Since the centrosome organizes microtubules for proper cell division, centrosome number fidelity is critical for equal partitioning of chromosomes into each daughter cell [52,53]. Cells burdened with extra centrosomes will often form three or more spindle poles during mitoses in lieu of a bipolar spindle [15,16]. This organellar abnormality and microtubule disorganization can result in cytoarchitectural alterations in tissue, leading to loss of cellular differentiation (anaplasia) [54]. Furthermore, this erroneous multipolar mitosis can lead to unequal segregation of chromosomes into each daughter cell and subsequently intolerable levels of aneuploidy. To avoid this error, cells will undergo mitotic arrest or mitotic catastrophe and activate apoptotic or necrotic cell death machinery [55,56]. However, KIFC1, which is a minus-end-directed microtubule binding protein that is activated by genomic instability signals, associates with the plus ends of microtubules and translocates to the nucleus, where it crosslinks and slides microtubules in an antiparallel fashion [57,58]. This antiparallel sliding allows extra centrosomes to aggregate at opposite poles of the cells to still form a “pseudo-bipolar spindle”, which results in the occurrence of lagging chromosomes [13,59,60]. This “pseudo-bipolar” spindle can thus allow premalignant or pre-invasive cancer cells to avert mitotic arrest and cell death so they can continue replicating as genomically unstable cells. This phenomenon can increase the genetic diversity among the cellular population [61]. This phenotypic heterogeneity in lesions can promote the selection of viable cellular subclones with advantageous traits and precipitate the expansion of more aggressive cellular phenotypes [61,62]. Hence, KIFC1-mediated centrosome clustering may be a key factor driving the survival of cells in benign lesions that are more likely to transform to a malignant state and progress.

CA has been shown to increase in normal HMECs with continued population doublings [63,64]. As previously discussed, normal HMECs that have bypassed the stasis barrier exhibit significant deregulation of the universal regulators of cell cycle control, Rb or p53. Centrosomes normally duplicate in the S phase of the cell cycle [65]. Activated Rb represses the E2F1-mediated gene transcription required for G1 to S phase transition, and p53 can halt cell division in the G1 phase by inhibiting CDK activity [66]. Centrosome duplication is tightly coordinated with genome duplication and cell division [16]. Thus, loss of Rb and p53 function along with downstream p16 and p21 loss has been shown to generate supernumerary centrosomes [63,67,68,69]. This induction of CA can lead to the selection of cells with upregulation of KIFC1 to prevent spindle multipolarity and death of progeny cells. KIFC1 expression was found to be higher in p53 mutant or null tumors compared to p53 wild-type tumors [70]. Inhibition of KIFC1 gene expression was shown to upregulate p21 and downregulate CDK2 to arrest cells in G2–M phases [32]. Furthermore, KIFC1 depletion in primary human fibroblast cells displayed features of senescence, such as β-galactosidase expression [71]. These findings suggest that KIFC1 may be upregulated as a result of Rb or p53 deregulation to allow genomically unstable cells to continue replicating and avoid cellular senescence. Thus, KIFC1 may play a key role in conferring normal HMECs, deregulated in Rb or p53, the ability to continue replicating and therefore successfully bypass the stasis barrier. An Rb loss-of-function signature was established in The Cancer Genome Atlas by identifying genes that correlate with E2F1 and E2F2 expression in breast cancer, and KIFC1 emerged as one of the top genes included in the signature [72].

KIFC1 may help pre-neoplastic cells bypass the stasis barrier through alternative mechanisms. KIFC1 may assist cells with CA that activate p53, as a result of causing mitotic defects, to avert apoptosis, allowing these cells to persist [61]. Additionally, KIFC1 has been shown to protect the apoptosis resistance protein survivin from degradation by E3 ligase APC/C [35]. KIFC1 overexpression in cancer cells was found to accelerate cell cycle kinetics, particularly from G2 to M phase. Specifically, KIFC1 overexpression upregulated survival signals such as phosphorylated Bcl2, Aurora B kinase, cyclin B1, D1, and A [35]. Cyclin D1 overexpression in particular has been shown to facilitate bypassing of the stasis barrier in normal HMECs [35,43].

