Mechanisms Promoting Escape from Mitotic Stress-Induced Tumor Cell Death

Non-small cell lung cancer (NSCLC) is notorious for its paltry responses to first-line therapeutic regimens. In contrast to acquired chemoresistance, little is known about the molecular underpinnings of the intrinsic resistance of chemo-naïve NSCLC. Here we report that intrinsic resistance to paclitaxel in NSCLC occurs at a cell-autonomous level due to the uncoupling of mitotic defects from apoptosis. To identify components that permit escape from mitotic stress-induced death, we employed a genome-wide RNAi-based strategy, which combines a high-throughput toxicity screen with a live-cell imaging platform to measure mitotic fate. This strategy revealed that prolonging mitotic arrest with a small molecule inhibitor of the APC/Cyclosome could sensitize otherwise paclitaxel-resistant NSCLC. We also defined novel roles for CASC1 and TRIM69 in supporting resistance to spindle poisons. CASC1, which is frequently co-amplified with KRAS in lung tumors, is essential for microtubule polymerization and satisfaction of the spindle assembly checkpoint. TRIM69, which associates with spindle poles and promotes centrosomal clustering, is essential for formation of a bipolar spindle. Notably, RNAi-mediated attenuation of CASC1 or TRIM69 was sufficient to inhibit tumor growth in vivo. On the basis of our results, we hypothesize that tumor evolution selects for a permissive mitotic checkpoint, which may promote survival despite chromosome segregation errors. Attacking this adaptation may restore the apoptotic consequences of mitotic damage to permit the therapeutic eradication of drug-resistant cancer cells

CUL9 Mediates the Functions of the 3M Complex and Ubiquitylates Survivin to Maintain Genome Integrity

The Cullin 9 (CUL9) gene encodes a putative E3 ligase that localizes in the cytoplasm. Cul9 null mice develop spontaneous tumors in multiple organs, however either the cellular or molecular mechanisms of CUL9 in tumor suppression are currently not known. We show here that deletion of Cul9 leads to abnormal nuclear morphology, increased DNA damage and aneuploidy. CUL9 knockdown rescues the microtubule and mitosis defects in cells depleted for CUL7 or OBSL1, two genes that are mutated in a mutually exclusive manner in 3M growth retardation syndrome and function in microtubule dynamics. CUL9 promotes the ubiquitylation and degradation of survivin and is inhibited by CUL7. Depletion of CUL7 decreases survivin level and overexpression of survivin rescues the defects caused by CUL7 depletion. We propose a 3M–CUL9-survivin pathway in maintaining microtubule and genome integrity, normal development and tumor suppression

Tumor Antigen Acrosin Binding Protein Normalizes Mitotic Spindle Function to Promote Cancer Cell Proliferation

Cancer cells manage to divide in the context of gross chromosomal abnormalities. These abnormalities can promote bypass of normal restraints on cell proliferation, but at a cost of mitotic vulnerabilities that can be attacked by chemotherapy. Determining how cancer cells balance these issues may permit chemotherapeutic sensitivity to be leveraged more efficiently. From a pan-genomic siRNA screen for modifiers of chemoresponsiveness, we identified the tumor antigen ACRBP/OY-TES-1 as a specifier of paclitaxel resistance. ACRBP expression is normally restricted to the testes but detected in a wide variety of cancers, including most ovarian cancers. We found that ACRBP is both necessary and sufficient for paclitaxel resistance in ovarian cancer cell lines and ovarian tumor explants. Moreover, high ACRBP expression correlated with reduced survival time and faster relapse among ovarian cancer patients. We identified the mitotic spindle protein NuMA as an ACRBP-interacting protein that could account for the effects of ACRBP on paclitaxel sensitivity. In cancer cells, ACRBP restricted a NuMA-dependent abrogation of mitotic spindle assembly that is otherwise pathologic. As a consequence, ACRBP depletion resulted in mitotic errors and reduced proliferative fitness that could be rescued by NuMA co-depletion. We propose that the co-dependent relationship of ACRBP and NuMA in cancer cells reflects their passage through a selection bottleneck during tumor evolution, one which requires the acquisition of traits which normalize mitotic perturbations that originally drove the plasticity of a pre-neoplastic genome. The molecular definition of such traits as defined by the ACRBP-NuMA complex may represent conceptually ideal intervention targets, based on the wide therapeutic windows they may offer

The 3M Complex Maintains Microtubule and Genome Integrity

CUL7, OBSL1, and CCDC8 genes are mutated in a mutually exclusive manner in 3M and other growth retardation syndromes. The mechanism underlying the function of the three 3M genes in development is not known. We found that OBSL1 and CCDC8 form a complex with CUL7 and regulate the level and centrosomal localization of CUL7, respectively. CUL7 depletion results in altered microtubule dynamics, prometaphase arrest, tetraploidy and mitotic cell death. These defects are recaptured in CUL7 mutated 3M cells and can be rescued by wild-type, but not 3M patients-derived CUL7 mutants. Depletion of either OBSL1 or CCDC8 results in similar defects and sensitizes cells to microtubule damage as loss of CUL7 function. Microtubule damage reduces the level of CCDC8 that is required for the centrosomal localization of CUL7. We propose that CUL7, OBSL1, and CCDC8 proteins form a 3M complex that functions in maintaining microtubule and genome integrity and normal development

