Mitochondrial Targeting of Adenomatous Polyposis Coli Protein Is Stimulated by Truncating Cancer Mutations

Abstract

The adenomatous polyposis coli (APC) protein tumor suppressor is mutated in the majority of colon cancers. Most APC gene mutations cause deletion of the C terminus and disrupt APC regulation of ␤-catenin turnover, microtubule dynamics, and chromosome segregation. Truncated APC mutant peptides may also gain unique properties, not exhibited by wild-type APC, which contribute to tumor cell survival and proliferation. Here we report a differential subcellular localization pattern for wild-type and mutant APC. A pool of APC truncation mutants was detected at mitochondria by cellular fractionation and confocal microscopy. In contrast, wild-type APC located poorly at mitochondria. Similar results were observed for endogenous and stably induced forms of APC, with the shortest N-terminal mutant peptides (N750, N853, N1309, N1337) displaying the strongest mitochondrial staining. The knock down of mutant APC(N1337) in SW480 tumor cells caused an increase in apoptosis and mitochondrial membrane permeability, and this correlated with reduced Bcl-2 protein levels in mitochondrial fractions. Interestingly, the silencing of APC did not alter expression of ␤-catenin or the apoptotic regulatory factors Bax, Bcl-xL, or survivin. APC formed a complex with Bcl-2 in mitochondrial fractions, and this may contribute to the APC-dependent regulation of Bcl-2. We propose that a subset of cancer mutations induce APC mitochondrial localization and that APC regulation of Bcl-2 at mitochondria may contribute to tumor cell survival. Mutations in the adenomatous polyposis coli (APC)4 tumor suppressor gene contribute to the pathogenesis of the benign polyp syndrome, familial adenomatous polyposis (FAP), and are responsible for the majority (Ͼ80%) of sporadic cases of colon cancer (1-3). Most APC gene mutations are detected within the central mutation cluster region (MCR) and are early events in the tumorigenic process. The mutations generate truncated APC peptides that lack a C terminus, causing loss of binding to several partners and to microtubules (4). APC is a large protein comprising 2843 amino acids, and interacts with proteins involved in the Wnt signaling pathway and cytoskeletal organization (5). Many APC mutations disrupt APC-dependent ␤-catenin turnover, causing oncogenic ␤-catenin to accumulate in the nucleus and activate transcription of genes that promote cell transformation, leading to tumor formation (1, 6). APC is multifunctional and localizes to several subcellular compartments (4). APC can translocate into and out of the nucleus (7), and in the cytoplasm APC accumulates at the ends of microtubule (MT) bundles in cortical clusters near the plasma membrane (8, 9). APC has also been detected at the mitotic spindle (10, 11), centrosomes (12), and at actin-dependent membrane regions (13,14).Truncating cancer mutations alter APC retention at microtubules and its movement to membrane clusters (9, 15), and disrupt several processes including regulation of ␤-catenin degradation and chromosome stability (3,4,6). APC mutants retain the a...

The microtubule poison vinorelbine kills cells independently of mitotic arrest and targets cells lacking the APC tumour suppressor more effectively

“…In HCT116 and SW480 cells, the increased killing is accompanied by increased BIM at mitochondria (data not shown). These data suggest that domains in N-terminal regions of APC directly contribute to events at mitochondria that affect apoptosis, consistent with the previous findings that N-terminal fragments of APC localize to mitochondria in tumour cells (Brocardo et al, 2008). These data, together with the increased sensitivity of colorectal tumour cells to vinorelbine, also suggest that changes in b-catenin or Wnt signalling are not required for the sensitivity to vinorelbine.…”

“…Some p53 and APC protein co-fractionated with mitochondria, suggesting that their anti-apoptotic function might involve mitochondrial processes (Brocardo et al, 2008;Marchenko et al, 2000). To examine the relationship between the changes induced by microtubule poisons and the mitochondrial recruitment of known apoptotic regulators, we chose to investigate BIM in this context (Figs 3, 4).…”

APC as a mobile scaffold: Regulation and function at the nucleus, centrosomes, and mitochondria

“…In particular, the Arm domain of APC was linked to apoptosis, although whether its accumulation triggers or prevents apoptosis is not clear (47). The survival activity might be partly attributed to modest regulation of b-catenin transcription activity (31,48); however, there is also evidence for b-catenin independent mechanisms (45,47,49). We identified APC at mitochondria and reported a novel gain of localization by APC cancer mutants, whose mitochondrial targeting was far more pronounced than the wild-type protein (49).…”

Dérèglements cellulaires et moléculaires à l’origine du phénotype métastatique

“…Aucun gène candidat n'a été clairement associé à cette délétion située à proximité du gène MSR1 bien que le gène FGF20, situé à 0,5 Mb, est associé à un plus haut risque de mauvais pronostic ou qu'un cluster de gènes impliqués dans l'apoptose a été proposé pour expliquer la variabilité du siège des mutations observées. Dans les deux hypothèses, dont la dernière paraît séduisante, les voies morphogéniques et leur modulation par APC seraient impliquées [14]. La perte du 8p est significativement associée à un gain du 8q, focalisée sur la région contenant le gène MYC [13].…”

“…L'absence de valeur pronostique indépendante du 8q s'explique par le lien étroit entre APC (gène effecteur) et MYC (gène cible) dans la voie Wnt. Notre hypothèse est qu'une coopération entre les altérations du 8q et du 5q optimise l'effet dose de la protéine Myc, essentielle dans la régulation de l'autonomie des cellules souches et l'apoptose, expliquant son haut pouvoir de transformation en cellule souche cancéreuse en cas de surdosage [10,14].…”