Multifunctional roles of urokinase plasminogen activator (uPA) in cancer stemness and chemoresistance of pancreatic cancer

Pancreatic ductal adenocarcinoma (PDAC) is almost always lethal. One of the underlying reasons for this lethality is believed to be the presence of cancer stem cells (CSC), which impart chemoresistance and promote recurrence, but the mechanisms responsible are unclear. Recently the poor prognosis of PDAC has been correlated with increased expression of urokinase plasminogen activator (uPA). In the present study we examine the role of uPA in the generation of PDAC CSC. We observe a subset of cells identifiable as a side population (SP) when sorted by flow cytometry of MIA PaCa-2 and PANC-1 pancreatic cancer cells that possess the properties of CSC. A large fraction of these SP cells are CD44 and CD24 positive, are gemcitabine resistant, possess sphere-forming ability, and exhibit increased tumorigenicity, known characteristics of cancer stemness. Increased tumorigenicity and gemcitabine resistance decrease after suppression of uPA. We observe that uPA interacts directly with transcription factors LIM homeobox-2 (Lhx2), homeobox transcription factor A5 (HOXA5), and Hey to possibly promote cancer stemness. uPA regulates Lhx2 expression by suppressing expression of miR-124 and p53 expression by repressing its promoter by inactivating HOXA5. These results demonstrate that regulation of gene transcription by uPA contributes to cancer stemness and clinical lethality

HLA Class I and II Blocks Are Associated to Susceptibility, Clinical Subtypes and Autoantibodies in Mexican Systemic Sclerosis (SSc) Patients

<div><p>Introduction</p><p>Human leukocyte antigen (HLA) polymorphism studies in Systemic Sclerosis (SSc) have yielded variable results. These studies need to consider the genetic admixture of the studied population. Here we used our previously reported definition of genetic admixture of Mexicans using HLA class I and II DNA blocks to map genetic susceptibility to develop SSc and its complications.</p><p>Methods</p><p>We included 159 patients from a cohort of Mexican Mestizo SSc patients. We performed clinical evaluation, obtained SSc-associated antibodies, and determined HLA class I and class II alleles using sequence-based, high-resolution techniques to evaluate the contribution of these genes to SSc susceptibility, their correlation with the clinical and autoantibody profile and the prevalence of Amerindian, Caucasian and African alleles, blocks and haplotypes in this population.</p><p>Results</p><p>Our study revealed that class I block HLA-C*12:03-B*18:01 was important to map susceptibility to diffuse cutaneous (dc) SSc, HLA-C*07:01-B*08:01 block to map the susceptibility role of HLA-B*08:01 to develop SSc, and the C*07:02-B*39:05 and C*07:02-B*39:06 blocks to map the protective role of C*07:02 in SSc. We also confirmed previous associations of HLA-DRB1*11:04 and –DRB1*01 to susceptibility to develop SSc. Importantly, we mapped the protective role of DQB1*03:01 using three Amerindian blocks. We also found a significant association for the presence of anti-Topoisomerase I antibody with HLA-DQB1*04:02, present in an Amerindian block (DRB1*08:02-DQB1*04:02), and we found several alleles associated to internal organ damage. The admixture estimations revealed a lower proportion of the Amerindian genetic component among SSc patients.</p><p>Conclusion</p><p>This is the first report of the diversity of HLA class I and II alleles and haplotypes Mexican patients with SSc. Our findings suggest that HLA class I and class II genes contribute to the protection and susceptibility to develop SSc and its different clinical presentations as well as different autoantibody profiles in Mexicans.</p></div

HLA class II genes associations with specific autoantibodies in SSc.

<p>anti-RNAP I/III: anti-RNA polymerase I/III.</p><p>HLA class II genes associations with specific autoantibodies in SSc.</p

Frequency of SSc-associated autoantibodies in our SSc patients.