Symplekin Specifies Mitotic Fidelity by Supporting Microtubule Dynamics ▿ †

Using a pangenomic loss-of-function screening strategy, we have previously identified 76 potent modulators of paclitaxel responsiveness in non-small-cell lung cancer. The top hit isolated from this screen, symplekin, is a well-established component of the mRNA polyadenylation machinery. Here, we performed a high-resolution phenotypic analysis to reveal the mechanistic underpinnings by which symplekin depletion collaborates with paclitaxel. We find that symplekin supports faithful mitosis by contributing to the formation of a bipolar spindle apparatus. Depletion of symplekin attenuates microtubule polymerization activity as well as expression of the critical microtubule polymerization protein CKAP5 (TOGp). Depletion of additional members of the polyadenylation complex induces similar phenotypes, suggesting that polyadenylation machinery is intimately coupled to microtubule function and thus mitotic spindle formation. Importantly, tumor cells depleted of symplekin display reduced fecundity, but the mitotic defects that we observe are not evident in immortalized cells. These results demonstrate a critical connection between the polyadenylation machinery and mitosis and suggest that tumor cells have an enhanced dependency on these components for spindle assembly

Mechanisms Promoting Escape from Mitotic Stress–Induced Tumor Cell Death

Non-small cell lung cancer (NSCLC) is notorious for its paltry responses to first-line therapeutic regimens. In contrast to acquired chemoresistance, little is known about the molecular underpinnings of the intrinsic resistance of chemo-naïve NSCLC. Here we report that intrinsic resistance to paclitaxel in NSCLC occurs at a cell-autonomous level due to the uncoupling of mitotic defects from apoptosis. To identify components that permit escape from mitotic stress-induced death, we employed a genome-wide RNAi-based strategy, which combines a high-throughput toxicity screen with a live-cell imaging platform to measure mitotic fate. This strategy revealed that prolonging mitotic arrest with a small molecule inhibitor of the APC/Cyclosome could sensitize otherwise paclitaxel-resistant NSCLC. We also defined novel roles for CASC1 and TRIM69 in supporting resistance to spindle poisons. CASC1, which is frequently co-amplified with KRAS in lung tumors, is essential for microtubule polymerization and satisfaction of the spindle assembly checkpoint. TRIM69, which associates with spindle poles and promotes centrosomal clustering, is essential for formation of a bipolar spindle. Notably, RNAi-mediated attenuation of CASC1 or TRIM69 was sufficient to inhibit tumor growth in vivo. On the basis of our results, we hypothesize that tumor evolution selects for a permissive mitotic checkpoint, which may promote survival despite chromosome segregation errors. Attacking this adaptation may restore the apoptotic consequences of mitotic damage to permit the therapeutic eradication of drug-resistant cancer cells

CUL9 Mediates the Functions of the 3M Complex and Ubiquitylates Survivin to Maintain Genome Integrity

The Cullin 9 (CUL9) gene encodes a putative E3 ligase that localizes in the cytoplasm. Cul9 null mice develop spontaneous tumors in multiple organs, however either the cellular or molecular mechanisms of CUL9 in tumor suppression are currently not known. We show here that deletion of Cul9 leads to abnormal nuclear morphology, increased DNA damage and aneuploidy. CUL9 knockdown rescues the microtubule and mitosis defects in cells depleted for CUL7 or OBSL1, two genes that are mutated in a mutually exclusive manner in 3M growth retardation syndrome and function in microtubule dynamics. CUL9 promotes the ubiquitylation and degradation of survivin and is inhibited by CUL7. Depletion of CUL7 decreases survivin level and overexpression of survivin rescues the defects caused by CUL7 depletion. We propose a 3M–CUL9-survivin pathway in maintaining microtubule and genome integrity, normal development and tumor suppression

The 3M Complex Maintains Microtubule and Genome Integrity

CUL7, OBSL1, and CCDC8 genes are mutated in a mutually exclusive manner in 3M and other growth retardation syndromes. The mechanism underlying the function of the three 3M genes in development is not known. We found that OBSL1 and CCDC8 form a complex with CUL7 and regulate the level and centrosomal localization of CUL7, respectively. CUL7 depletion results in altered microtubule dynamics, prometaphase arrest, tetraploidy and mitotic cell death. These defects are recaptured in CUL7 mutated 3M cells and can be rescued by wild-type, but not 3M patients-derived CUL7 mutants. Depletion of either OBSL1 or CCDC8 results in similar defects and sensitizes cells to microtubule damage as loss of CUL7 function. Microtubule damage reduces the level of CCDC8 that is required for the centrosomal localization of CUL7. We propose that CUL7, OBSL1, and CCDC8 proteins form a 3M complex that functions in maintaining microtubule and genome integrity and normal development