Purpose Continuous chromosome missegregation over successive mitotic divisions, known as chromosomal instability (CIN), is common in cancer. Though it has been associated with treatment resistance and poor prognosis, increasing CIN above a maximally tolerated threshold leads to cell death because of loss of essential chromosomes. Because radiation causes CIN, we hypothesize that pre-existing CIN sensitizes tumor cells to radiation therapy. Methods and Materials We induced mitotic defects that lead to CIN in FaDu (head and neck cancer, HNC) and HeLa (cervical) cells by knocking down or overexpressing the mitotic checkpoint protein mitotic arrest deficient 1 (Mad1), which induces lagging chromosomes. Radiation sensitivity was tested with clonogenic assays in vitro and tumor regression in patient-derived xenografts in vivo. MTT assays were used to determine the sensitivity of human papillomavirus (HPV) positive and HPV-negative HNC cells to docetaxel, and mitotic defects were quantified using immunofluorescence microscopy. Docetaxel-induced mitotic errors and tumor growth delay were evaluated in vivo. Six-chromosome fluorescence in situ hybridization was used to quantify CIN in a cohort of patients with laryngeal cancer treated with definitive radiation. Results Here, we show in two tissue contexts using engineered isogenic cancer cell lines that higher rates of chromosome missegregation sensitize to ionizing radiation, which itself induces mitotic errors. Consistent with this result, higher rates of anaphase defects in HPV-positive and HPV-negative HNC patient-derived xenograft tumors correlate with response to radiation. Moreover, laryngeal tumors with higher CIN before treatment tend to have an improved response to radiation therapy in the clinic. Furthermore, we show that docetaxel, a microtubule-stabilizing drug commonly used in combination with radiation, causes cell death and radiosensitizes cells by inducing abnormal multipolar spindles rather than causing mitotic arrest. Conclusions These results mechanistically implicate CIN as an inducer of radiation response and provide evidence that increasing the rate of CIN is a rational method to enhance radiation sensitivity, which has significant implications for personalized therapy.

Continuous chromosome missegregation over successive mitotic divisions, known as chromosomal instability (CIN), is common in cancer. Increasing CIN above a maximally tolerated threshold leads to cell death due to loss of essential chromosomes. Here, we show in two tissue contexts that otherwise isogenic cancer cells with higher levels of CIN are more sensitive to ionizing radiation, which itself induces CIN. CIN also sensitizes HPV-positive and HPV-negative head and neck cancer patient derived xenograft (PDX) tumors to radiation. Moreover, laryngeal cancers with higher CIN prior to treatment show improved response to radiation therapy. In addition, we reveal a novel mechanism of radiosensitization by docetaxel, a microtubule stabilizing drug commonly used in combination with radiation. Docetaxel causes cell death by inducing CIN due to abnormal multipolar spindles rather than causing mitotic arrest, as previously assumed. Docetaxel-induced CIN, rather than mitotic arrest, is responsible for the enhanced radiation sensitivity observed in vitro and in vivo, challenging the mechanistic dogma of the last 40 years. These results implicate CIN as a potential biomarker and inducer of radiation response, which could provide valuable cancer therapeutic opportunities. Statement of Significance:Cancer cells and laryngeal tumors with higher chromosome missegregation rates are more sensitive to radiation therapy, supporting chromosomal instability as a promising biomarker of radiation response.

Mitotic Arrest Deficient 1 (gene name MAD1L1), an essential component of the mitotic spindle assembly checkpoint, is frequently overexpressed in colon cancer, which correlates with poor disease-free survival. MAD1 upregulation induces two phenotypes associated with tumor promotion in tissue culture cells-low rates of chromosomal instability (CIN) and destabilization of the tumor suppressor p53. Using CRISPR/Cas9 gene editing, we generated a novel mouse model by inserting a doxycycline (dox)-inducible promoter and HA tag into the endogenous mouse Mad1l1 gene, enabling inducible expression of HA-MAD1 following exposure to dox in the presence of the reverse tet transactivator (rtTA). A modest 2-fold overexpression of MAD1 in murine colon resulted in decreased p53 expression and increased mitotic defects consistent with CIN. After exposure to the colon-specific inflammatory agent dextran sulfate sodium (DSS), 31% of mice developed colon lesions, including a mucinous adenocarcinoma, while none formed in control animals. Lesion incidence was particularly high in male mice, 57% of which developed at least one hyperplastic polyp, adenoma or adenocarcinoma in the colon. Notably, mice expressing HA-MAD1 also developed lesions in tissues in which DSS is not expected to induce inflammation. These findings demonstrate that MAD1 upregulation is sufficient to promote colon tumorigenesis in the context of inflammation in immune-competent mice.

