDN5390-resistant cells but also in cells made resistant to cisplatin, topotecan, and doxorubicin, whereas no changes were noticed in paclitaxel-and gemcitabine-resistant cells. Acute treatment with IDN5390 was able to down-regulate SEMA6A in cells unselected for drug resistance. TUBB3 expression was assessed in A2780 clones with stable overexpression of SEMA6A and in a panel of clones in which silencing of the protein was obtained. Quantitative PCR was then used to check the modulation of SEMA6A as well as to assess the expression of TUBB3. TUBB3 was increased (median value, 5.4) and reduced (median value, 0.47) in cells with overexpression and silencing of SEMA6A, respectively. Thus, the findings indicate a correlation between the expression of SEMA6A and TUBB3. Then, we found that a form of 83 kDa of SEMA6A is expressed in the cytoskeleton in association with B-actin. These findings suggest for SEMA6A a novel function in the cytoskeleton and a role in modulating tubulin isotype composition and microtubule dynamics.

Hypoxia selects the most aggressive and drug-resistant clones in solid malignancies. One of the pivotal transcription factors induced by hypoxia is Hif-1α. However, in serous ovarian cancer (SEOC), Hif-1α expression is not a prognostic biomarker. This study aims to assess the hypothesis that the serine-threonine kinase Nek6 functions as a downstream effector cooperating with Hif-1α in driving ovarian cancer aggressiveness. Nek6 was overexpressed and Hif-1α was silenced in A2780 cells. Nek6 was also stably silenced in Hey cells. The dependence of Nek6 expression on Hif-1α was assayed as a function of hypoxic growth conditions. Nek6 interaction with the cytoskeletal gateway of drug resistance was investigated with far western blot. The co-expression of NEK6, HIF1A, TUBB3 and GBP1 transcripts was quantified with qPCR in two cohorts of SEOC patients (346 locally treated patients and 344 from the TCGA dataset). Nek6 expression is induced by hypoxia in a Hif-1α dependent fashion. Nek6 directly interacts with GBP-1, thus being a component of the cytoskeletal gateway of drug resistance. Nek6 overexpression increases and silencing decreases the anchorage-independent growth of cultured cells. In SEOC patients, NEK6 expression is significantly correlated with HIF1A. Co-expression of NEK6, HIF1A, TUBB3 and GBP1 transcripts identifies a subset of SEOC patients characterized by poor outcome and drug resistance. This study demonstrates the functional relevance of Nek6 in the context of the adaptive response to hypoxia in SEOC. This finding may help identify a sub-population of patients at high risk of relapse to standard first-line chemotherapy. Citation Format: Marta De Donato, Mara Fanelli, Marisa Mariani, Giuseppina Raspaglio, Deep Pandya, Shiquan He, Paul Fiedler, Marco Petrillo, Giovanni Scambia, Cristiano Ferlini. Nek6 and Hif-1α cooperate with the cytoskeletal gateway of drug resistance to drive outcome in serous ovarian cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4327. doi:10.1158/1538-7445.AM2015-4327