TPX2 overexpression promotes sensitivity to dasatinib in breast cancer by activating YAP transcriptional signaling.

Chromosomal instability (CIN) is a hallmark of cancer aggressiveness, providing genetic plasticity and tumor heterogeneity that allows the tumor to evolve and adapt to stress conditions. CIN is considered a cancer therapeutic biomarker because healthy cells do not exhibit CIN. Despite recent efforts to identify therapeutic strategies related to CIN, the results obtained have been very limited. CIN is characterized by a genetic signature where a collection of genes, mostly mitotic regulators, are overexpressed in CIN-positive tumors, providing aggressiveness and poor prognosis. We attempted to identify new therapeutic strategies related to CIN genes by performing a drug screen, using cells that individually express CIN-associated genes in an inducible manner. We find that the overexpression of targeting protein for Xklp2 (TPX2) enhances sensitivity to the proto-oncogene c-Src (SRC) inhibitor dasatinib due to activation of the Yes-associated protein 1 (YAP) pathway. Furthermore, using breast cancer data from The Cancer Genome Atlas (TCGA) and a cohort of cancer-derived patient samples, we find that both TPX2 overexpression and YAP activation are present in a significant percentage of cancer tumor samples and are associated with poor prognosis; therefore, they are putative biomarkers for selection for dasatinib therapy.

Nuclear VANGL2 Inhibits Lactogenic Differentiation.

Planar cell polarity (PCP) proteins coordinate tissue morphogenesis by governing cell patterning and polarity. Asymmetrically localized on the plasma membrane of cells, transmembrane PCP proteins are trafficked by endocytosis, suggesting they may have intracellular functions that are dependent or independent of their extracellular role, but whether these functions extend to transcriptional control remains unknown. Here, we show the nuclear localization of transmembrane, PCP protein, VANGL2, in the HCC1569 breast cancer cell line, and in undifferentiated, but not differentiated, HC11 cells that serve as a model for mammary lactogenic differentiation. The loss of Vangl2 function results in upregulation of pathways related to STAT5 signaling. We identify DNA binding sites and a nuclear localization signal in VANGL2, and use CUT&RUN to demonstrate recruitment of VANGL2 to specific DNA binding motifs, including one in the Stat5a promoter. Knockdown (KD) of Vangl2 in HC11 cells and primary mammary organoids results in upregulation of Stat5a, Ccnd1 and Csn2, larger acini and organoids, and precocious differentiation; phenotypes are rescued by overexpression of Vangl2, but not Vangl2ΔNLS. Together, these results advance a paradigm whereby PCP proteins coordinate tissue morphogenesis by keeping transcriptional programs governing differentiation in check.

Nuclear VANGL2 Inhibits Lactogenic Differentiation.

Planar cell polarity (PCP) proteins coordinate tissue morphogenesis by governing cell patterning and polarity. Asymmetrically localized on the plasma membrane of cells, PCP proteins are also trafficked by endocytosis, suggesting they may have intracellular functions that are dependent or independent of their extracellular role, but whether these functions extend to transcriptional control remains unknown. Here, we show the nuclear localization of transmembrane, PCP protein, VANGL2, in undifferentiated, but not differentiated, HC11 cells, which serve as a model for mammary lactogenic differentiation. Loss of Vangl2 function results in upregulation of pathways related to STAT5 signaling. We identify DNA binding sites and a nuclear localization signal in VANGL2, and use CUT&RUN to demonstrate direct binding of VANGL2 to specific DNA binding motifs, including one in the Stat5a promoter. Knockdown (KD) of Vangl2 in HC11 cells and primary mammary organoids results in upregulation of Stat5a , Ccnd1 and Csn2 , larger acini and organoids, and precocious differentiation; phenotypes rescued by overexpression of Vangl2 , but not Vangl2 ΔNLS . Together, these results advance a paradigm whereby PCP proteins coordinate tissue morphogenesis by keeping transcriptional programs governing differentiation in check.

Whole-Genome Doubling as a source of cancer: how, when, where, and why?