This file contains 5 supplemental figures and 1 supplemental table

Chromosome segregation during mitosis is highly regulated to ensure production of genetically identical progeny. Recurrent mitotic errors cause chromosomal instability (CIN), a hallmark of tumors. The E6 and E7 oncoproteins of high-risk human papillomavirus (HPV), which causes cervical, anal, and head and neck cancers (HNC), cause mitotic defects consistent with CIN in models of anogenital cancers, but this has not been studied in the context of HNC. Here, we show that HPV16 induces a specific type of CIN in patient HNC tumors, patient-derived xenografts, and cell lines, which is due to defects in chromosome congression. These defects are specifically induced by the HPV16 oncogene E6 rather than E7. We show that HPV16 E6 expression causes degradation of the mitotic kinesin CENP-E, whose depletion produces chromosomes that are chronically misaligned near spindle poles (polar chromosomes) and fail to congress. Though the canonical oncogenic role of E6 is the degradation of the tumor suppressor p53, CENP-E degradation and polar chromosomes occur independently of p53. Instead, E6 directs CENP-E degradation in a proteasome-dependent manner via the E6-associated ubiquitin protein ligase E6AP/UBE3A. This study reveals a mechanism by which HPV induces CIN, which may impact HPV-mediated tumor initiation, progression, and therapeutic response.

Table S1 shows expected and observed birth frequencies.

Figure S3 shows that reduction of CENP-E extends tumor latency only in tumors where it causes high CIN.

Increased Aurora B protein expression, which is common in cancers, is expected to increase Aurora B kinase activity, yielding elevated phosphorylation of Aurora B substrates. In contrast, here we show that elevated expression of Aurora B reduces phosphorylation of six different Aurora B substrates across three species and causes defects consistent with Aurora B inhibition. Complexes of Aurora B and its binding partner INCENP autophosphorylate in trans to achieve full Aurora B activation. Increased expression of Aurora B mislocalizes INCENP, reducing the local concentration of Aurora B:INCENP complexes at the inner centromere/kinetochore. Co-expression of INCENP rescues Aurora B kinase activity and mitotic defects caused by elevated Aurora B. However, INCENP expression is not elevated in concert with Aurora B in breast cancer, and increased expression of Aurora B causes resistance rather than hypersensitivity to Aurora B inhibitors. Thus, increased Aurora B expression reduces, rather than increases, Aurora B kinase activity.

Spontaneous exocytosis of single synaptic vesicles generates miniature synaptic currents, which provide a window into the dynamic control of synaptic transmission. To resolve the impact of different factors on the dynamics and variability of synaptic transmission, we recorded miniature excitatory postsynaptic currents (mEPSCs) from cocultures of mouse hippocampal neurons with HEK cells expressing the postsynaptic proteins GluA2, neuroligin 1, PSD-95, and stargazin. Synapses between neurons and these heterologous cells have a molecularly defined postsynaptic apparatus, while the compact morphology of HEK cells eliminates the distorting effect of dendritic filtering. HEK cells in coculture produced mEPSCs with a higher frequency, larger amplitude, and more rapid rise and decay than neurons from the same culture. However, mEPSC area indicated that nerve terminals in synapses with both neurons and HEK cells release similar populations of vesicles. Modulation by the glutamate receptor ligand aniracetam revealed receptor contributions to mEPSC shape. Dendritic cable effects account for the slower mEPSC rise in neurons, whereas the slower decay also depends on other factors. Lastly, expression of synaptobrevin transmembrane domain mutants in neurons slowed the rise of HEK cell mEPSCs, thus revealing the impact of synaptic fusion pores. In summary, we show that cocultures of neurons with heterologous cells provide a geometrically simplified and molecularly defined system to investigate the time course of synaptic transmission and to resolve the contribution of vesicles, fusion pores, dendrites, and receptors to this process.