As previously discussed, the ability of normal or post-stasis HMECs to correct telomere dysfunction or continue replicating with critically shortened telomeres can allow this subset of cells to bypass the second and one of the most important senescence barriers to immortalization. Telomere dysfunction as a result of telomere attrition (critical shortening of chromosomal telomeres), also known as replicative senescence, has been shown to elicit genomic instability/CIN and activation of DNA damage response repair pathways. Specifically, telomere dysfunction can induce breakage–fusion–bridge cycles that can cause structural and numerical chromosomal aberrations resulting in CIN and aneuploidy [23,73].

However, evidence exists suggesting that centrosomal aberrations precede the emergence of TP53 mutations and end-to-end fusions during early carcinogenesis [16,74]. Telomere dysfunction has been suggested to induce supernumerary centrosomes and shown to directly correlate with increased levels of CA in HMECs, cancer cell lines, and tumor tissue. Specifically, genotoxic stress-mediated telomere dysfunction via perturbation of p16 activity in post-stasis HMECs induced the presence of centriole overduplication [64]. This stimulation of telomere dysfunction via genotoxic stress promoted localization of telomerase transcriptional elements-interacting factor (TEIF), a transactivator of human telomerase reverse transcriptase subunit, to the centrosome and induced CA [24]. TEIF was found to positively correlate with CA in colorectal tumors [75]. Telomere dysfunction induced in Drosophila oogenesis caused deregulation of centrosome biogenesis, leading to embryonic lethality [22]. Since CA also elicits CIN and genomic instability, telomere dysfunction may cause CIN and aneuploidy to arise in pre-neoplastic HMECs or early stage lesions.

KIFC1 is possibly upregulated upon induction of CA as a result of telomere dysfunction. This upregulation can confer a survival advantage among cells with dysfunctional telomeres. Thus, KIFC1 may promote the survival of cells with loss of telomere function. Therefore, KIFC1 may act as a key player in facilitating pre-neoplastic cells that have acquired telomere abnormalities to bypass the replicative cellular senescence barrier. Inhibiting KIFC1, and subsequently declustering extra centrosomes, could potentially induce apoptosis of telomere-dysfunctional cells. KIFC1 phosphorylation induced upon DNA damaging agent treatment conferred cancer cell resistance to this therapy, and inhibition of this KIFC1 phosphorylation repressed centrosome clustering and tumor recurrence [76]. An alternative theory on KIFC1′s potential role in bypassing this senescence barrier is that through its involvement in promoting phenotypic diversity, it may be fostering the survival of subcellular clones that selectively reactivate telomerase.

The mechanisms underlying oncogene-induced senescence remain poorly elucidated. However, HMECs that have attained immortality via reactivation of telomerase are not vulnerable to this senescence barrier but exhibit the acquisition of malignant properties after overexpression of Raf-1, Ras, or ErbB2 [42]. Induction of K-Ras, alone or co-expressed with c-Myc (transcription factor downstream of Ras signaling), was shown to induce CA in premalignant human mammary glands and HMECs via dysregulation of key regulators of the centrosome duplication cycle, Cyclin D1/CDK4 and Nek2 [77]. Transduction of post-stasis HMECs with c-myc was shown to promote bypassing of the replicative senescence barrier [43]. Thus, KIFC1 may be upregulated upon Ras-induced CA and thereby confer survival of these cells. Knockdown of KIFC1 was found to inhibit the MAPK signaling cascade and downstream signaling, suggesting that KIFC1 inhibition may abrogate Ras-induced CA. These findings suggest that HMECs upregulated in oncogenic Ras signaling may be bypassing the oncogenic-induced senescence barrier by upregulating KIFC1 as a mechanism to cope with Ras-stimulated CA.

The epithelial–mesenchymal transition (EMT) process confers mesenchymal characteristics that impart invasive capabilities [78]. The EMT process is marked by loss of the epithelial cell junction protein E-cadherin and upregulation of the transcriptional factors N-cadherin, Snail, and ZEB1. Downregulation of E-cadherin is necessary for KIFC1 to efficiently cluster extra centrosomes by increasing cortical contractility [79]. Thus, low clustering capacity was found to correlate with high levels of E-cadherin. Knockdown of KIFC1 increased expression of E-cadherin and decreased expression of N-cadherin, Snail, and ZEB1 in cancer cells [80]. Thus, the existence of CA and concomitant upregulation of KIFC1 in pre-invasive lesions may also be accompanied by loss of E-cadherin at cell–cell junctions, thereby additionally imparting the ability to invade surrounding tissue to these lesions. Hence, KIFC1-mediated centrosome clustering may impart invasive capabilities to pre-invasive cells via downregulation of E-cadherin.