Chromosome instability is a well-known hallmark of cancer, leading to increased genetic plasticity of tumoral cells, which favors cancer aggressiveness, and poor prognosis. One of the main sources of chromosomal instability are events that lead to a Whole-Genome Duplication (WGD) and the subsequently generated cell polyploidy. In recent years, several studies showed that WGD occurs at the early stages of cell transformation, which allows cells to later become aneuploid, thus leading to cancer progression. On the other hand, other studies convey that polyploidy plays a tumor suppressor role, by inducing cell cycle arrest, cell senescence, apoptosis, and even prompting cell differentiation, depending on the tissue cell type. There is still a gap in understanding how cells that underwent WGD can overcome the deleterious effect on cell fitness and evolve to become tumoral. Some laboratories in the chromosomal instability field recently explored this paradox, finding biomarkers that modulate polyploid cells to become oncogenic. This review brings a historical view of how WGD and polyploidy impact cell fitness and cancer progression, and bring together the last studies that describe the genes helping cells to adapt to polyploidy.

DNA damage triggers squamous metaplasia in human lung and mammary cells via mitotic checkpoints.

Epithelial transdifferentiation is frequent in tissue hyperplasia and contributes to disease in various degrees. Squamous metaplasia (SQM) precedes epidermoid lung cancer, an aggressive and frequent malignancy, but it is rare in the epithelium of the mammary gland. The mechanisms leading to SQM in the lung have been very poorly investigated. We have studied this issue on human freshly isolated cells and organoids. Here we show that human lung or mammary cells strikingly undergo SQM with polyploidisation when they are exposed to genotoxic or mitotic drugs, such as Doxorubicin or the cigarette carcinogen DMBA, Nocodazole, Taxol or inhibitors of Aurora-B kinase or Polo-like kinase. To note, the epidermoid response was attenuated when DNA repair was enhanced by Enoxacin or when mitotic checkpoints where abrogated by inhibition of Chk1 and Chk2. The results show that DNA damage has the potential to drive SQM via mitotic checkpoints, thus providing novel molecular candidate targets to tackle lung SCC. Our findings might also explain why SCC is frequent in the lung, but not in the mammary gland and why chemotherapy often causes complicating skin toxicity.

Squamous differentiation requires G2/mitosis slippage to avoid apoptosis.

The cellular mechanisms controlling cell fate in self-renewal tissues remain unclear. Cell cycle failure often leads to an apoptosis anti-oncogenic response. We have inactivated Cdk1 or Polo-like-1 kinases, essential targets of the mitotic checkpoints, in the epithelia of skin and oral mucosa. Here, we show that inactivation of the mitotic kinases leading to polyploidy in vivo, produces a fully differentiated epithelium. Cells within the basal layer aberrantly differentiate and contain large or various nuclei. Freshly isolated KO cells were also differentiated and polyploid. However, sustained metaphase arrest downstream of the spindle anaphase checkpoint (SAC) due to abrogation of CDC20 (essential cofactor of anaphase-promoting complex), impaired squamous differentiation and resulted in apoptosis. Therefore, upon prolonged arrest keratinocytes need to slip beyond G2 or mitosis in order to initiate differentiation. The results altogether demonstrate that mitotic checkpoints drive squamous cell fate towards differentiation or apoptosis in response to genetic damage.

Keratinocyte Differentiation by Flow Cytometry.

The epidermis is continuously exposed to environmental hazard and undergoes continuous cell renewal. The maintenance of the epidermal balance between proliferation and differentiation is essential for the homeostasis of the skin. Proliferation and terminal differentiation are compartmentalized in basal and suprabasal layers, respectively. These compartments can be identified by different patterns of protein expression that can be used as differentiation markers. For instance, components of the intermediate filament cytoskeleton keratins K5 and K14 are confined to the proliferative basal layer, while keratins K1 and K10, keratins K6 and K16, or precursors of the cornified envelope such as involucrin are expressed by suprabasal terminally differentiating cells. The analysis of the expression of these markers allows studying the imbalance typical of disease. Although these markers have been traditionally analyzed on skin microsections, on attached cells by immunostaining or by western blotting, it is possible and advantageous to quantify them by flow cytometry. We have extensively applied this technology onto human and mouse keratinocytes. Here we describe detailed flow cytometry methods to determine the differentiation status of keratinocyte populations.