Abstract Chromosomal instability (CIN) is a hallmark of cancer. While low levels of CIN can be tumor promoting, high levels of CIN cause cell death and tumor suppression. The widely used chemotherapeutic, paclitaxel (Taxol), exerts its anticancer effects by increasing CIN above a maximally tolerated threshold. One significant outstanding question is whether the p53 tumor suppressor is required for the cell death and tumor suppression caused by high CIN. Both p53 loss and reduction of the mitotic kinesin, centromere-associated protein-E, cause low CIN. Combining both genetic insults in the same cell leads to high CIN. Here, we test whether high CIN causes cell death and tumor suppression even in the absence p53. Despite a surprising sex-specific difference in tumor spectrum and latency in p53 heterozygous animals, these studies demonstrate that p53 is not required for high CIN to induce tumor suppression. Pharmacologic induction of high CIN results in equivalent levels of cell death due to loss of essential chromosomes in p53+/+ and p53−/− cells, further demonstrating that high CIN elicits cell death independently of p53 function. Implications: These results provide support for the efficacy of anticancer therapies that induce high CIN, even in tumors that lack functional p53.

Paclitaxel (Taxol) is a cornerstone of cancer treatment. However, its mechanism of cytotoxicity is incompletely understood and not all patients benefit from treatment. We show that patients with breast cancer did not accumulate sufficient intratumoral paclitaxel to induce mitotic arrest in tumor cells. Instead, clinically relevant concentrations induced multipolar mitotic spindle formation. However, the extent of early multipolarity did not predict patient response. Whereas multipolar divisions frequently led to cell death, multipolar spindles focused into bipolar spindles before division at variable frequency, and maintaining multipolarity throughout mitosis was critical to induce the high rates of chromosomal instability necessary for paclitaxel to elicit cell death. Increasing multipolar divisions in paclitaxel resulted in improved cytotoxicity. Conversely, decreasing paclitaxel-induced multipolar divisions reduced paclitaxel efficacy. Moreover, we found that preexisting chromosomal instability sensitized breast cancer cells to paclitaxel. Both genetic and pharmacological methods of inducing chromosomal instability were sufficient to increase paclitaxel efficacy. In patients, the amount of pretreatment chromosomal instability directly correlated with taxane response in metastatic breast cancer such that patients with a higher rate of preexisting chromosomal instability showed improved response to taxanes. Together, these results support the use of baseline rates of chromosomal instability as a predictive biomarker for paclitaxel response. Furthermore, they suggest that agents that increase chromosomal instability or maintain multipolar spindles throughout mitosis will improve the clinical utility of paclitaxel.

Chromosome instability (CIN) generates genetic and karyotypic diversity that is common in hematological malignancies. Low to moderate levels of CIN are well tolerated and can promote cancer proliferation. However, high levels of CIN are lethal. Thus, CIN may serve both as a prognostic factor to predict clinical outcome and as a predictive biomarker. A retrospective study was performed to evaluate CIN in acute myeloid leukemia (AML). Chromosome mis-segregation frequency was correlated with clinical outcome in bone marrow core biopsy specimens from 17 AML cases. Additionally, we induced chromosome segregation errors in AML cell lines with AZ3146, an inhibitor of the Mps1 mitotic checkpoint kinase, to quantify the phenotypic effects of high CIN. We observed a broad distribution of chromosome mis-segregation frequency in AML bone marrow core specimens. High CIN correlated with complex karyotype in AML, as expected, although there was no clear survival effect. In addition to CIN, experimentally inducing chromosome segregation errors by Mps1 inhibition in AML cell lines causes DNA damage, micronuclei formation, and upregulation of interferon stimulated genes. High levels of CIN appear to be immunostimulatory, suggesting an opportunity to combine mitotic checkpoint inhibitors with immunotherapy in treatment of AML.

Polo-like kinase 1 has hundreds of substrates and multiple functions that operate within the ∼60 min of mitosis. Herein, we describe a chemical-genetic system that allows particular substrates to be "toggled" into or out of chemical control using engineered phosphoacceptor selectivity. Biochemical assays and phosphoproteomic analysis of mitotic cell extracts showed that Plk1S (L197F) and Plk1T (L197S/L211A) selectively phosphorylate Ser and Thr, respectively. Plk1S but not Plk1T sustains mitotic progression to anaphase, affording the opportunity to toggle substrate residues between Ser and Thr to place them under chemical control. Using this system, we evaluated Kif2b, a known substrate of Plk1 that regulates chromosome alignment. Toggling Ser to Thr on Kif2b places these phosphorylation sites under reversible chemical control, as indicated by a sharp increase in the frequency of misaligned chromosomes and prometaphase arrest. Thus, we demonstrate the ability to chemically control a single substrate by a genetic Ser/Thr toggle.