<p>dcSSc: diffuse cutaneous systemic sclerosis, lcSSc: limited cutaneous systemic sclerosis, Anti-RNA Pol III: anti-RNA polymerase I/III.</p><p>Frequency of SSc-associated autoantibodies in our SSc patients.</p

Demographic data and organ damage according to the modified Medsger’s Severity Scale.

<p>dcSSc: diffuse cutaneous systemic sclerosis, lcSSc: limited cutaneous systemic sclerosis, ILD: interstitial lung disease; PAH: pulmonary arterial hypertension.</p><p>Demographic data and organ damage according to the modified Medsger’s Severity Scale.</p

Principal component analysis (PCA) plot reveals a close genetic relationship of Mexican admixed SSc patients and healthy controls (HC) from Mexico City to Native American groups.

<p>Native American populations are represented in the upper left of the graphic and Caucasian components in the right bottom area of the graphic. Amerindian components are represented in the left bottom area. Red and blue dots represent difusse and limited SSc patients respectively and the total group in represented in green. The different populations included in the PCA analysis were: Ire: Ireland, Eng: England, Ger: Germany, Aus: Austria; Spa: Spain, Ita: Italy, UK: United Kingdom, Fra: France, Azo: Azores, Sao: São Tomé Island, Cam: Cameroon, Mal: Mali, Zam: Zambia, KLu: Luo from Kenia, KNa: Nandi from Kenia, Sen: Senegal, Gui: Guinea Bissau, Tar: Tarahumara, Gil: Native Americans from Gila River, Yup: Yu’pik from Alaska, Mit: Mixtec from Oaxaca, Zap: Zapotec from Oaxaca, Mix: Mixe from Oaxaca, Ser: Seri from Sonora, Nav: Navajo from New Mexico, HC: “Mexican Admixed controls” [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0126727#pone.0126727.ref026" target="_blank">26</a>].</p

Frequencies of <i>HLA-DRB1-DQB1</i> blocks in 234 Mexican admixed individuals (468 haplotypes).

<p>Blocks of each ancestry (Amerindian, Caucasian, Caucasian shared with other populations, African and Asian) were defined as those found in original populations with H.F. >1,0%, and not found in other native human groups in frequencies higher than 1,0%. We consider <i>t</i> value must be ≥2.0 to denote statistically significant association and thus validate Δ′ (shaded values).^† Similar to DQB1*05∶02 with a silent mutation at codon 133 cgg>cga.</p

Measures of genetic diversity at the allele level for HLA system in a Mexican admixed population.

<p>O.H.: Observed heterozygosity. E.H.: Expected Heterozygosity. <i>p</i> values <0.05 are considered statistically significative and thus reflect differences between O.H. and E.H. PIC: Polymorphism Information Contents. PD: Power of Discrimination.</p

Extension of HLA Conserved Extended Haplotypes to <i>HLA-A</i> in 234 Mexican Admixed individuals.

<p>Blocks of each ancestry (Amerindian, Caucasian, Caucasian shared with other populations, African and Asian) were defined as those found in original populations with H.F. >1,0%, and not found in other native human groups in frequencies higher than 1,0%. We consider <i>t</i> value must be ≥2.0 to denote statistically significant association and thus validate Δ′ (shaded values).</p

Frequencies of <i>HLA-C-B</i> blocks in 234 admixed Mexican individuals (468 haplotypes).

<p>Blocks of each ancestry (Amerindian, Caucasian, Caucasian shared with other populations, African, and Asian) were defined as those found in original populations with H.F. >1,0%, and not found in other native human groups in frequencies higher than 1,0%. We consider <i>t</i> value must be ≥2.0 to denote statistically significant association and thus validate Δ′ (shaded values).</p>*<p>Similar to B*35∶01 with a mutation at codon 207 ggc>tgc (Gly>Cys).</p>§<p>Similar to C*03∶04 with a mutation at codon 189 gtg>atg (Val>Met).</p