Genetic Modification of Human Primary Keratinocytes by Lentiviral Vectors.

Keratinocytes are hard to transfect. Viral vectors are a good alternative to genetically modify primary keratinocytes. A classical method is the use of retroviral vectors by co-culture of keratinocytes with virus-producer cells. This method is efficient in high-calcium conditions with feeder cells. However, sometimes co-culture is not possible and is more laborious as producer cells need to be replaced by feeder cells. Our solution is the use of lentiviral vectors, far more efficient as supernatant on keratinocytes. In this chapter we describe improved detailed protocols for stable genetic modification of human primary keratinocytes of the skin or head and neck, in both low- and high-calcium conditions by lentiviral vectors.

Response of head and neck epithelial cells to a DNA damage-differentiation checkpoint involving polyploidization.

BACKGROUND: Squamous epithelia of the head and neck undergo continuous cell renewal and are continuously exposed to mutagenic hazard, the main cause of cancer. How they maintain homeostasis upon cell cycle deregulation is unclear. METHODS: To elucidate how head and neck epithelia respond to cell cycle stress, we studied human keratinocytes from various locations (oral mucosa, tonsil, pharynx, larynx, and trachea). We made use of genotoxic or mitotic drugs (doxorubicin [DOXO], paclitaxel, and nocodazole), or chemical inhibitors of the mitotic checkpoint kinases, Aurora B and polo-like-1. We further tested the response to inactivation of p53, ectopic cyclin E, or to the chemical carcinogen 7,12-dimethylbenz[a]anthracene (DMBA). RESULTS: All treatments provoked DNA damage or mitosis impairment and strikingly triggered squamous differentiation and polyploidization, resulting in irreversible loss of clonogenic capacity. CONCLUSION: Keratinocytes from head and neck epithelia share a cell-autonomous squamous DNA damage-differentiation response that is common to the epidermis and might continuously protect them from cancer.

Sublethal UV irradiation induces squamous differentiation via a p53-independent, DNA damage-mitosis checkpoint.

The epidermis is a self-renewal epithelium continuously exposed to the genotoxic effects of ultraviolet (UV) light, the main cause of skin cancer. Therefore, it needs robust self-protective mechanisms facing genomic damage. p53 has been shown to mediate apoptosis in sunburn cells of the epidermis. However, epidermal cells daily receive sublethal mutagenic doses of UV and massive apoptosis would be deleterious. We have recently unravelled an anti-oncogenic keratinocyte DNA damage-differentiation response to cell cycle stress. We now have studied this response to high or moderate single doses of UV irradiation. Whereas, as expected, high levels of UV induced p53-dependent apoptosis, moderate levels triggered squamous differentiation. UV-induced differentiation was not mediated by endogenous p53. Overexpression of the mitosis global regulator FOXM1 alleviated the proliferative loss caused by UV. Conversely, knocking-down the mitotic checkpoint protein Wee1 drove UV-induced differentiation into apoptosis. Therefore, the results indicate that mitosis checkpoints determine the response to UV irradiation. The differentiation response was also found in cells of head and neck epithelia thus uncovering a common regulation in squamous tissues upon chronic exposure to mutagens, with implications into homeostasis and disease.

Mammalian endoreplication emerges to reveal a potential developmental timer.

Among the most intriguing and relevant questions in physiology is how developing tissues correctly coordinate proliferation with differentiation. Endoreplication, in a broad sense, is a consequence of a cell division block in the presence of an active cell cycle, and it typically occurs as cells differentiate terminally to fulfill a specialised function. Until recently, endoreplication was thought to be a rare variation of the cell cycle in mammals, more common in invertebrates and plants. However, in the last years, endoreplication has been uncovered in various tissues in mammalian organisms, including human. A recent report showing that cells in the mammary gland become binucleate at lactation sheds new insight into the importance of mammalian polyploidisation. We here propose that endoreplication is a widespread phenomenon in mammalian developing tissues that results from an automatic, robust and simple self-limiting mechanism coordinating cell multiplication with differentiation. This mechanism might act as a developmental timer. The model has implications for homeostasis control and carcinogenesis.