Mitotic arrest deficient 1 (Mad1) plays a well-characterized role in the mitotic checkpoint. However, interphase roles of Mad1 that do not impact mitotic checkpoint function remain largely uncharacterized. Here we show that upregulation of Mad1, which is common in human breast cancer, prevents stress-induced stabilization of the tumor suppressor p53 in multiple cell types. Upregulated Mad1 localizes to ProMyelocytic Leukemia (PML) nuclear bodies in breast cancer and cultured cells. The C-terminus of Mad1 directly interacts with PML, and this interaction is enhanced by sumoylation. PML stabilizes p53 by sequestering MDM2, an E3 ubiquitin ligase that targets p53 for degradation, to the nucleolus. Upregulated Mad1 displaces MDM2 from PML, freeing it to ubiquitinate p53. Upregulation of Mad1 accelerates growth of orthotopic mammary tumors, which show decreased levels of p53 and its downstream effector p21. These results demonstrate an unexpected interphase role for Mad1 in tumor promotion via p53 destabilization.

Abstract Mitotic arrest deficient 1 (Mad1) plays a well-characterized role in the mitotic checkpoint. However, interphase roles of Mad1 that do not impact mitotic checkpoint function are still largely uncharacterized. We have found that upregulation of Mad1, which is common in human breast cancer, results in decreased protein stability of the tumor suppressor p53 in multiple cell types. To gain mechanistic insight into this process, we first determined whether increasing expression of Mad1 alters its localization. Upregulated Mad1 localizes to puncta within interphase nuclei in both breast cancer tissue and cultured cells. We found that upregulated Mad1 localizes to ProMyelocytic Leukemia Nuclear Bodies (PML NBs), which have been implicated in the stabilization of p53. Immunoprecipitation results indicate that Mad1 and PML interact and that the C-terminal domain (CTD) of Mad1 and the N-terminal domain of PML are required for this interaction. Within the CTD of Mad1, the SUMO-Interacting Motif (SIM) is critical for the localization of Mad1 to PML NBs. MDM2 is an E3 ubiquitin ligase that targets p53 for degradation. In response to DNA damage, PML sequesters Mdm2 to the nucleolus, which stabilizes p53. In cells with elevated levels of Mad1, Mad1 interrupts the interaction between PML and MDM2. Mad1, rather than MDM2, is sequestered to the nucleolus after DNA damage, and Mad1 displaces MDM2 from PML. The displaced MDM2 is no longer sequestered in the nucleolus, freeing it to ubiquitinate p53, resulting in p53 destabilization. Upregulation of Mad1 promotes the growth of orthotopic mammary tumors. Mammary tumors with upregulated Mad1 show decreased levels of p53 and its downstream effector p21. Our results show that, in addition to causing a low rate of chromosome missegregation, Mad1 upregulation has an unexpected interphase role in promoting tumor formation and progression by destabilizing p53. Thus, Mad1 upregulation promotes tumors in through at least two distinct pathways. Citation Format: Jun Wan, Beth A. Weaver. Mad1 promotes tumor progression through destabilization of p53 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4290.

Activation of I kappa B kinase (IKK) and NF-kappa B by genotoxic stresses modulates apoptotic responses and production of inflammatory mediators, thereby contributing to therapy resistance and premature aging. We previously reported that genotoxic agents induce nuclear localization of NF-kappa B essential modulator (NEMO) via an undefined mechanism to arbitrate subsequent DNA damage-dependent IKK/NF-kappa B signaling. Here we show that a nonclassical nuclear import pathway via IPO3 (importin 3, transportin 2) mediates stress-induced NEMO nuclear translocation. We found putative nuclear localization signals in NEMO whose mutations disrupted stress-inducible nuclear translocation of NEMO and IKK/NF-kappa B activation in stably reconstituted NEMO-deficient cells. RNAi screening of both importin alpha and beta family members, as well as co-immunoprecipitation analyses, revealed that a nonclassical importin beta family member, IPO3, was the only importin that was able to associate with NEMO and whose reduced expression prevented genotoxic stress-induced NEMO nuclear translocation, IKK/NF-kappa B activation, and inflammatory cytokine transcription. Recombinant IPO3 interacted with recombinant NEMO but not the nuclear localization signal mutant version and induced nuclear import of NEMO in digitonin-permeabilized cells. We also provide evidence that NEMO is disengaged from IKK complex following genotoxic stress induction. Thus, the IPO3 nuclear import pathway is an early and crucial determinant of the IKK/NF-kappa B signaling arm of the mammalian DNA damage response.

It is often overlooked that genes that play well-characterized, essential roles during one stage of the cell cycle may also perform completely unrelated, but still critical functions during other cell cycle stages. Alternate functions of well-studied proteins are often difficult to define because they are outside the area of expertise of laboratories that focus on pathways and processes in which the protein serves its canonical role. However, a full understanding of the roles a protein plays throughout the cell cycle is critical for accurate interpretation of loss of function and overexpression studies that occur over multiple cell cycles, particularly experiments in whole animals during development and aging. Mitotic arrest deficient 1 (Mad1) was first identified as one of the genes essential for the mitotic checkpoint (also known as the spindle assembly checkpoint), which prevents chromosome missegregation and aneuploidy by delaying anaphase onset until all chromosomes have made stable attachments to spindle microtubules. We have focused our attention on this protein because overexpression of Mad1 is common in tumors and is a marker of poor prognosis.1 The function of Mad1 during mitosis has been well studied and is largely dependent on its association with another mitotic checkpoint component, Mad2. Mad1 accumulates at kinetochores on unattached chromosomes that have not yet made stable attachments to spindle microtubules and would therefore be randomly segregated if the cells entered anaphase. At unattached kinetochores, Mad1 recruits and converts Mad2 from an inactive, open form into an active, closed form that inhibits the Anaphase Promoting Complex/Cyclosome (APC/C) bound to its specificity factor Cdc20.2 Although the function of Mad1 in mitosis has been well studied, Mad1 is expressed throughout the cell cycle and its protein levels do not exhibit cell cycle regulation.1 Previous evidence indicated that Mad1 interacts with Mad2 throughout the cell cycle.2 In interphase, both Mad1 and Mad2 are associated with the nuclear pore complex. Nuclear pore binding stabilizes both proteins and helps to scaffold production of APC/C-Cdc20 inhibitors during interphase, which delays activation of APC/C-Cdc20 in mitosis.3 It remains unclear whether nuclear pore-associated pools of Mad1 and Mad2 perform functional roles during interphase in vertebrates. Recently we identified an unsuspected Golgi-localized pool of Mad1 (Fig. 1).4 Golgi localization of Mad1 was confirmed by immunofluorescence experiments and cell fractionation. The perinuclear Mad1 signal dispersed after treatment with the microtubule poison vinblastine or with an inhibitor of protein trafficking, Brefeldin A, both of which cause disassembly of the Golgi. Transient and stable depletion of Mad1 removed the Golgi localized pool. Interestingly, unlike kinetochore and nuclear pore bound pools of Mad1, Golgi associated Mad1 is independent of Mad2 (Fig. 1). Figure 1. Mad1 localizes to the Golgi where it regulates secretion of α5 integrin and cell migration. (Left) Unlike Mad1 localization to the nucleus and nuclear envelope, the localization of Mad1 on the Golgi is independent of Mad2. Golgi-associated Mad1 ... To determine whether Mad1 functions in secretion at the Golgi, we generated several cell lines in which Mad1 expression was stably knocked down (Mad1-KD cells). Previous studies have identified the proteins required for global secretion, which did not include Mad1.5 Consistent with this, we found that the depletion of Mad1 did not affect secretion of VSVG or EGFR. However, testing of a variety of additional secretory proteins revealed that Mad1 knockdown results in impaired secretion of α5 integrin. In complex with β1 integrin, α5 integrin serves as a key molecule on the plasma membrane to anchor cells to the extracellular matrix (ECM) component fibronectin. In Mad1-KD cells, the α5 integrin subunit was enriched in the Golgi and showed less accumulation at the cell surface than in wild type cells (Fig. 1). The defects in α5 integrin secretion suggested that Mad1-KD cells exhibit impaired cellular adhesion and migration on fibronectin. Consistent with this, fewer Mad1-KD cells adhered to and spread on fibronectin coated plates compared to wild type cells. Mad1-KD cells also exhibited impaired migration on fibronectin in cell culture wounding and transwell migration assays.4 These effects were not due to decreased proliferation, and were also apparent in single cell migration tracking assays. Overexpression of Mad1 enhanced migration on fibronectin, further supporting a role for Mad1 in secretion of α5 integrin. Notably, cells depleted of Mad2 did not show defects in secretion of α5 integrin or spreading on fibronectin.4 In the future, it will be important to gain a mechanistic understanding of Mad1 localization to the Golgi and its regulation of integrin secretion. It will also be of interest to determine whether Mad1 upregulation, which frequently occurs in human breast cancer, potentiates cellular migration and metastasis. Since Mad1 plays a well-established role in the mitotic checkpoint, mouse models expressing altered levels of Mad1 (and multiple other mitotic checkpoint genes) have been used to assess the effects of chromosome missegregation and aneuploidy on tumorigenesis. Our data demonstrating a role for Mad1 in secretion and cellular motility highlight the fact that proteins that are present throughout the cell cycle have the potential to participate in additional functions that may influence tumor phenotype. With respect to aneuploidy, certain animals with mitotic checkpoint defects are tumor prone, others are not, and some actually exhibit fewer tumors than controls, depending on the specific genetic mutations involved.6,7 This highlights the more general caveat that unknown functions during other stages of the cell cycle may have a significant impact on phenotypic outcomes of targeted genetic manipulations.

Jun Wan,1,2 Fen Zhu,1 Lauren M. Zasadil,1,3 Jiaquan Yu,4 Lei Wang,5 Adam Johnson,5 Erwin Berthier,4 David J. Beebe,4,6 Anjon Audhya,5,6 and Beth A. Weaver1,6,* 1Department of Cell and Regenerative Biology 2Physiology Training Program 3Molecular and Cellular Pharmacology Training Program 4Department of Biomedical Engineering 5Department of Biomolecular Chemistry 6Carbone Cancer Center University of Wisconsin, Madison, WI 53705, USA

The ARF tumor suppressor is part of the CDKN2A locus and is mutated or undetectable in numerous cancers. The best-characterized role for ARF is in stabilizing p53 in response to cellular stress. However, ARF has tumor suppressive functions outside this pathway that have not been fully defined. Primary mouse embryonic fibroblasts (MEFs) lacking the ARF tumor suppressor contain abnormal numbers of chromosomes. However, no role for ARF in cell division has previously been proposed. Here we demonstrate a novel, p53-independent role for ARF in the mitotic checkpoint. Consistent with this, loss of ARF results in aneuploidy in vitro and in vivo. ARF(-/-) MEFs exhibit mitotic defects including misaligned and lagging chromosomes, multipolar spindles, and increased tetraploidy. ARF(-/-) cells exhibit overexpression of Mad2, BubR1, and Aurora B, but only overexpression of Aurora B phenocopies mitotic defects observed in ARF(-/-) MEFs. Restoring Aurora B to near-normal levels rescues mitotic phenotypes in cells lacking ARF. Our results define an unexpected role for ARF in chromosome segregation and mitotic checkpoint function. They further establish maintenance of chromosomal stability as one of the additional tumor-suppressive functions of ARF and offer a molecular explanation for the common up-regulation of Aurora B in human cancers.

Mitotic arrest deficient 1 (Mad1) plays a well-characterized role in the major cell-cycle checkpoint that regulates chromosome segregation during mitosis, the mitotic checkpoint (also known as the spindle assembly checkpoint). During mitosis, Mad1 recruits Mad2 to unattached kinetochores [1, 2], where Mad2 is converted into an inhibitor of the anaphase-promoting complex/cyclosome bound to its specificity factor, Cdc20 [1, 3-6]. During interphase, Mad1 remains tightly bound to Mad2 [2, 3, 7, 8], and both proteins localize to the nucleus and nuclear pores [9, 10], where they interact with Tpr (translocated promoter region). Recently, it has been shown that interaction with Tpr stabilizes both proteins [11] and that Mad1 binding to Tpr permits Mad2 to associate with Cdc20 [12]. However, interphase functions of Mad1 that do not directly affect the mitotic checkpoint have remained largely undefined. Here we identify a previously unrecognized interphase distribution of Mad1 at the Golgi apparatus. Mad1 colocalizes with multiple Golgi markers and cosediments with Golgi membranes. Although Mad1 has previously been thought to constitutively bind Mad2, Golgi-associated Mad1 is Mad2 independent. Depletion of Mad1 impairs secretion of alpha 5 integrin and results in defects in cellular attachment, adhesion, and FAK activation. Additionally, reduction of Mad1 impedes cell motility, while its overexpression accelerates directed cell migration. These results reveal an unexpected role for a mitotic checkpoint protein in secretion, adhesion, and motility. More generally, they demonstrate that, in addition to generating aneuploidy, manipulation of mitotic checkpoint genes can have unexpected interphase effects that influence tumor phenotypes.